DcR3 variants

Abstract

The present invention relates to a DcR3 variant, DNA encoding the DcR3 variant, a vector containing the DNA, a transformant obtained by introducing the vector, a method for producing a variant using the transformant, and a prophylactic or therapeutic agent for an autoimmune disease, an inflammatory disease or an allergic disease comprising the variant as an active ingredient. The present invention relates to a DcR3 variant having binding activity (preferably neutralizing activity) against a ligand of DcR3 while showing reduced production of aggregates and / or improved in vivo kinetics compared to wild-type DcR3 when prepared with a mammalian-derived cell as a host, and provides a DcR3 variant comprising a portion of DcR3 and a portion of a TNF superfamily molecule.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on September 11, 2020, with Chinese application number 202080064092.3 and invention title "DcR3 variant". Technical Field

[0002] This invention relates to DcR3 variants, which are variants of wild-type DcR3. More specifically, this invention relates to DcR3 variants that have binding activity (preferably neutralizing activity) against ligands of DcR3, and, when prepared using mammalian cells as a host, exhibit reduced aggregate formation and / or improved in vivo kinetics compared to wild-type DcR3. Background Technology

[0003] The tumor necrosis factor (TNF) superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) form structurally similar families of 18 ligands and 29 receptors, respectively. Antibodies and Fc fusion proteins targeting multiple molecules included in these families have been developed and marketed, showing therapeutic effects in the treatment of various autoimmune diseases (Non-Patent Literature 1).

[0004] Many TNFRSFs are expressed on the cell membrane and transmit signals downstream via ligand binding, but some molecules are decoy receptors (DcRs) that do not participate in signal transduction. To date, four decoy receptors have been identified: DcR1, DcR2, DcR3, and OPG (osteoprotegerin).

[0005] OPG is a soluble trapping receptor for RANKL and TRAIL, inhibiting signal transduction by competitively binding to ligands of the RANKL and TRAIL receptors. On the other hand, DcR1 and DcR2 are trapping receptors for TRAIL, and DcR3 is a trapping receptor for LIGHT, TL1A, and FasL, all of which neutralize ligands by competitively inhibiting the binding of the receptors that transmit signals (Non-Patent Literature 2).

[0006] DcR3 is a soluble molecule composed of 300 amino acid residues. It has a signal peptide at the N-terminus, followed by four cysteine-rich domains (CRDs) characteristic of TNFRSF (CRD1, CRD2, CRD3, and CRD4), and a heparan sulfate-binding region (HBD) rich in basic amino acids, containing a heparan sulfate-binding motif. Any of LIGHT, TL1A, and FasL are mediated by the binding of CRD2 and CRD3 of DcR3 (Non-Patent Literature 3, 4, 5).

[0007] In addition to its function as a trapping receptor based on ligand neutralization, DcR3 also functions as an immunomodulatory molecule based on HBD activity. For example, it has been reported that direct binding of glycosaminoglycans (GAGs), represented by heparan sulfate on the cell membranes of monocytes, macrophages, or dendritic cells, via HBD induces various immunosuppressive and immunoactivating effects, such as Th2 induction based on dendritic cell differentiation, M2 macrophage induction, increased monocyte adhesion, osteoclast differentiation, or decreased expression of MHC type II molecules (Non-Patent Literature 6, 12).

[0008] The involvement of DcR3 ligands in autoimmune diseases, inflammatory diseases, allergies, cancer, infectious diseases, and various other inflammatory responses has been reported. For example, LIGHT, TL1A, and FasL are all included in inflammatory bowel disease (IBD)-sensitive loci, and multiple gene polymorphisms associated with TL1A are known to be relevant to the disease. Furthermore, reports have documented increased expression of DcR3 ligands in the blood or tissues of IBD patients, and disease improvement resulting from DcR3 ligand inhibition in mouse models of enteritis (Non-Patent Literature 6-9).

[0009] DcR3 expression is very low in normal human tissues, but its expression is induced by infection or tissue damage. Elevated blood levels of DcR3 are also known in various autoimmune or inflammatory diseases such as IBD, SLE, atopic dermatitis (AD), or rheumatoid arthritis (RA). Furthermore, although no DcR3 homologs have been identified in mice, disease improvement in transgenic mice with human DcR3 and efficacy based on plasmid or recombinant DcR3 administration have been confirmed in mouse disease models such as type 1 diabetes, multiple sclerosis, and nephritis (Non-Patent Literature 6, 10).

[0010] Genentech cloned the human DcR3 gene and demonstrated that the fusion of DcR3 with the Fc region of human IgG1 binds to soluble human FasL and inhibits human FasL-dependent apoptosis in vitro (Patent Document 1).

[0011] Eli Lilly obtained FLINT, a protease-resistant DcR3 mutant, through a single amino acid mutation (R218Q) in wild-type DcR3, and reported improved in vivo pharmacokinetics in mice and monkeys compared to wild-type DcR3. However, the blood half-lives of wild-type DcR3 and FLINT administered intravenously at 0.5 mg / kg to cynomolgus monkeys were extremely short, at 9 hours and 12.3 hours, respectively (Patent Literature 2, 3, Non-Patent Literature 11).

[0012] Existing technical documents

[0013] Patent documents Patent Document 1: Japanese Patent No. 4303883 Patent Document 2: US Patent 6,835,814 B1 Patent Document 3: US Patent 6,965,012 B1

[0014] Non-patent literature Non-patent literature 1: Nature Reviews Drug Discovery, 2013, 12: pp. 147-168 Non-patent literature 2: Nature Reviews Cancer, 2002, 2: pp. 420-430 Non-patent literature 3: Structure, 2011, 19: pp. 162-171 Non-patent literature 4: Structure, 2014, 22: pp. 1252-1262 Non-patent literature 5: Structure, 2016, 24: pp. 2016-2023 Non-patent literature 6: Biochemical Pharmacology, 2011, 81: pp. 838-847 Non-patent literature 7: Immunology, 2009, 128: pp. 451-458 Non-Patent Literature 8: PNAS, 2006, 103: pp. 8441-8446 Non-patent literature 9: Am.J. Physiol. Gastrointest. Liver Physiol., 2003, 285: p. G754-G760 Non-patent literature 10: Journal of Biomedical Science, 2017, 24: 39 Non-patent literature 11: Drug Metabolism and Disposition, 2003, 31: pp. 502-507 Non-patent literature 12: J. Immunol., 2006, 176: pp. 173-180 Summary of the Invention

[0015] The problem that the invention aims to solve

[0016] FLINT, an amino acid variant of wild-type DcR3, exhibits significantly poor in vivo pharmacokinetics. As a recombinant formulation with a ligand-neutralizing mechanism of action, it requires multiple administrations, making it unpromising as a drug. Therefore, it is hoped that DcR3 variants with improved in vivo pharmacokinetics, thereby ensuring a consistent dosing interval, can be used as drugs.

[0017] Furthermore, to date, no functional DcR3 variant has been found that exhibits reduced clotting volume and neutralizing activity against DcR3 ligands when expressed, isolated, and purified using mammalian cells as hosts.

[0018] The present invention aims to provide the following DcR3 variants, DNA encoding the DcR3 variants, vectors containing the DNA, transformants obtained by introducing the vectors, methods for manufacturing variants using the transformants, and pharmaceutical compositions and preventive or therapeutic agents for autoimmune diseases, inflammatory diseases, or allergies using the variants as active ingredients. The DcR3 variants have binding activity (preferably neutralizing activity) against ligands of DcR3, and when DcR3 proteins are prepared using cells derived from mammals as hosts, the amount of lectin formation is reduced and / or improved in vivo kinetics are observed compared to wild-type DcR3.

[0019] Methods for solving problems

[0020] To address the aforementioned issues, the present invention provides the following invention.

[0021] [1] The DcR3 variant is a variant of the wild-type Decoy Receptor 3 (hereinafter referred to as DcR3), which exhibits improved in vivo dynamics compared to the previously mentioned wild-type DcR3.

[0022] [2] The DcR3 variant described in [1] has a complex sugar chain with more than one N-glycosidic bond.

[0023] [3] The DcR3 variants described in [1] or [2] have neutralizing activity against at least one of LIGHT, TL1A and FasL.

[0024] [4] The DcR3 variants described in any of [1] to [3] have neutralizing activity against LIGHT, TL1A and FasL.

[0025] [5] The DcR3 variant described in any one of [1] to [3] has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A or more.

[0026] [6] The DcR3 variants described in any of [1] to [3] and [5] have no neutralizing activity against FasL and have neutralizing activity against LIGHT and TL1A.

[0027] [7] A DcR3 variant comprising a first chimeric cysteine ​​enriched region or a second chimeric cysteine ​​enriched region, wherein the first chimeric cysteine ​​enriched region comprises an amino acid sequence obtained by replacing at least a portion of the cysteine ​​enriched domain of the wild-type DcR3 with at least a portion of the cysteine ​​enriched domain of a TNF receptor superfamily molecule other than DcR3 (hereinafter referred to as CRD), and the second chimeric cysteine ​​enriched region comprises an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 30 amino acids in the amino acid sequence of the first chimeric cysteine ​​enriched region.

[0028] [8] The DcR3 variant described in [7] has a complex glycan chain with more than one N-glycosidic bond.

[0029] [9] The DcR3 variants described in [7] or [8] have neutralizing activity against at least one of LIGHT, TL1A and FasL.

[0030]

[10] The DcR3 variants described in any of [7] to [9] have neutralizing activity against LIGHT, TL1A and FasL.

[0031]

[11] The DcR3 variant described in any of [7] to [9] has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A or more.

[0032]

[12] The DcR3 variants described in any of [7] to [9] and

[11] have no neutralizing activity against FasL and have neutralizing activity against LIGHT and TL1A.

[0033]

[13] The DcR3 variant as described in any one of [7] to

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

[0034]

[14] The DcR3 variant as described in any one of [7] to

[13] , wherein at least a portion of the cysteine-enriched domain of the aforementioned wild-type DcR3 is selected from all or a portion of CRD1, all or a portion of CRD2, all or a portion of CRD3, and all or a portion of CRD4.

[0035] At least a portion of the cysteine-enriched domains of the aforementioned TNF receptor superfamily molecules are selected from all or a portion of CRD1, all or a portion of CRD2, all or a portion of CRD3, and all or a portion of CRD4.

[0036]

[15] The DcR3 variant as described in

[14] , wherein the aforementioned first chimeric cysteine ​​enrichment region has one or more substitutions selected from the following: A portion of the CRD1 of the aforementioned wild-type DcR3 was replaced by a portion of the CRD1 of the aforementioned TNF receptor superfamily molecule that corresponds to a portion of the CRD1 of the aforementioned wild-type DcR3. The entire CRD1 of the aforementioned wild-type DcR3 was replaced by the entire CRD1 of the aforementioned TNF receptor superfamily molecules; A portion of the CRD2 of the aforementioned wild-type DcR3 was replaced by a portion of the CRD2 of the aforementioned TNF receptor superfamily molecule that corresponds to a portion of the CRD2 of the aforementioned wild-type DcR3. The entire CRD2 of the aforementioned wild-type DcR3 was replaced by the entire CRD2 of the aforementioned TNF receptor superfamily molecules; A portion of the CRD3 of the aforementioned wild-type DcR3 was replaced by a portion of the CRD3 of the aforementioned TNF receptor superfamily molecule that corresponds to a portion of the CRD3 of the aforementioned wild-type DcR3. The entire CRD3 of the aforementioned wild-type DcR3 was replaced by the entire CRD3 of the aforementioned TNF receptor superfamily molecules; A portion of the CRD4 of the aforementioned wild-type DcR3 was replaced by a portion of the CRD4 of the aforementioned TNF receptor superfamily molecule that corresponds to a portion of the CRD4 of the aforementioned wild-type DcR3; and All of the aforementioned wild-type DcR3 CRD4s were replaced by all of the aforementioned TNF receptor superfamily CRD4s.

[0037]

[16] The DcR3 variant described in

[15] retains part or all of the aforementioned wild-type DcR3 CRD2 in the aforementioned first chimeric cysteine ​​enrichment region.

[0038]

[17] The DcR3 variant as described in

[15] or

[16] retains part or all of the aforementioned wild-type DcR3 CRD3 in the aforementioned first chimeric cysteine ​​enrichment region.

[0039]

[18] The DcR3 variant as described in any of

[14] to

[17] , wherein the aforementioned first chimeric cysteine-rich region comprises the amino acid sequence of (a), (b), (c), or (d) below, and the aforementioned second chimeric cysteine-rich region comprises the amino acid sequence of (e) below: (a) The amino acid sequence in the cysteine-rich region of the aforementioned wild-type DcR3 where the CRD1 of wild-type DcR3 is replaced by the CRD1 of OPG. (b) In the amino acid sequence of the cysteine-rich region of the aforementioned wild-type DcR3, the amino acid sequence in which the CRD4 of wild-type DcR3 is replaced by the CRD4 of OPG. (c) In the amino acid sequence of the cysteine-rich region of the aforementioned wild-type DcR3, the amino acid sequence in which the CRD1 of wild-type DcR3 is replaced by the CRD1 of OPG and the CRD4 of wild-type DcR3 is replaced by the CRD4 of OPG. (d) In the amino acid sequences of (a), (b), or (c) above, the portion from position 103 to position 123 from the N-terminus is replaced by the corresponding portion of the amino acid sequence of the cysteine ​​enrichment domain of OPG. (e) An amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 30 amino acids in the amino acid sequences of (a), (b), (c) or (d) above.

[0040]

[19] The DcR3 variant as described in

[18] , wherein, The amino acid sequence mentioned above (a) is the amino acid sequence containing the amino acids from the N-terminus 1 to 164 of the amino acid sequence recorded in sequence number 26 or 50. The amino acid sequence mentioned above (b) is the amino acid sequence containing the amino acids from the N-terminus 1 to 164 of the amino acid sequence recorded in sequence number 28 or 52. The amino acid sequence mentioned above (c) is the amino acid sequence containing the amino acids from position 1 to position 164 from the N-terminus of the amino acid sequence recorded in sequence number 30 or 54. The amino acid sequence mentioned above (d) is the amino acid sequence containing the amino acid from position 1 to position 164 from the N-terminus of the amino acid sequence recorded in sequence number 32 or 56.

[0041]

[20] The DcR3 variant as described in

[18] or

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

[0042]

[21] The DcR3 variant as described in any of

[18] to

[20] , wherein the amino acid sequence of (e) is such that Glu at position 57 and Arg at position 58 of the amino acid sequence of (a), (b), (c) or (d) are replaced by other amino acid substitutions.

[0043]

[22] The DcR3 variant as described in any of

[18] to

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

[0044]

[23] The DcR3 variant as described in any of

[18] to

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

[0045]

[24] The DcR3 variant as described in any of

[18] to

[23] , wherein the amino acid sequence of (e) above has substitutions selected from (f) to (i): (f) The Asn at positions 131 and 144 from the N-terminus of the amino acid sequences in (b), (c), or (d) above is replaced with other amino acids. (g) The Asn at positions 131, 144, and 157 from the N-terminus of the amino acid sequences in (b), (c), or (d) above is replaced with other amino acids. (h) The amino acid sequences in (b), (c), or (d) above are modified by replacing Thr at position 133 and Ser at position 146 from the N-terminus with other amino acids. (i) The amino acid sequences in (b), (c) or (d) above are replaced by other amino acids, with Thr at position 133, Ser at position 146 and Thr at position 159 from the N-terminus.

[0046]

[25] The DcR3 variant as described in any of

[18] to

[24] , wherein the amino acid sequence of the preceding (e) has substitutions selected from the following (f') to (i'):

[0047] (f') The Asn at positions 131 and 144 from the N-terminus of the amino acid sequences in (b), (c), or (d) above is replaced with Ser.

[0048] (g') The Asn at positions 131, 144, and 157 from the N-terminus of the amino acid sequences in (b), (c), or (d) above is replaced with Ser.

[0049] (h') The amino acid sequence in (b), (c), or (d) above is modified so that Thr at position 133 and Ser at position 146 from the N-terminus to Ala.

[0050] (i') The amino acid sequences in (b), (c) or (d) above are replaced with Ala at position 133 (Thr), position 146 (Ser), and position 159 (Thr) from the N-terminus.

[0051]

[26] The DcR3 variant as described in any of

[18] to

[25] , wherein the amino acid sequence of (e) above is an amino acid sequence comprising the amino acid from the N-terminus to the 164th amino acid in the amino acid sequence described in sequence numbers 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286.

[0052]

[27] A DcR3 variant as described in any of [7] to

[26] , wherein the aforementioned DcR3 variant comprises the aforementioned first or second chimeric cysteine-rich region and part or all of the heparan sulfate-binding region of the aforementioned wild-type DcR3 bound to the C-terminus of the aforementioned first or second chimeric cysteine-rich region; or The aforementioned DcR3 variant includes the aforementioned first or second chimeric cysteine-rich region and does not contain the aforementioned heparan sulfate-binding region of wild-type DcR3.

[0053]

[28] The DcR3 variant as described in

[27] , wherein the aforementioned DcR3 variant comprises an amino acid sequence selected from any of the following:

[0054] (I) The amino acid sequence described in sequence numbers 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 30 amino acids, and

[0055] (II) The amino acid sequence recorded in sequence numbers 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 obtained by deleting, substituting, inserting or adding 1 to 30 amino acids.

[0056]

[29] The DcR3 variants as described in [7] to

[28] , wherein the aforementioned DcR3 variants comprise an Fc region derived from human IgG1, IgG2 or IgG4 antibodies or a mutant Fc region described below, wherein the mutant Fc region comprises an amino acid sequence obtained by deleting, substituting, inserting or adding one or more amino acids in the amino acid sequence of the Fc region.

[0057]

[30] The DcR3 variant as described in

[29] , wherein the aforementioned Fc region or the aforementioned mutant Fc region is connected to the C-terminal side of the aforementioned first or second chimeric cysteine-rich region via other regions or linkers.

[0058]

[31] The DcR3 variant as described in

[29] or

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

[0059]

[32] The DcR3 variant as described in

[31] , wherein the aforementioned mutant Fc region has substitutions in the amino acid sequence of the heavy chain of human IgG1, wherein Leu at position 234 is replaced with Ala, Leu at position 235 is replaced with Ala, and Gly at position 237 is replaced with Ala according to the EU index.

[0060]

[33] The DcR3 variant as described in

[31] or

[32] , wherein the aforementioned mutant Fc region has a substitution of Asn to Ala at position 434 according to the EU index in the amino acid sequence of the heavy chain of human IgG1.

[0061]

[34] The DcR3 variant as described in

[31] , wherein the aforementioned mutant Fc region has substitutions in the amino acid sequence of the heavy chain of human IgG1, wherein Met at position 252 is replaced with Tyr, Ser at position 254 is replaced with Thr, and Thr at position 256 is replaced with Glu according to the EU index.

[0062]

[35] The DcR3 variant as described in

[29] or

[30] , wherein the aforementioned mutant Fc region has substitutions in the amino acid sequence of the heavy chain of human IgG4, wherein Ser at position 228 is replaced with Pro, Leu at position 235 is replaced with Glu, and Arg at position 409 is replaced with Lys.

[0063]

[36] The DcR3 variant as described in

[29] or

[30] , wherein the aforementioned DcR3 variant comprises a mutant Fc region comprising the amino acid sequence described in sequence numbers 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313.

[0064]

[37] The DcR3 variant as described in any of

[29] to

[36] , wherein the aforementioned DcR3 variant includes serial numbers 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, 2 The amino acid sequences described in 48, 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 amino acid sequences obtained by deleting, substituting, inserting or adding 1 to 30 amino acids in such amino acid sequences.

[0065]

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

[37] .

[0066]

[39] The composition as described in

[38] contains a DcR3 variant having one or more N-glycosidic complex sugar chains and a DcR3 variant not having an N-glycosidic complex sugar chain.

[0067]

[40] DNA that encodes any of the DcR3 variants described in [1] to

[37] .

[0068]

[41] A recombinant gene vector containing the DNA described in

[40] .

[0069]

[42] Transformant, which is obtained by introducing the recombinant vector of

[41] into a host cell.

[0070]

[43] The transformant as described in

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

[0071]

[44] The transformant as described in

[43] , wherein the cell derived from a mammal is a CHO cell.

[0072]

[45] A method for manufacturing a DcR3 variant or a DcR3 variant composition, characterized in that the transformant described in any one of

[42] to

[44] is cultured in a culture medium to generate and accumulate the DcR3 variant described in any one of [1] to

[37] , and the aforementioned DcR3 variant is purified from the obtained culture medium.

[0073]

[46] DcR3 variants or DcR3 variant compositions, which are manufactured using the manufacturing method described in

[45] .

[0074]

[47] A pharmaceutical composition comprising, as an active ingredient, any one of [1] to

[39] and

[46] a DcR3 variant or a DcR3 variant composition.

[0075]

[48] ​​The pharmaceutical composition described in

[47] is a preventive or therapeutic agent for autoimmune diseases, inflammatory diseases or allergic diseases.

[0076]

[49] A method for the prevention or treatment of autoimmune diseases, inflammatory diseases or allergic diseases, the method comprising administering the pharmaceutical composition described in

[47] or

[48] to a patient who requires the prevention or treatment of autoimmune diseases, inflammatory diseases or allergic diseases.

[0077] Invention Effects

[0078] According to the present invention, the following are provided: a DcR3 variant, DNA encoding the DcR3 variant, a vector containing the DNA, a transformant obtained by introducing the vector, a method for manufacturing a variant using the transformant, and a pharmaceutical composition and a preventive or therapeutic agent for autoimmune diseases, inflammatory diseases, or allergies using the variant as an active ingredient, wherein the DcR3 variant has binding activity (preferably neutralizing activity) against a ligand of DcR3, and when preparing DcR3 protein using cells derived from mammals as a host, the amount of lectin formation is reduced compared to wild-type DcR3, and / or it exhibits improved in vivo kinetics. Attached Figure Description

[0079] [ Figure 1 ] Figure 1Figure A shows the SDS-PAGE results of various wild-type DcR3 controls prepared using mammalian cells. Lanes 1 and 3 are DcR3 FL-Fc, lanes 2 and 4 are DcR3 FL-FLAG, and lanes 5 and 6 are S195-Fc (g1S), which were electrophoresed under non-reducing or reducing conditions, respectively. Figure 1 Figure B shows commercially available human DcR3-Fc (lane 7) prepared using HEK293 cells under non-reducing conditions by electrophoresis and detected by Western blotting based on anti-human IgG antibodies. Figure 1 Figure C shows the electrophoresis of commercially available DcR3 FL-Fc (lanes 8, 9), S195-Fc(g1S) (lanes 10, 11), DcR3 FL-Fc(g1S) (lanes 14, 15, 18, 19), and R218Q-Fc(g1S) (lanes 12, 13, 16, 17) prepared from insect cells under non-reducing or reducing conditions. M indicates the molecular weight label (Bio Rad).

[0080] [ Figure 2 ] Figure 2 This is a diagram showing the cysteine-enriched domains represented as CRD1, CRD2, CRD3, and CRD4, respectively, in the comparison of immature amino acid sequences of human DcR3 and human OPG.

[0081] [ Figure 3 ] Figure 3 The structural domain structures of various wild-type DcR3 controls, various DcR3 variants, and human OPG are schematically shown. A: DcR3 FL-Fc, B: S195-Fc, C: Chimeric A-Fc, D: 103-123OPG-Fc, E: N-type glycan 2-substituted form-Fc, F: N-type glycan 3-substituted form-Fc, G: Chimeric B-Fc, H: Chimeric C-Fc, I: OPG. The vertical lines of CRD4 in E and F indicate that there are 2 or 3 substitutions of N-type glycan residues, respectively. DD in I indicates the death domain.

[0082] [ Figure 4 ] Figure 4 The results of SDS-PAGE for various DcR3 variants prepared using mammalian cells are shown. Lanes 1 and 4 show chimeric A-Fc (IEGRMD g1S) prepared using Expi293 cells; lanes 2 and 5 show chimeric A-Fc (IEGRMD g1S) prepared using CHO-S cells; lanes 3 and 6 show chimeric B-Fc (g1S) prepared using Expi293 cells; and lanes 7 and 8 show chimeric C-Fc (IEGRMD g1S) prepared using Expi293 cells, all electrophoresed under non-reducing or reducing conditions. M indicates the molecular weight label (Bio-Rad).

[0083] [ Figure 5 ] Figure 5 Melting curves based on DSF are shown for R218Q-Fc, S195-Fc, and the chimeric A-Fc (IEGRMD g1S). The vertical axis represents the fluorescence intensity RFU (10⁻¹⁰). 3 The horizontal axis represents temperature (°C).

[0084] [ Figure 6 ] Figure 6 The graph shows the binding of various wild-type DcR3 controls and various DcR3 variants to human primary cells and CHO cells. Each DcR3 variant was reacted with cells at 10 μg / mL, followed by staining with 0.1 μg / mL PE-labeled anti-human antibody. The fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the graph represents the geometric mean of PE. The upper segment represents HUVECs, the middle segment represents hepatocytes, and the lower segment represents the staining results of CHO cells.

[0085] [ Figure 7 ] Figure 7 The changes in serum concentrations of S195-Fc and chimeric A-Fc (IEGRMD g1S) after intravenous (iv) administration of 10 mg / kg are shown in BALB / c mice. The vertical axis represents serum concentration (ng / mL), and the horizontal axis represents the time elapsed after administration (hr).

[0086] [ Figure 8 ] Figure 8 Inset A shows the binding of various wild-type DcR3 controls and various DcR3 variants to RANKL (OPG ligand). Figure 8 Inset Figure B shows the binding of various wild-type DcR3 controls and variants to TRAIL (OPG ligand). After capturing the wild-type DcR3 controls and variants onto plates immobilized with anti-human antibodies, RANKL or TRAIL diluted to various concentrations was added, and binding was evaluated. Detection was performed using biotinylated anti-RANKL antibody or biotinylated anti-TRAIL antibody, and streptavidin-HRP. The horizontal axis shows the concentration (pg / mL) of RANKL or TRAIL, and the vertical axis shows the absorbance (the absorbance at 450 nm minus the absorbance at 570 nm).

[0087] [ Figure 9 ] Figure 9The neutralizing activity against LIGHT is shown for various wild-type DcR3 controls and various DcR3 variants. IL-8 production from HT-29 cells is shown with the addition of LIGHT 100 ng / mL and with the addition of various wild-type DcR3 controls and various DcR3 variants at 0.1, 1, or 10 μg / mL. The vertical axis shows IL-8 concentration (pg / mL), and the horizontal axis shows various DcR3 variants added as inhibitors.

[0088] [ Figure 10 ] Figure 10 The neutralizing activities of various wild-type DcR3 controls and various DcR3 variants against TL1A are shown. The production of IFN-γ from human T cells is shown with the addition of TL1A 100 ng / mL and with the addition of various wild-type DcR3 controls and various DcR3 variants at 0.1, 1, or 10 μg / mL. The vertical axis shows IFN-γ concentration (pg / mL), and the horizontal axis shows various DcR3 variants added as inhibitors.

[0089] [ Figure 11 ] Figure 11 The neutralizing activity against FasL is shown for various wild-type DcR3 and control DcR3 variants. ATP production from live Jurkat cells is shown in RLU at the addition of 100 ng / mL FasL and at the addition of 0.01, 0.1, or 1 μg / mL of various wild-type DcR3 controls and DcR3 variants. The vertical axis shows cell viability (RLU x 10) as an indicator of ATP-dependent chemiluminescence. 6 The horizontal axis shows the various DcR3 variants that are added as inhibitors.

[0090] [ Figure 12A ] Figure 12A This diagram illustrates the binding of chimeric A-Fc (g4PEK) and the FasL-reduced binding variant (g4PEK) to various DcR3 ligands. It also shows a sensor chip immobilized with anti-human antibodies, capturing chimeric A-Fc (g4PEK) or the FasL-reduced binding variants, and displaying the DcR3 ligands (human FasL, human LIGHT, human TL1A) as analyte aging data. The vertical axis represents binding amount (RU), and the horizontal axis represents time (Sec).

[0091] [ Figure 12B ] Figure 12B This diagram illustrates the binding of the FasL-debinding variant (g4PEK) to each DcR3 ligand. It also shows a sensor plot capturing each FasL-debinding variant onto a sensor chip immobilized with anti-human antibodies, with the DcR3 ligands (human FasL, human LIGHT, human TL1A) as the analyte and aging process. The vertical axis represents binding amount (RU), and the horizontal axis represents time (Sec).

[0092] [ Figure 12C ] Figure 12C This diagram illustrates the binding of the FasL-debinding variant (g4PEK) to each DcR3 ligand. It also shows a sensor plot capturing each FasL-debinding variant onto a sensor chip immobilized with anti-human antibodies, with the DcR3 ligands (human FasL, human LIGHT, human TL1A) as the analyte and aging process. The vertical axis represents binding amount (RU), and the horizontal axis represents time (Sec).

[0093] [ Figure 13 ] Figure 13 The results of SDS-PAGE of wild-type DcR3 controls prepared using various mammalian cells are shown. SDS-PAGE was performed by electrophoresis of commercially available human DcR3-Fc prepared using various mammalian cells under reducing or non-reducing conditions. HEK293 cells (Abcam) were used as mammalian cells in lanes 1 and 4, CHO cells (AdipoGen) in lanes 2 and 5, and HEK293 cells (Enzo) in lanes 3 and 6. M indicates the molecular weight marker (Bio Rad).

[0094] [ Figure 14A ] Figure 14A The percentages (%) of monomers, aggregates, and decomposition products are calculated from the peak areas of the FasL-reduced binding variant purified from Protein A by SEC-HPLC or SEC-UPLC. Figure 14A The results for all FasL-binding-reducing variants (g4PEK) prepared are shown.

[0095] [ Figure 14B ] Figure 14B The percentages (%) of monomer, aggregate, and decomposition products are calculated from the peak areas of the reduced-binding variant of FasL purified from Protein A by SEC-HPLC or SEC-UPLC. Figure 14B The results for various mutant Fc fusions of the selected FasL binding-reducing variants are shown.

[0096] [ Figure 14C ] Figure 14C The percentages (%) of monomers, aggregates, and decomposition products are shown, calculated from the peak areas of various chimeric A-mutant Fc fusions purified from Protein A by SEC-HPLC or SEC-UPLC.

[0097] [ Figure 15A ] Figure 15AThe results show the binding activity of various DcR3 variants against their respective DcR3 ligands as determined by BIAcore. The diagram illustrates the capture of various DcR3 variants onto a sensor chip immobilized with anti-human antibodies, using trimer DcR3 ligands (human FasL, human LIGHT, or human TL1A) as the analyte, and the various kinetic constants (ka, kd, K) of the analyte. D ). Figure 15A The results of various chimeric A-Fc sequences with different Fc sequences are shown.

[0098] [ Figure 15B ] Figure 15B The results show the binding activity of various DcR3 variants against their respective DcR3 ligands as determined by BIAcore. The results also show the capture of various DcR3 variants onto a sensor chip immobilized with anti-human antibodies, with the trimer DcR3 ligands (human FasL, human LIGHT, or human TL1A) used as various kinetic constants (ka, kd, KD) for the analyte stream. Figure 15B The results for the FasL binding-reduced variant are shown.

[0099] [ Figure 16A ] Figure 16A The results show the kinetic constants (KD values) for each DcR3 ligand calculated by BIAcore when selecting FasL-reduced variants, compared with the chimeric A-Fc (g4PEK). Figure 16A The results for single amino acid substitutes are shown.

[0100] [ Figure 16B ] Figure 16B The results show the kinetic constants (KD values) for each DcR3 ligand calculated by BIAcore when selecting FasL-reduced variants, compared with the chimeric A-Fc (g4PEK). Figure 16B The results for the 2-amino acid substitute are shown.

[0101] [ Figure 17A ] Figure 17A The results of the neutralizing activity evaluation of various DcR3 variants against soluble LIGHT are shown. Figure 17A The diagram illustrates the inhibitory activity against LIGHT-dependent CXCL10 production from IFN-γ-stimulated enterofibroblasts induced by various chimeric A-Fc variants with different Fc sequences. The vertical axis represents the CXCL10 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0102] [ Figure 17B ] Figure 17B The results of the neutralizing activity evaluation of various DcR3 variants against soluble LIGHT are shown. Figure 17BThis illustrates the inhibitory activity against LIGHT-dependent CXCL10 from IFN-γ-stimulated enterofibroblasts, induced by chimeric A-Fc cells with Fc sequences containing different mutations. The vertical axis represents the CXCL10 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0103] [ Figure 17C ] Figure 17C The results of the neutralizing activity evaluation of various DcR3 variants against soluble LIGHT are shown. Figure 17C The diagram shows the inhibitory activity against LIGHT-dependent IL-8 production from HT-29 cells induced by a single amino acid-substituted FasL-binding variant. The vertical axis represents the IL-8 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0104] [ Figure 17D ] Figure 17D The results of the neutralizing activity evaluation of various DcR3 variants against soluble LIGHT are shown. Figure 17D The diagram shows the inhibitory activity against LIGHT-dependent CXCL10 production from IFN-γ-stimulated enterofibroblasts induced by a 2-amino acid substitution variant with reduced FasL binding. The vertical axis represents the CXCL10 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0105] [ Figure 18A ] Figure 18A The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble TL1A are shown. Figure 18A This diagram illustrates the inhibitory activity against TL1A-dependent IFN-γ production from IL-12 and IL-18-stimulated human T cells induced by various chimeric A-Fc variants with different Fc sequences. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0106] [ Figure 18B ] Figure 18B The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble TL1A are shown. Figure 18B This diagram illustrates the inhibitory activity against TL1A-dependent IFN-γ production from IL-12 and IL-18-stimulated human T cells induced by chimeric A-Fc cells with Fc sequences containing different mutations. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0107] [ Figure 18C ] Figure 18CThe results of the evaluation of the neutralizing activity of various DcR3 variants against soluble TL1A are shown. Figure 18C The diagram shows the inhibitory activity against TL1A-dependent IFN-γ production from IL-12 and IL-18-stimulated human T cells induced by a single amino acid-substituted FasL-binding variant. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0108] [ Figure 18D ] Figure 18D The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble TL1A are shown. Figure 18D The diagram shows the inhibitory activity against TL1A-dependent IFN-γ production from IL-12 and IL-18-stimulated human T cells induced by a 2-amino acid substitution variant with reduced FasL binding. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0109] [ Figure 19A ] Figure 19A The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble FasL are shown. Figure 19A The inhibitory activity against cell death in A3 cells induced by various chimeric A-Fcs with different Fc sequences is shown. The vertical axis shows cell survival (RLU x 10) as an indicator of ATP-dependent chemiluminescence. 6 The horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0110] [ Figure 19B ] Figure 19B The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble FasL are shown. Figure 19B This illustrates the inhibitory activity against cell death in Jurkat cells induced by chimeric A-Fc cells with Fc sequences containing different mutations. The vertical axis represents cell survival (RLU x 10) as an indicator of ATP production. 6 The horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0111] [ Figure 19C ] Figure 19C The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble FasL are shown. Figure 19C The diagram shows the inhibitory activity against cell death in Jurkat cells induced by variants with reduced FasL binding due to single amino acid substitution. The vertical axis represents cell survival (RLU x 10) as an indicator of ATP production. 6 The horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0112] [ Figure 19D ] Figure 19D The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble FasL are shown. Figure 19D The inhibitory activity against cell death in Jurkat cells induced by a variant with reduced FasL binding due to 2-amino acid substitution is shown. The vertical axis represents cell survival (RLU x 10) as an indicator of ATP production. 6 The horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0113] [ Figure 20A ] Figure 20A The results show the binding activity evaluation of various chimeric A-Fc strains with different S195-Fc and Fc sequences against the membrane-bound LIGHT-forced expression strain. Various DcR3 variants were reacted with cells, stained with PE-labeled anti-human antibody, and the fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the figure represents the geometric mean of PE.

[0114] [ Figure 20B ] Figure 20B The results show the evaluation of the binding activity of various chimeric A-Fc strains with different S195-Fc and Fc sequences against membrane-bound TL1A and membrane-bound FasL forced expression lines. Various DcR3 variants were reacted with cells, stained with PE-labeled anti-human antibody, and the fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the figure represents the geometric mean of PE.

[0115] [ Figure 21A ] Figure 21A The results of evaluating the binding activity of S195-Fc, various chimeric A-Fc, and FasL-binding-reduced variants against the membrane-bound LIGHT-forced expression strain are shown. Various DcR3 variants were reacted with cells, stained with PE-labeled anti-human antibodies, and the fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the figure represents the geometric mean of PE.

[0116] [ Figure 21B ] Figure 21B The results of evaluating the binding activity of S195-Fc, various chimeric A-Fc, and FasL-reduced binding variants against membrane-bound TL1A-forced expression strains are shown. Various DcR3 variants were reacted with cells, stained with PE-labeled anti-human antibodies, and the fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the figure represents the geometric mean of PE.

[0117] [ Figure 21C ] Figure 21CThe results of evaluating the binding activity of chimeric A-Fc and FasL-reduced binding variants (g4PEK) against membrane-bound FasL-forced expression strains are shown. Various DcR3 variants were reacted with cells, stained with PE-labeled anti-human antibodies, and the fluorescence intensity of PE was measured by flow cytometry. The vertical axis of the figure represents the geometric mean of PE.

[0118] [ Figure 22 ] Figure 22 The results of the evaluation of the binding activity of chimeric A-Fc against the membrane-bound LIGHT of primary cells are shown. Figure 22 Figure A shows the results of measuring the expression of membrane-bound light on activated human T cells using PE-labeled anti-light antibody. Figure 22 Inset B shows the geometric mean (Geo.Mean) obtained by measuring and calculating the binding of Alexa Fluor 488-labeled chimeric A-Fc to membrane-bound light on activated human T cells using flow cytometry. Each vertical axis represents the geometric mean (Geo.Mean) of fluorescence intensity.

[0119] [ Figure 23 ] Figure 23 The results of evaluating the binding activity of chimeric A-Fc against membrane-bound TL1A in primary cells are shown. The geometric mean (Geo.Mean) is calculated by measuring the binding of Alexa Fluor 647-labeled chimeric A-Fc against membrane-bound TL1A on HUVEC cells under flow cytometry conditions in the presence or absence of competing proteins.

[0120] [ Figure 24 ] Figure 24 The results show the results obtained by sandwich ELISA of AICD-induced human T cell culture supernatant to measure the binding activity of chimeric A-Fc against FasL derived from primary cells. Figure 24 Figure A shows the absorbance at 450 nm when culture supernatant was added to a plate containing chimeric A-Fc cells and detected with anti-FasL antibody. Figure 24 Figure B shows the absorbance at 450 nm when culture supernatant was added to a plate containing Fas-Fc and detected with anti-FasL antibody.

[0121] [ Figure 25 ] Figure 25 The results of dissolution times (in minutes) in hydrophobic interaction chromatography (HIC) for various chimeric A-Fc and FasL-reduced variants with different Fc sequences are shown.

[0122] [ Figure 26 ] Figure 26The results show the Tm values ​​(°C) of various chimeric A-Fc and FasL-reduced binding variants with different Fc sequences, calculated by differential scanning fluorescence (DSF).

[0123] [ Figure 27 ] Figure 27 The values ​​of plasma half-life (h) and area under the plasma concentration-time curve (AUC0-∞) up to infinity are shown for the elimination phase after intravenous administration of chimeric A-Fc (Eg1S) and FasL-reduced variants with different Fc sequences at 10 mg / kg in BALB / c mice.

[0124] [ Figure 28A ] Figure 28A The gross disease score is shown in the efficacy test of chimeric A-Fc using a mouse acute xenogeneic GVHD model.

[0125] [ Figure 28B ] Figure 28B The chart shows the individual scores for each group and the average disease score for each group. The vertical axis represents the disease score, and the horizontal axis represents the treatment groups.

[0126] [ Figure 29 ] Figure 29 For chimeric A-Fc cells in a mouse acute xenogeneic GVHD model, the number of cells in each spleen is shown as per individual and the mean for each group in a pharmacodynamic assay using a human CD45-positive, human CD3 and CD4-positive, and human CD3 and CD8-positive cell types. Group 1 represents the group without cell translocation, Group 2 represents the group treated with DNP antibody and human cell translocation, and Group 3 represents the group treated with chimeric A-Fc and human cell translocation. The vertical axis represents the number of cells positive for each surface marker, and the horizontal axis represents each treatment group.

[0127] [ Figure 30A ] Figure 30A The results of BIAcore measurements on the binding activity of various DcR3 variants against human DcR3 ligands are shown. Various kinetic constants (ka, kd, K) are presented when using trimers of human FasL, human LIGHT, or human TL1A as human DcR3 ligands. D ).

[0128] [ Figure 30B ] Figure 30B The results of BIAcore measurements on the binding activity of various DcR3 variants against cynomolgus DcR3 ligands are shown. Various kinetic constants (ka, kd, K) are presented when the trimers of cynomolgus FasL, cynomolgus LIGHT, or cynomolgus TL1A are used as cynomolgus DcR3 ligands.D ).

[0129] [ Figure 31 ] Figure 31 The results of the neutralizing activity evaluation of various DcR3 variants against soluble LIGHT are shown. Figure 31 The inhibitory activity of chimeric A-Fc with various mutant Fc and FasL-reduced variants against LIGHT-dependent CXCL10 production in enteric myofibroblasts is shown. The vertical axis represents the CXCL10 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0130] [ Figure 32 ] Figure 32 The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble TL1A are shown. Figure 32 The diagram shows the inhibitory activity of chimeric A-Fc with various mutant Fc variants and FasL-reduced variants against TL1A-dependent IFN-γ production in human T cells. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0131] [ Figure 33 ] Figure 33 The results of the evaluation of the neutralizing activity of various DcR3 variants against soluble FasL are shown. Figure 33 The inhibitory activity against Jurkat cell death induced by chimeric A-Fc variants with various mutant Fc and variants with reduced FasL binding is shown. The vertical axis represents cell survival (RLU x 10) as an indicator of ATP-dependent chemiluminescence. 6 The horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0132] [ Figure 34A ] Figure 34A The results of flow cytometry evaluation of the binding activity of various DcR3 variants against the membrane-bound LIGHT-forced expression strain are shown. The vertical axis of the figure represents the geometric mean of PE.

[0133] [ Figure 34B ] Figure 34B The results of flow cytometry evaluation of the binding activity of various DcR3 variants against membrane-bound TL1A-forced expression strains are shown. The vertical axis of the figure represents the geometric mean of PE.

[0134] [ Figure 34C ] Figure 34C The results of flow cytometry evaluation of the binding activity of various DcR3 variants against membrane-bound FasL-forced expression strains are shown. The vertical axis of the figure represents the geometric mean of PE.

[0135] [ Figure 35 ] Figure 35 The results of neutralizing activity evaluations for various DcR3 variants against the membrane-bound LIGHT are shown. Figure 35 The inhibitory activity of chimeric A-Fc with various mutant Fc and FasL-reduced variants against membrane-type LIGHT-dependent CXCL10 production in enteric myofibroblasts is shown. The vertical axis represents the CXCL10 concentration (ng / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0136] [ Figure 36 ] Figure 36 The results of neutralizing activity evaluations of various DcR3 variants against membrane-bound TL1A are shown. Figure 36 The diagram illustrates the inhibitory activity against membrane-bound TL1A-dependent IFN-γ production in human CD4-positive T cells induced by chimeric A-Fc variants with various mutant Fc and FasL-reduced variants. The vertical axis represents the IFN-γ concentration (pg / mL), and the horizontal axis represents the concentration of the added DcR3 variant (ng / mL).

[0137] [ Figure 37 ] Figure 37 The results of the neutralizing activity evaluation of various DcR3 variants against membrane-bound FasL are shown. Figure 37 The figure illustrates the inhibitory activity against membrane-type FasL-dependent cell death in Jurkat cells induced by chimeric A-Fc with various mutant Fc variants and variants with reduced FasL binding. The vertical axis of the figure shows the percentage (%) of Annexin V-positive dead cells, and the horizontal axis shows the concentration (ng / mL) of the various DcR3 variants added.

[0138] [ Figure 38A ] Figure 38A The results show the results obtained by sandwich ELISA measuring the binding activity of various DcR3 variants against recombinant human LIGHT. The vertical axis shows the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis shows the concentration (ng / mL) of the added recombinant human LIGHT.

[0139] [ Figure 38B ] Figure 38BThe results show the assay of binding activity of various DCR3 variants against LIGHT derived from primary human cells using a sandwich ELISA targeting soluble LIGHT in the culture supernatant of human T cells. White bars represent results obtained using culture supernatant from unstimulated human T cells, while black bars represent results using culture supernatant from human T cells stimulated with anti-CD3 and anti-CD28 antibodies. The vertical axis represents the absorbance at 450 nm minus the absorbance at 570 nm.

[0140] [ Figure 38C ] Figure 38C The results show the results obtained by sandwich ELISA measuring the binding activity of various DcR3 variants against recombinant human TL1A. The vertical axis shows the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis shows the concentration of recombinant TL1A (ng / mL).

[0141] [ Figure 38D ] Figure 38D The results show the results of measuring the binding activity of various DCR3 variants against TL1A derived from primary human cells using a sandwich ELISA targeting soluble TL1A in the culture supernatant of human PBMCs. White bars represent results obtained using culture supernatant from unstimulated human PBMCs, while black bars represent results obtained using culture supernatant from human PBMCs stimulated by an immune complex. The vertical axis represents the absorbance at 450 nm minus the absorbance at 570 nm.

[0142] [ Figure 38E ] Figure 38E The results show the results obtained by sandwich ELISA measuring the binding activity of various DcR3 variants against recombinant human FasL. The vertical axis shows the absorbance at 450 nm minus the absorbance at 570 nm, and the horizontal axis shows the human FasL concentration (ng / mL).

[0143] [ Figure 38F ] Figure 38F The results show the binding activity of various DCR3 variants against FasL derived from primary human cells, obtained by sandwich ELISA targeting soluble FasL in the culture supernatant of AICD-induced human T cells. White bars represent results obtained using culture supernatant of uninduced human T cells, and black bars represent results obtained using culture supernatant of AICD-induced human T cells. The vertical axis represents the absorbance at 450 nm minus the absorbance at 570 nm.

[0144] [ Figure 39 ] Figure 39The values ​​of plasma half-life (h) and area under the plasma concentration-time curve (AUC0-∞) up to infinity are shown for the elimination phase after intravenous administration of various DcR3 variants at 10 mg / kg to BALB / c mice.

[0145] [ Figure 40 ] Figure 40 The results are shown by immunoblotting of various DcR3 variants of transiently expressed, unfused Fc in mammalian cells. The following electrophoresis was performed under both reducing and non-reducing conditions, and the results were detected by immunoblotting based on an anti-6x-His tag antibody: lanes 1 and 5 were 30-fold concentrates of culture supernatant expressing S195-His6; lanes 2 and 6 were 6-fold dilutions of culture supernatant expressing chimeric A-His6; lanes 3 and 7 were 6-fold dilutions of culture supernatant expressing E57K-His6; and lanes 4 and 8 were 6-fold dilutions of culture supernatant expressing 45-18-His6. Detailed Implementation

[0146] The preferred embodiments for carrying out the present invention will be described below. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention and are not intended to narrowly define the scope of the present invention.

[0147] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0148] This invention relates to DcR3 variants, which are variants of wild-type DcR3. Specifically, this invention relates to DcR3 variants that have DcR3 ligand-binding (or neutralizing) activity, exhibit improved in vivo kinetics compared to wild-type DcR3, and / or exhibit reduced agglutination compared to wild-type DcR3 when produced in mammalian cells, and that the DcR3 variants include a cysteine-rich domain obtained by introducing a mutation into the cysteine-rich domain of wild-type DcR3 by means of one or more amino acids.

[0149] 1. Wild-type DCR3

[0150] DcR3 is also commonly known as 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 that lacks a membrane-bound domain. It competitively inhibits the binding of each of its three ligands—LIGHT, TL1A, and FasL—to the receptor, thereby neutralizing the ligands. In addition, as a function different from ligand neutralization, it has been reported that through the heparan sulfate binding domain (HBD), it directly binds to glycosaminoglycans (GAGs), represented by heparan sulfate, on the cell membranes of monocytes, macrophages, or dendritic cells, causing various immunosuppressive and immunoactivating effects [Biochemical. Pharmacology, 2011, 81: p.838-847, J. Immunol., 2006, 176: p.173-180].

[0151] Naturally occurring DcR3 contains CRD1, CRD2, CRD3, CRD4, and HBD sequentially from the N-terminus. Naturally occurring DcR3 also contains regions between CRD1 and CRD2, between CRD2 and CRD3, between CRD3 and CRD4, and between CRD4 and HBD. The cysteine-rich region of naturally occurring DcR3 extends from the N-terminus of CRD1 to the C-terminus of CRD4, encompassing CRD1, CRD2, CRD3, and CRD4, and including the regions between CRD1 and CRD2, between CRD2 and CRD3, and between CRD3 and CRD4. "Wild-type DcR3" is a molecule containing both the cysteine-rich region and HBD, meaning that the cysteine-rich region and HBD are wild-type (i.e., the cysteine-rich region and HBD are the same as those in naturally occurring DcR3). Therefore, in addition to naturally occurring DcR3, naturally occurring DcR3 mutants, such as those with gene polymorphisms or isotypes, are also included in "wild-type DcR3" as long as they contain the cysteine-rich region and HBD of naturally occurring DcR3. Furthermore, immature and mature DcR3 are also included in "wild-type DcR3." "Immature DcR3" refers to DcR3 with a signal peptide, while "mature DcR3" refers to DcR3 with its signal peptide cleaved. The signal peptide can be any sequence derived from naturally occurring DcR3, an artificial sequence, a sequence derived from an expression vector, or a sequence from other proteins in host cells suitable for expressing naturally occurring DcR3. Additionally, where the cleavage site varies depending on the signal peptide used, the different amino acid sequences at the N-terminus of mature DcR3 are also included in "wild-type DcR3." The CRD of naturally occurring DcR3 and the CRD of wild-type DcR3 are also called "wild-type CRD". The cysteine ​​enrichment region of naturally occurring DcR3 and the cysteine ​​enrichment region of wild-type DcR3 are also called "wild-type cysteine ​​enrichment region".

[0152] The wild-type DcR3 in this invention is not limited by its source and can be derived from various eukaryotic organisms. Examples include 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 cattle. When using the DcR3 variant of this invention in humans, it is preferable to use human-derived DcR3 as the wild-type DcR3.

[0153] The cDNA sequence of human DcR3 is represented by sequence number 1, and the corresponding mRNA sequence is accessed in GenBank (NCBI) as accession number NM_003823.3. The amino acid sequence of human DcR3 is represented by sequence number 2, and is accessed in GenBank (NCBI) as 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 a basic amino acid-rich HBD at the C-terminus. Mature human DcR3 is obtained by cleaving the signal peptide from the immature form. The amino acid sequence of mature human DcR3 is represented by, for example, sequence number 4, and the base sequence of the DNA encoding the amino acid sequence of mature human DcR3 is represented by, for example, sequence number 3. In this invention, in the amino acid sequence of human DcR3 (Sequence No. 2), the region from the N-terminus 30 to 70 is defined as CRD1 (Sequence No. 6), the region from the N-terminus to the N-terminus is defined as CRD2 (Sequence No. 8), the region from the N-terminus to the N-terminus is defined as CRD3 (Sequence No. 10), the region from the N-terminus to the N-terminus is defined as CRD4 (Sequence No. 12), and the region from the N-terminus to the N-terminus is defined as HBD (Sequence No. 48). Figure 2 The base sequences of the DNA encoding the amino acid sequences of CRD1, CRD2, CRD3, CRD4 and HBD of human DcR3 are represented, for example, by sequence numbers 5, 7, 9, 11 and 47. In addition, the amino acid sequence of CRD has several definitions besides the above. The amino acid sequence of any CRD can be defined using known information for the DcR3 variants of the present invention [UniProt O95407, GenBank NP_003814.1, Structure, 2011, 19: p.162-171].

[0154] As mentioned above, examples of ligands for DcR3 include LIGHT, TL1A, and FasL, all of which belong to TNFSF.

[0155] LIGHT (lymphotoxin-like, exhibiting inducible expression and competing with herpes simplex virus (HSV) glycoprotein D (gD) for the HVEM receptor expressed on T lymphocytes) is also commonly known as TNFSF14 (Tumor necrosis factor superfamily member 14), LTg, HVEM-L, or CD258. The mRNA sequence and corresponding cDNA sequence of human LIGHT are accessed in GenBank (NCBI) under accession number NM_003807.4, and the amino acid sequence under accession number NP_003798.2. Soluble LIGHT is generated by expressing the membrane-bound LIGHT onto the cell membrane, followed by shedding of the extracellular region by proteases. The cleavage site in the membrane-bound LIGHT is between amino acids 82 and 83 of NP_003798.2. Both the soluble and membrane-bound forms are functional.

[0156] TL1A (Tumor necrosis factor-like cytokine 1A) is also commonly known as TNFSF15 (TNF superfamily member 15), TL1, VEGI, or VEGI-251. The mRNA sequence and corresponding cDNA sequence of human TL1A are accessed in GenBank (NCBI) under accession number NM_005118.3, and the amino acid sequence under accession number NP_005109.2. Soluble TL1A is generated by the detachment of the extracellular domain from the membrane-bound TL1A after expression on the cell membrane. The cleavage site in membrane-bound TL1A is between amino acids 71 ​​and 72 in NP_005109.2. Both the soluble and membrane-bound forms are functional.

[0157] FasL (FaS ligand) is also commonly known as FASLG, TNFSF6 (Tumor necrosis factor superfamily member 6), CD178, or APT1LG1. The mRNA sequence and corresponding cDNA sequence of human FasL are accessed in GenBank (NCBI) under accession number NM_000639.2, and the amino acid sequence under accession number NP_000630.1. Soluble FasL is generated by the detachment of the extracellular region of membrane-bound FasL from the cell membrane via protease. The cleavage sites in membrane-bound FasL are between amino acids 81 and 82, or between amino acids 129 and 130 in NP_000630.1. Membrane-bound FasL has been reported to be the major functional ligand in vivo.

[0158] It should be noted that genes encoding proteins in eukaryotes often exhibit polymorphism or isomorphism. For the genes used in this invention, those with mutations in their base or amino acid sequences due to such polymorphism are also included in the genes encoding LIGHT, TL1A, or FasL in this invention.

[0159] 2. DCR3 variant

[0160] The DcR3 variant of the present invention contains a chimeric cysteine-rich region.

[0161] 2-1. Chimeric cysteine ​​enrichment region

[0162] The chimeric cysteine-rich region of this invention comprises an amino acid sequence obtained by introducing mutations into one or more amino acids in the cysteine-rich region of wild-type DcR3. It should be noted that "introducing mutations into an amino acid / base sequence" means substituting, deleting, inserting, or adding one or more amino acids / bases to 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 the cysteine-rich region of wild-type DcR3 from at least one or two CRDs selected from CRD1, CRD2, CRD3, and CRD4. Mutations may or may not be introduced into the regions between CRD1 and CRD2, between CRD2 and CRD3, and between CRD3 and CRD4 within the cysteine-rich region of wild-type DcR3. When introducing mutations into one or more regions selected from those between CRD1 and CRD2, between CRD2 and CRD3, and between CRD3 and CRD4, the number of amino acids constituting each region after mutation introduction is typically 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 mutation introduction is not particularly limited. Mutations introduced into the cysteine-rich regions of wild-type DcR3 include any type of mutation, both naturally occurring and artificial. Examples of chimeric cysteine-rich regions in this invention include, for example, chimeric cysteine-rich regions containing amino acid sequences obtained by substituting, deleting, inserting, or adding one or more amino acids to the amino acid sequence of a cysteine-rich region of wild-type DcR3. Examples of such chimeric cysteine-rich regions include the first and second chimeric cysteine-rich regions described later.

[0163] 2-1-1. First chimeric cysteine ​​enrichment region

[0164] The first chimeric cysteine-enriched region comprises an amino acid sequence obtained by replacing at least a portion of the CRD of wild-type DcR3 with other peptides or proteins from the cysteine-enriched region of wild-type DcR3. That is, the first chimeric cysteine-enriched region comprises an amino acid sequence derived from the cysteine-enriched region of wild-type DcR3 and an amino acid sequence derived from other peptides or proteins.

[0165] The portion of the cysteine-rich region of wild-type DcR3 that is replaced by other peptides or proteins is preferably selected from at least a portion of at least one of CRDs selected from CRD1, CRD2, CRD3, and CRD4. Therefore, the portion of the cysteine-rich region of wild-type DcR3 that is replaced by other peptides or proteins can be selected from all or a portion of CRD1, all or a portion of CRD2, all or a portion of CRD3, and all or a portion of CRD4.

[0166] The amino acid sequence of the first chimeric cysteine-rich region also includes the following amino acid sequence: In the amino acid sequence of the cysteine-rich region of wild-type DcR3, in addition to at least a portion of the wild-type CRD, the portion other than the wild-type CRD is replaced by other peptides or proteins. The portion other than the CRD that is replaced by other peptides or proteins can be selected from the region between CRD1 and CRD2, the region between CRD2 and CRD3, and the region between CRD3 and CRD4. The portion other than the CRD that is replaced by other peptides or proteins can be one portion or two or more portions. For example, the amino acid sequence of the first chimeric cysteine-rich region also includes an amino acid sequence obtained by replacing the amino acid sequence in the region between CRD1 and CRD2, the region between CRD2 and CRD3, or the region between CRD3 and CRD4 with other peptides or proteins.

[0167] The portion of the cysteine-rich region of wild-type DcR3 that is replaced by other peptides or proteins can be one or more portions. When at least a portion of the CRD in the cysteine-rich region of wild-type DcR3 is replaced, the replaced portion can be all or part of one CRD, or all or part of multiple CRDs, but more preferably, it retains all or part of the regions of wild-type DcR3 CRD2 and / or all or part of CRD3 involved in binding with LIGHT, TL1A, and FasL, while all or part of the other CRDs are replaced by other peptides or proteins.

[0168] The other peptide or protein to be substituted can be any natural or artificial peptide or protein, such as at least a portion of a CRD derived from a protein other than DcR3. In a preferred embodiment, the other peptide or protein to be substituted is at least a portion of a CRD of a TNF receptor superfamily (TNFRSF) molecule other than DcR3. That is, in a preferred embodiment, the first chimeric cysteine-rich region comprises the amino acid sequence of the cysteine-rich region of wild-type DcR3, obtained by replacing at least a portion of the cysteine-rich domain of wild-type DcR3 with at least a portion of the cysteine-rich domain of a TNFRSF molecule other than DcR3. At least a portion of the CRD of the TNFRSF molecule can be selected from all or a portion of CRD1, all or a portion of CRD2, all or a portion of CRD3, and all or a portion of CRD4. Therefore, the first chimeric cysteine ​​enrichment region may have one or more substitutions selected from the following: a portion of the CRD1 of wild-type DcR3 is replaced by a portion of the CRD1 of the TNFRSF molecule corresponding to a portion of the CRD1 of wild-type DcR3; the entire CRD1 of wild-type DcR3 is replaced by the entire CRD1 of the TNFRSF molecule; a portion of the CRD2 of wild-type DcR3 is replaced by a portion of the CRD2 of the TNFRSF molecule corresponding to a portion of the CRD2 of wild-type DcR3; the entire CRD2 of wild-type DcR3 is replaced by a portion of the CRD2 of the TNFRSF molecule corresponding to a portion of the CRD2 of wild-type DcR3; or the entire CRD2 of wild-type DcR3 is replaced by a portion of the CRD1 .... The substitution of all CRD2 in the F molecule, the substitution of a portion of the CRD3 of wild-type DcR3 with the portion of the CRD3 of the TNFRSF molecule corresponding to a portion of the CRD3 of wild-type DcR3, the substitution of all CRD3 in the wild-type DcR3 with all CRD3 of the TNFRSF molecule, the substitution of a portion of the CRD4 of the wild-type DcR3 with the portion of the CRD4 of the TNFRSF molecule corresponding to a portion of the CRD4 of wild-type DcR3, and the substitution of all CRD4 in the wild-type DcR3 with all CRD4 of the TNFRSF molecule.

[0169] Examples of first chimeric cysteine ​​enrichment regions include: chimeric cysteine ​​enrichment regions where CRD1 of wild-type DcR3 is substituted to CRD1 of TNFRSF molecules (preferably, other CRDs of wild-type DcR3 are retained in such chimeric cysteine ​​enrichment regions); chimeric cysteine ​​enrichment regions where CRD4 of wild-type DcR3 is substituted to CRD4 of TNFRSF molecules (preferably, other CRDs of wild-type DcR3 are retained in such chimeric cysteine ​​enrichment regions); or chimeric cysteine ​​enrichment regions where CRD1 of wild-type DcR3 is substituted to CRD1 of TNFRSF molecules and CRD4 of wild-type DcR3 is substituted to CRD4 of TNFRSF molecules (preferably, other CRDs of wild-type DcR3 are retained in such chimeric cysteine ​​enrichment regions). In addition, the first chimeric cysteine ​​enrichment region also includes the following chimeric cysteine ​​enrichment regions, which, in addition to one or more of the above-mentioned substitutions, also have a substitution in which a portion of the CRD2 of wild-type DcR3 is substituted to a portion of the CRD2 of the corresponding TNFRSF molecule, and / or a substitution in which a portion of the CRD3 of wild-type DcR3 is substituted to a portion of the CRD3 of the corresponding TNFRSF molecule.

[0170] 29 receptors belong to human TNFRSF. Each receptor has a CRD in its N-terminal extracellular domain. Typically, for one CRD1, three disulfide bonds are formed by six Cys residues, so each receptor has one to four CRDs [Trends Biochem Sci, 2002, 27: p-19-26.].

[0171] Examples of human TNFRSFs include DcR1 (TNFRSF10C, TRAIL-R3, LIT, TRID, CD263) and DcR2 (TNFRSF10D, TRAIL-R4, TRUNDD).CD264), Type I TNFR (TNFRSF1A, TNF-R, CD120a, TNFAR, TNF-R55, TNFR60), Type II TNFR (TNFRSF1B, TNFBR, CD120b, TNFR80, p75, TNF-R75), LTBR (lymphotoxin β 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), DR 4 (TNFRSF10A, Apo2, TRAILR-1, CD261), DR5 (TNFRSF10B, TRAIL-R2, KILLER, TRICK2A, TRICKB, CD262), RANK (TNFRSF11A, C D265, FEO), FN14 (TNFRSF12A, TweakR, CD266), TACI (TNFRSF13B, CD267, IGAD2), BAFFR (TNFRSF13C, CD268), HVEM (TNFRSF 14. 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, CD270), NGFR (nerve growth factor receptor, TNFRSF16, p75NTR, CD271), BCMA (TNFRSF17, BCM, CD269, TNFRSF13A), DR6 (death receptor 6, TNFRSF21, CD270), NGFR (nerve growth factor receptor, TNFRSF16, p75NTR, CD271 ... 358), DR3 (death receptor 3, TNFRSF25, TRAMP, WSL-1, LARD, WSL-LR, DDR3, TR3, APO-3), EDAR (ectodysplasin A receptor, ED3, DL, ED5, EDA3, Edar, ED1R, EDA1R), EDAR2R (ectodysplasin A2 receptor, XEDAR, EDAA2R, EDA-A2R, TNFRSF27), or osteoprotegerin (OPG, TNFRSF11B, TR1, OCIF).

[0172] In the first chimeric cysteine ​​enrichment region, there are no restrictions on other peptides or proteins that replace at least a portion of wild-type DcR3, with OPG being particularly preferred in TNFRSF.

[0173] The source of OPG is not limited, and examples of OPG from various eukaryotic organisms can be cited. Examples include OPG from amphibians such as frogs, birds such as chickens, or mammals such as primates including humans, even-toed ungulates such as pigs and cattle, or rodents including mice.

[0174] The cDNA sequence of human OPG is represented by sequence number 13, and the corresponding mRNA sequence is accessed in GenBank (NCBI, USA) as accession number NM_002546.3. The amino acid sequence of human OPG is represented by sequence number 14, and is accessed in GenBank (NCBI, USA) as accession number NP_002537.3.

[0175] Immature human OPG (Sequence No. 14) has a signal peptide at its N-terminus. Mature human OPG is obtained by cleaving the signal peptide from the immature form. The amino acid sequence of mature human OPG is represented, for example, by Sequence No. 16, and the base sequence of the DNA encoding the amino acid sequence of mature human OPG is represented, for example, by Sequence No. 15. In this invention, the region from position 22 to 62 of the amino acid sequence of immature human OPG (Sequence No. 14) from the N-terminus is defined as CRD1 (Sequence No. 18), the region from position 65 to 105 is defined as CRD2 (Sequence No. 20), the region from position 107 to 142 is defined as CRD3 (Sequence No. 22), and the region from position 145 to 185 is defined as CRD4 (Sequence No. 24). The base sequences of the DNA encoding the amino acid sequences of CRD1, CRD2, CRD3, and CRD4 of human OPG are represented, for example, by Sequence Nos. 17, 19, 21, and 23, respectively. In addition to the above, there are several other definitions for the amino acid sequence of CRD. Any definition of the amino acid sequence of CRD can be used with known information for the DcR3 variant of the present invention [UniProt O00300, GenBank NP_002537.3].

[0176] It should be noted that for genes encoding proteins in eukaryotes, gene polymorphism and isotypes can often be identified. For the genes used in this invention, genes with mutations in their base or amino acid sequences due to such polymorphism are also included in the gene encoding OPG in this invention.

[0177] OPG binds to RANKL and inhibits osteoclast-based bone destruction by neutralizing its activity [J. Immunol., 2012, 189: p. 245-252]. Additionally, OPG binds to TRAIL and inhibits TRAIL-mediated apoptosis by neutralizing its activity [Am. J. Cancer. Res., 2012, 2: p. 45-64]. Neutralization by either ligand can potentially lead to undesirable activity; therefore, it is desirable that the DcR3 variant of the present invention does not exhibit neutralizing activity against either RANKL or TRAIL.

[0178] In a preferred embodiment, the first chimeric cysteine ​​enrichment region comprises, or is composed of, the amino acid sequence of (a), (b), (c), or (d) below.

[0179] (a) The amino acid sequence obtained by replacing the CRD1 of wild-type DcR3 with the CRD1 of OPG in the cysteine-rich region of wild-type DcR3 (preferably retaining other CRDs of wild-type DcR3 in this amino acid sequence).

[0180] (b) The amino acid sequence obtained by replacing the CRD4 of wild-type DcR3 with the CRD4 of OPG in the cysteine-rich region of wild-type DcR3 (preferably retaining other CRDs of wild-type DcR3 in this amino acid sequence).

[0181] (c) An amino acid sequence obtained by replacing the CRD1 of wild-type DcR3 with the CRD1 of OPG and the CRD4 of wild-type DcR3 with the CRD4 of OPG in the cysteine-rich region of wild-type DcR3 (preferably retaining the other CRDs of wild-type DcR3 in this amino acid sequence).

[0182] (d) An amino acid sequence obtained by replacing a portion of the CRD2 of wild-type DcR3 with the corresponding portion of the CRD2 of OPG, and / or replacing a portion of the CRD3 of wild-type DcR3 with the corresponding portion of the CRD3 of OPG in the amino acid sequence described in (a), (b) or (c) above (preferably retaining the other CRDs of wild-type DcR3).

[0183] As an example of the amino acid sequence described in (d) above, one could be an amino acid sequence in which, for instance, in the amino acid sequences of (a), (b), or (c), positions 103 to 123 from the N-terminus are replaced by the corresponding portion of the amino acid sequence of the OPG CRD. In this case, the amino acid sequence containing positions 18 to 36 of the wild-type DcR3 CRD3 and the two amino acid residues adjacent to the C-terminus of the CRD3 amino acid sequence are replaced by a portion of the OPG corresponding to that amino acid sequence.

[0184] As a specific example of the amino acid sequence in (a) above, an amino acid sequence containing amino acids from position 1 to 164 from the N-terminus of the amino acid sequence described in sequence number 26 or 50 can be given. As a specific example of the amino acid sequence in (b) above, an amino acid sequence containing amino acids from position 1 to 164 from the N-terminus of the amino acid sequence described in sequence number 28 or 52 can be given. As a specific example of the amino acid sequence in (c) above, an amino acid sequence containing amino acids from position 1 to 164 from the N-terminus of the amino acid sequence described in sequence number 30 or 54 can be given. As a specific example of the amino acid sequence in (d) above, an amino acid sequence containing amino acids from position 1 to 164 from the N-terminus of the amino acid sequence described in sequence number 32 or 56 can be given.

[0185] It should be noted that sequence number 26 represents the amino acid sequence of chimeric B-HBD (a DcR3 variant obtained by replacing CRD1 with CRD1 of OPG in wild-type DcR3 (sequence number 4)); sequence number 28 represents the amino acid sequence of chimeric C-HBD (a DcR3 variant obtained by replacing CRD4 with CRD4 of OPG in wild-type DcR3 (sequence number 4)); sequence number 30 represents the amino acid sequence of chimeric A-HBD (a DcR3 variant obtained by replacing CDR1 and CDR4 with CDR1 and CDR4 of OPG, respectively, in wild-type DcR3 (sequence number 4)); sequence number 32 represents the amino acid sequence of 103-123OPG-HBD (a DcR3 variant obtained by replacing the amino acid sequence containing the portion of CRD3 from position 18 to position 36 and the two amino acids on the C-terminus with human OPG in chimeric A-HBD); and sequence number 50 represents the amino acid sequence of chimeric B-HBD (a DcR3 variant obtained by replacing the amino acid sequence containing the portion of CRD3 from position 18 to position 36 and the two amino acids on the C-terminus with human OPG). The amino acid sequence of B (a DcR3 variant obtained by replacing CRD1 with CRD1 of OPG in wild-type DcR3 (Sequence No. 4) and deleting the heparan sulfate binding region), sequence No. 52 represents the amino acid sequence of chimeric C (a DcR3 variant obtained by replacing CRD4 with CRD4 of OPG in wild-type DcR3 (Sequence No. 4) and deleting the heparan sulfate binding region), sequence No. 54 represents the amino acid sequence of chimeric A (a DcR3 variant obtained by replacing CDR1 and CDR4 with CDR1 and CDR4 of OPG in wild-type DcR3 (Sequence No. 4) and deleting the heparan sulfate binding region), and sequence No. 56 represents the amino acid sequence of 103-123OPG (a DcR3 variant obtained by replacing the amino acid sequence of adult OPG in chimeric A, which includes the portion of CRD3 from position 18 to position 36 and the two amino acids on the C-terminus).

[0186] In a preferred embodiment, the DcR3 variant containing the first chimeric cysteine ​​enrichment region may include a DcR3 variant that is active against at least one of LIGHT, TL1A, and FasL; a DcR3 variant that is active against all of LIGHT, TL1A, and FasL; a DcR3 variant that is not active against FasL but is active against either LIGHT or TL1A; or a DcR3 variant that is not active against FasL but is active against both LIGHT and TL1A.

[0187] In this invention, "having binding activity against a ligand" is used to mean that: the binding activity of the DcR3 variant containing the first chimeric cysteine ​​enrichment region against the ligand is equivalent to, and not significantly reduced compared to, the binding activity of the wild-type DcR3 against the ligand; and that it is significantly enhanced compared to the binding activity of the wild-type DcR3 against the ligand. For example, when measured by surface plasmon resonance (SPR), binding activity against a ligand can be determined if the dissociation constant (KD) value of the DcR3 variant is less than 3 times that of the wild-type DcR3.

[0188] In this invention, the statement that a DcR3 variant "has no binding activity against the ligand" is used to mean that: the binding activity of the DcR3 variant containing the first chimeric cysteine ​​enrichment region against the ligand was not detected, and that the binding activity against the ligand was significantly reduced compared to that of wild-type DcR3. For example, when measured by the SPR method, if the KD value of the DcR3 variant is greater 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 against the ligand is significantly reduced, and the DcR3 variant can be defined as having no binding activity against the ligand.

[0189] In a particularly preferred embodiment, examples of DcR3 variants containing a first chimeric cysteine-rich region include DcR3 variants with reduced binding activity to FasL. "Reduced FasL binding variant" means a DcR3 variant containing a chimeric cysteine-rich region that has no binding activity to FasL but has binding activity to either or more of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region that has no binding activity to FasL but has binding activity to both LIGHT and TL1A.

[0190] In a preferred embodiment, the DcR3 variant of the present invention is a DcR3 variant that has neutralizing activity against at least one of LIGHT, TL1A and FasL, a DcR3 variant that has neutralizing activity against all of LIGHT, TL1A and FasL, a DcR3 variant that has no neutralizing activity against FasL but has neutralizing activity against any of LIGHT and TL1A, or a DcR3 variant that has no neutralizing activity against FasL but has neutralizing activity against LIGHT and TL1A.

[0191] In this invention, the term "neutralizing activity against a ligand" includes: inhibiting the binding of the ligand to a receptor on the cell membrane surface by binding the ligand to a DcR3 variant, and inhibiting the cell function caused by the binding of the ligand to the receptor on the cell membrane surface, i.e., the biological activity of the ligand (e.g., biological activities against cytokine production, hyperproliferation, apoptosis induction, etc.) by inhibiting the binding of the ligand to the receptor on the cell membrane surface.

[0192] In this invention, the expression "having neutralizing activity" for a DcR3 variant is used to mean that the neutralizing activity of the DcR3 variant against the ligand is not significantly different from that of wild-type DcR3 against the ligand, and that the neutralizing activity of the DcR3 variant against the ligand is significantly enhanced compared to that of wild-type DcR3.

[0193] In this invention, the expression "no neutralizing activity" for the DcR3 variant is used to mean that the neutralizing activity of the DcR3 variant against the ligand is significantly reduced compared to the neutralizing activity of wild-type DcR3 against the ligand.

[0194] In a particularly preferred embodiment, the DcR3 variant of the present invention is a DcR3 variant with reduced binding to FasL. "Reduced FasL binding variant" means a DcR3 variant that has no neutralizing activity against FasL but has neutralizing activity against either or more of LIGHT and TL1A, or a DcR3 variant that has no neutralizing activity against FasL but has neutralizing activity against LIGHT and TL1A.

[0195] 2-1-2. Second chimeric cysteine ​​enrichment region

[0196] The second chimeric cysteine ​​enrichment region is obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids in the amino acid sequence of the first chimeric cysteine ​​enrichment region.

[0197] As a method for obtaining a polypeptide having an amino acid sequence in which one or more amino acids are missing, substituted, inserted or added in the amino acid sequence of the first chimeric cysteine ​​enrichment region, the site-specific mutation introduction method can be cited [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)].

[0198] The mutations (modifications) that can be added to the first chimeric cysteine-rich region include any of the following: natural mutations and artificial substitutions, deletions, insertions, or additions of amino acids. Examples of amino acid sequences for the second chimeric cysteine-rich region include those obtained by substituting, deleting, inserting, or adding one or more, preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3 amino acids in the amino acid sequence of the first chimeric cysteine-rich region; or amino acid sequences that have at least 80%, preferably 85%, more preferably 90%, for example 93%, 95%, 97%, 98%, or 99% identity with the amino acid sequence of the first chimeric cysteine-rich region. As a method of describing the amino acid substitute, for example, when Asn is replaced with Ser at position 131 from the N-terminus of the amino acid sequence to which the amino acid is substituted, it can be denoted as N131S.

[0199] In a preferred embodiment, the second chimeric cysteine ​​enrichment region comprises, or is composed of, the amino acid sequence of (e).

[0200] (e) An amino acid sequence obtained by deleting, substituting, inserting or adding 1 to 30 amino acids in the amino acid sequences of (a), (b), (c) or (d) above.

[0201] Examples of mutations (modifications) that can be added to the first chimeric cysteine-rich region include the addition or deletion of glycan addition sites. By adding or deleting glycan addition sites in the first chimeric cysteine-rich region, the biological activity or properties of the DcR3 variant of the present invention, in vivo kinetics such as blood half-life, or physical or chemical properties such as protein stability can be controlled.

[0202] Glycan addition generally refers to the N-glycosidic bonding of a glycan to an asparagine residue in a peptide or protein and / or the O-glycosidic bonding of a glycan to a serine or threonine residue. Examples of O-type glycans added to DcR3 variants include Core1 and Core2, while examples of N-type glycans include high-mannose, hybrid, or complex glycans, with complex glycans being preferred.

[0203] As a mutation (modification) that can be added to the first chimeric cysteine ​​enrichment region, examples include replacing at least one amino acid in the amino acid sequence of the first chimeric cysteine ​​enrichment region with an amino acid that can add a sugar chain through an N-glycosidic bond or an O-glycosidic bond, and adding a sugar chain, with the addition of an N-glycosidic bond type sugar chain being particularly preferred.

[0204] Additionally, the present invention also includes, for example, replacing at least one, preferably two or more, amino acids involved in N-glycosidic bonding in the amino acid sequence of the chimeric cysteine-rich region with other amino acids, and removing the glycan. Generally, when expressing peptides or proteins using yeast, insect cells, or mammalian cells, the N-glycosidic bond of the glycan is generated by recognizing the Asn-X-Thr / Ser sequence (here, X is any amino acid residue other than Pro). For example, the N-glycosidic glycan can be removed by replacing Asn, Ser, or Thr in the Asn-X-Thr / Ser sequence present in the DcR3 variant with other amino acids.

[0205] As a second chimeric cysteine ​​enrichment region, in order to reduce the condensation of the DcR3 variant of the present invention, a chimeric cysteine ​​enrichment region that maintains an N-glycosidic bond between the amino acid sequence of the first chimeric cysteine ​​enrichment region (for example, an amino acid sequence consisting of amino acids from the N-terminus 1 to 164 of sequence number 30) and the Asn at position 157 from the N-terminus is particularly preferred.

[0206] In a preferred embodiment, the amino acid sequence of the second chimeric cysteine-rich region from which the glycan addition site has been removed (an embodiment of the amino acid sequence in (e) above) has substitutions selected from the following:

[0207] (f) The amino acid sequences in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) have their Asn at positions 131 and 144 from the N-terminus replaced with other amino acids.

[0208] (g) The amino acid sequences in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) have their Asn positions at positions 131, 144, and 157 from the N-terminus replaced with other amino acids.

[0209] (h) The amino acid sequences described in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from positions 1 to 164 from the N-terminus in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) are substituted with other amino acids at positions 133 (Thr) and 146 (Ser) from the N-terminus.

[0210] (i) The Thr at position 133, the Ser at position 146, and the Thr at position 159 of the amino acid sequence (b), (c), or (d) above (e.g., an amino acid sequence consisting of amino acids from position 1 to position 164 from the N-terminus of the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) are replaced with other amino acids.

[0211] In a further preferred embodiment, the amino acid sequence from which the glycan addition site has been removed (one embodiment of the amino acid sequence in (e) above) has substitutions selected from the following:

[0212] (f') The amino acid sequences in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) have their Asn positions at positions 131 and 144 from the N-terminus replaced with Ser.

[0213] (g') The amino acid sequences in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) have their Asn positions at positions 131, 144, and 157 replaced with Ser from the N-terminus.

[0214] (h') The amino acid sequences in (b), (c), or (d) above (e.g., amino acid sequences consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54, or 56) are replaced with Ala at position 133 (Thr) and position 146 (Ser) from the N-terminus.

[0215] (i') The amino acid sequence in (b), (c) or (d) above (e.g., an amino acid sequence consisting of amino acids from position 1 to position 164 from the N-terminus in the amino acid sequences shown in sequence numbers 28, 30, 32, 52, 54 or 56) is replaced with Ala at position 133, position 146, and position 159 from the N-terminus.

[0216] Examples of amino acid sequences having the above-mentioned (f') substitutions include, for instance, the amino acid sequence formed by replacing Asn at positions 131 and 144 from the N-terminus with Ser (N131S / N144S) in the amino acid sequence described in sequence number 30 or 54 (the amino acid sequence formed by replacing Asn at positions 1 to 164 from the N-terminus in the amino acid sequence described in sequence number 34 or 58).

[0217] It should be noted that sequence number 34 represents the amino acid sequence of N131S / N144S-HBD (a DcR3 variant in which Asn at positions 131 and 144 from the N-terminus of chimeric A-HBD is replaced with Ser), and sequence number 58 represents the amino acid sequence of N131S / N144S (a DcR3 variant in which Asn at positions 131 and 144 from the N-terminus of chimeric A is replaced with Ser).

[0218] Examples of amino acid sequences having the above-mentioned (h') substitutions include, for instance, the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence described in sequence number 30 or 54, where Thr at position 133 and Ser at position 146 from the N-terminus are replaced by Ala (T133A / S146A), and the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence described in sequence number 36 or 60.

[0219] It should be noted that sequence number 36 represents the amino acid sequence of T133A / S146A-HBD (a DcR3 variant obtained by replacing Thr at position 133 and Ser at position 146 from the N-terminus in chimeric A-HBD with Ala), and sequence number 60 represents the amino acid sequence of T133A / S146A (a DcR3 variant obtained by replacing Thr at position 133 and Ser at position 146 from the N-terminus in chimeric A with Ala).

[0220] Examples of amino acid sequences having the above-mentioned (g') substitutions include, for instance, the amino acid sequence consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequence described in sequence number 30 or sequence number 54, where Asn at positions 131, 144, and 157 from the N-terminus is replaced by Ser (N131S / N144S / N157S), and the amino acid sequence consisting of amino acids from the N-terminus 1 to 164 in the amino acid sequence described in sequence number 38 or sequence number 62.

[0221] It should be noted that sequence number 38 represents the amino acid sequence of N131S / N144S / N157S-HBD (a DcR3 variant in which Asn at positions 131, 144, and 157 from the N-terminus of chimeric A-HBD is replaced with Ser), and sequence number 62 represents the amino acid sequence of N131S / N144S / N157S (a DcR3 variant in which Asn at positions 131, 144, and 157 from the N-terminus of chimeric A is replaced with Ser).

[0222] Examples of amino acid sequences having the above-mentioned (i') substitutions include, for instance, the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence described in sequence number 30 or sequence number 54, where Thr at position 133, Ser at position 146, and Thr at position 159 from the N-terminus are replaced by Ala (T133A / S146A / T159A), and the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence described in sequence number 40 or sequence number 64.

[0223] It should be noted that sequence number 40 represents the amino acid sequence of T133A / S146A / T159A-HBD (a DcR3 variant in which the Thr at position 133, Ser at position 146, and Thr at position 159 of the chimeric A-HBD are replaced with Ala), and sequence number 64 represents the amino acid sequence of T133A / S146A / T159A (a DcR3 variant in which the Thr at position 133, Ser at position 146, and Thr at position 159 of the chimeric A are replaced with Ala).

[0224] Among the mutations (modifications) that can be added to the first chimeric cysteine-rich region, for example, by not introducing mutations into CRD2 and CRD3 of the first chimeric cysteine-rich region involved in the binding of LIGHT, TL1A, and FasL, but introducing mutations into CRD1 and / or CRD4 of the first chimeric cysteine-rich region, a DcR3 variant containing a chimeric cysteine-rich region in which the binding activity of LIGHT, TL1A, and FasL to the chimeric cysteine-rich region is not reduced can be obtained. On the other hand, by introducing mutations into CRD2 and / or CRD3 of the first chimeric cysteine-rich region involved in the binding of LIGHT, TL1A, or FasL, a DcR3 variant containing a chimeric cysteine-rich region in which the binding activity of LIGHT, TL1A, or FasL to the chimeric cysteine-rich region is altered can be obtained. That is, by introducing the above mutations, a DcR3 variant containing a chimeric cysteine-rich region with the desired LIGHT, TL1A or FasL binding properties can be obtained.

[0225] In a preferred embodiment, examples of DcR3 variants comprising a second chimeric cysteine ​​enrichment region include: DcR3 variants comprising a chimeric cysteine ​​enrichment region having binding activity against at least one of LIGHT, TL1A, and FasL; DcR3 variants comprising a chimeric cysteine ​​enrichment region having binding activity against all of LIGHT, TL1A, and FasL; DcR3 variants comprising a chimeric cysteine ​​enrichment region having no binding activity against FasL but having binding activity against any of LIGHT and TL1A; or DcR3 variants comprising a chimeric cysteine ​​enrichment region having no binding activity against FasL but having binding activity against LIGHT and TL1A, etc. The meanings of the expressions "having binding activity against the ligand" and "having no binding activity against the ligand" for the DcR3 variants of the present invention are as described above.

[0226] In a particularly preferred embodiment, examples of DcR3 variants containing a second chimeric cysteine-rich region include DcR3 variants with reduced binding activity to FasL. "Reduced FasL binding variant" means a DcR3 variant containing a chimeric cysteine-rich region that has no binding activity to FasL but has binding activity to either or more of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-rich region that has no binding activity to FasL but has binding activity to both LIGHT and TL1A.

[0227] In a preferred embodiment, examples of DcR3 variants comprising a second chimeric cysteine ​​enrichment region include: DcR3 variants comprising a chimeric cysteine ​​enrichment region having neutralizing activity against at least one of LIGHT, TL1A, and FasL; DcR3 variants comprising a chimeric cysteine ​​enrichment region having neutralizing activity against all of LIGHT, TL1A, and FasL; DcR3 variants comprising a chimeric cysteine ​​enrichment region having no neutralizing activity against FasL but having neutralizing activity against any of LIGHT and TL1A; or DcR3 variants comprising a chimeric cysteine ​​enrichment region having no neutralizing activity against FasL but having neutralizing activity against LIGHT and TL1A, etc. The meanings of the expressions "having neutralizing activity against the ligand" and "having no neutralizing activity against the ligand" for the DcR3 variants of the present invention are as described above.

[0228] In a particularly preferred embodiment, examples of DcR3 variants containing a second chimeric cysteine-enriched region include DcR3 variants with reduced neutralizing activity against FasL. "Reduced FasL-binding variant" means a DcR3 variant containing a chimeric cysteine-enriched region that has no neutralizing activity against FasL but has neutralizing activity against either or more of LIGHT and TL1A, or a DcR3 variant containing a chimeric cysteine-enriched region that has no neutralizing activity against FasL but has neutralizing activity against LIGHT and TL1A.

[0229] For example, DcR3 variants containing chimeric cysteine-rich regions, which are FasL-binding-reducing variants, can be obtained by: preparing modified versions of DcR3 or DcR3 variants whose binding sites with each ligand are substituted with Ala or other amino acids, based on crystal structure analysis, and measuring their binding and neutralizing activities against LIGHT, TL1A, or FasL ligands. Alternatively, they can be obtained by: preparing a gene library by randomly altering the ligand-binding sites of DcR3 or DcR3 variants with other amino acids, displaying the library in bacteriophages, yeast, mammalian cells, etc., and screening for binding and neutralizing activities against LIGHT, TL1A, or FasL ligands.

[0230] As an amino acid sequence (one of the amino acid sequences in (e) above) contained in the chimeric cysteine ​​enrichment region of the FasL-binding reduced variant, examples include, for instance, an amino acid sequence in which one or more amino acids selected from the group consisting of Glu at position 57, Arg at position 58, and Arg at position 60 from the N-terminus of (a), (b), (c), or (d) above are substituted with other amino acids.

[0231] There are no particular limitations on the other amino acids that can replace Glu at position 57. Suitable choices can be made from 19 amino acids other than Glu among the 20 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 preferred to choose from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met. Choosing from Lys, Leu, Arg, and Val is further preferred. Choosing from Lys, Arg, and Val is even more preferred. Choosing from Lys and Arg is still even more preferred.

[0232] There are no particular limitations on the other amino acids that can replace Arg at position 58. Suitable choices can be made from 19 amino acids other than Arg out of 20 amino acids, but selection from Asp, Glu and Thr is preferred, and selection from Asp and Glu is even more preferred.

[0233] There are no particular restrictions on the other amino acid that can replace Arg at position 60. Suitable choices can be made from the 19 amino acids other than Arg out of the 20 amino acids, but Lys is preferred.

[0234] In a preferred embodiment, one or more amino acids selected from the group consisting of Glu at position 57, Arg at position 58, and Arg at position 60 are one amino acid consisting of Glu at position 57.

[0235] In other preferred embodiments, one or more amino acids selected from the group consisting of Glu at position 57, Arg at position 58, and Arg at position 60 are two amino acids consisting of Glu at position 57 and Arg at position 58. In this embodiment, it is preferred to combine substitution of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with substitution of Arg at position 58 with Asp, Glu, or Thr; it is further preferred to combine substitution of Glu at position 57 with Lys, Leu, Arg, or Val with substitution of Arg at position 58 with Asp or Glu; and it is even more preferred to combine substitution of Glu at position 57 with Lys or Arg with substitution of Arg at position 58 with Asp or Glu.

[0236] The second chimeric cysteine ​​enrichment region may have one or more amino acids other than Glu at position 57, Arg at position 58, and Arg at position 60 substituted with other amino acids. Examples of amino acids other than Glu at position 57, Arg at position 58, and Arg at position 60 include Trp at position 53 from the N-terminus, Asn at position 54, Tyr at position 55, and Leu at position 56 in the amino acid sequences (a), (b), (c), or (d) above.

[0237] There are no particular limitations on the other amino acids that can replace Trp at position 53. Suitable choices can be made from the 19 amino acids other than Trp out of the 20 amino acids, but selection from Asp and Asn is preferred. The substitution of Trp at position 53 with other amino acids can be combined with, for example, the substitution of Glu at position 57 with other amino acids.

[0238] The other amino acid that replaces Asn at position 54 is not particularly limited, and can be suitably selected from the 19 amino acids other than Asn out of the 20 amino acids, preferably Asp. The substitution of Asn at position 54 with other amino acids can be combined with, for example, the substitution of Glu at position 57 with other amino acids.

[0239] There are no particular limitations on the other amino acids that can replace Tyr at position 55. Suitable choices can be made from the 19 amino acids other than Tyr out of the 20 amino acids, but selection from Thr, Asp, Gln, and Glu is preferred. The substitution of Tyr at position 55 with other amino acids can be combined with, for example, the substitution of Glu at position 57 with other amino acids.

[0240] The other amino acid that replaces Leu at position 56 is not particularly limited, and can be suitably selected from the 19 amino acids other than Leu out of the 20 amino acids, but selection from Asp, Gln, Thr, Glu, Gly, Asn, and Pro is preferred. The substitution of Leu at position 56 with other amino acids can be combined with, for example, the substitution of Glu at position 57 with other amino acids.

[0241] An amino acid sequence in which one or more amino acids from the group consisting of Glu at position 57, Arg at position 58, and Arg at position 60, selected from the amino acid sequences (a), (b), (c), or (d) above, are substituted with other amino acids, can be specifically exemplified as follows:

[0242] The amino acid sequence obtained by replacing Glu at position 57 from the N-terminus with Lys in the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 30 (the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 42).

[0243] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with Lys in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 66).

[0244] The amino acid sequence obtained by replacing Glu at position 57 from the N-terminus with Leu in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 30 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 44).

[0245] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with Leu in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 68).

[0246] The amino acid sequence obtained by replacing Arg at position 60 from the N-terminus with Lys in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 30 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 46).

[0247] The amino acid sequence obtained by replacing Arg at position 60 from the N-terminus with Lys in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 70).

[0248] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus, wherein Glu at position 57 from the N-terminus is replaced by Arg, is the amino acid sequence obtained.

[0249] The amino acid sequence obtained by replacing Glu at position 57 from the N-terminus with Arg in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 180).

[0250] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with Val.

[0251] The amino acid sequence obtained by replacing Glu at position 57 from the N-terminus with Val in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 182).

[0252] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with Ala.

[0253] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with Ala in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 270).

[0254] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with Phe.

[0255] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with Phe in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 272).

[0256] In the amino acid sequence described in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with His.

[0257] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with His in the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 274).

[0258] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with Ile.

[0259] The amino acid sequence obtained by replacing Glu at position 57 from the N-terminus with Ile in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 276).

[0260] In the amino acid sequence recorded in sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus, with Glu at position 57 from the N-terminus replaced by Met, yields the following amino acid sequence.

[0261] The amino acid sequence obtained by replacing Glu at position 57 (from the N-terminus) with Met in the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 278).

[0262] The amino acid sequence obtained by replacing Glu at position 57 with Lys and Arg at position 58 with Asp in the amino acid sequence from position 1 to 164 starting from the N-terminus in the amino acid sequence recorded by sequence number 30.

[0263] The amino acid sequence obtained by replacing Glu at position 57 with Lys and Arg at position 58 with Asp in the amino acid sequence recorded in sequence number 54, which consists of amino acids from position 1 to 164 from the N-terminus.

[0264] In the amino acid sequence recorded by sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 (N-terminus) with Lys and Arg at position 58 with Glu.

[0265] The amino acid sequence obtained by replacing Glu at position 57 with Lys and Arg at position 58 with Glu in the amino acid sequence recorded in sequence number 54 (the amino acid sequence composed of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 186).

[0266] In the amino acid sequence described by sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 (N-terminus) with Arg and Arg at position 58 with Asp.

[0267] The amino acid sequence obtained by replacing Glu at position 57 with Arg and Arg at position 58 with Asp in the amino acid sequence recorded in sequence number 188, consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54.

[0268] The amino acid sequence obtained by replacing Glu at position 57 with Lys and Arg at position 58 with Thr in the amino acid sequence from position 1 to 164 starting from the N-terminus in the amino acid sequence recorded by sequence number 30.

[0269] The amino acid sequence obtained by replacing Glu at position 57 with Lys and Arg at position 58 with Thr in the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 280).

[0270] In the amino acid sequence recorded by sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 (N-terminus) with Leu and Arg at position 58 with Glu.

[0271] The amino acid sequence obtained by replacing Glu at position 57 with Leu and Arg at position 58 with Glu in the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 282).

[0272] In the amino acid sequence described by sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 (N-terminus) with Val and Arg at position 58 with Thr.

[0273] The amino acid sequence obtained by replacing Glu at position 57 with Val and Arg at position 58 with Thr in the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 54 (the amino acid sequence from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 284).

[0274] In the amino acid sequence recorded by sequence number 30, the amino acid sequence consisting of amino acids 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 (N-terminus) with Val and Arg at position 58 with Glu.

[0275] In the amino acid sequence recorded in sequence number 54, the amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus is obtained by replacing Glu at position 57 from the N-terminus with Val and Arg at position 58 with Glu, etc. (Amino acid sequence consisting of amino acids from position 1 to 164 from the N-terminus in the amino acid sequence recorded in sequence number 286).

[0276] It should be noted that sequence number 42 represents the amino acid sequence of chimeric A-E57K-HBD (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A-HBD with Lys), sequence number 44 represents the amino acid sequence of chimeric A-E57L-HBD (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A-HBD with Leu), sequence number 46 represents the amino acid sequence of chimeric A-R60K-HBD (a DcR3 variant obtained by replacing Arg at position 60 from the N-terminus of chimeric A with Lys), and sequence number 66 represents the amino acid sequence of chimeric A-E57K (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with... The amino acid sequences of the DcR3 variants derived from Lys are as follows: sequence number 68 represents the amino acid sequence of chimeric A-E57L (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Leu); sequence number 70 represents the amino acid sequence of chimeric A-R60K (the DcR3 variant obtained by replacing Arg at position 60 from the N-terminus of chimeric A with Lys); sequence number 180 represents the amino acid sequence of chimeric A-E57R (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Arg); and sequence number 182 represents the amino acid sequence of chimeric A-E57V (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Val). The amino acid sequences of the chimeric A-E57K_R58D (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus with Lys and Arg at position 58 with Asp in chimeric A) are shown in sequence number 184. Sequence number 186 represents the amino acid sequence of the chimeric A-E57K_R58E (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus with Lys and Arg at position 58 with Glu in chimeric A) is shown in sequence number 188. Sequence number 188 represents the DcR3 variant obtained by the chimeric A-E57R_R58D (the DcR3 variant obtained by replacing Glu at position 57 from the N-terminus with Arg and Arg at position 58 with Asp in chimeric A). The amino acid sequences are as follows: sequence number 270 represents the amino acid sequence of chimeric A-E57A (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Ala); sequence number 272 represents the amino acid sequence of chimeric A-E57F (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Phe); sequence number 274 represents the amino acid sequence of chimeric A-E57H (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with His); and sequence number 276 represents the amino acid sequence of chimeric A-E57I (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Ile).Serial number 278 represents the amino acid sequence of chimeric A-E57M (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Met), serial number 280 represents the amino acid sequence of chimeric A-E57K_R58T (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Lys and Arg at position 58 with Thr), and serial number 282 represents the amino acid sequence of chimeric A-E57K_R58T (a DcR3 variant obtained by replacing Glu at position 57 from the N-terminus of chimeric A with Lys and Arg at position 58 with Thr). The amino acid sequences of the following chimeric variants are given: A-E57V_R58T (a DcR3 variant obtained by replacing Glu at position 57 (N-terminus) with Val and Arg at position 58 with Thr), and A-E57V_R58E (a DcR3 variant obtained by replacing Glu at position 57 (N-terminus) with Val and Arg at position 58 with Glu).

[0277] 2-2. Other areas

[0278] The DcR3 variant of the present invention may include one or more other regions bound to the C-terminus of the first or second chimeric cysteine-rich region, or may not include said other regions. Examples of other regions include, for instance, a portion or all of the region present between CRD4 and HBD in wild-type DcR3, a portion or all of the region at the C-terminus of the amino acid sequence adjacent to CRD4 in TNF receptor superfamily (TNFRSF) molecules other than DcR3, a portion or all of the HBD in wild-type DcR3, etc. The expression “other regions bound to the C-terminus of the first or second chimeric cysteine-rich region” is used to mean that: the other region is directly bound to the C-terminus of the first or second chimeric cysteine-rich region, and that the other region is further bound to the C-terminus of the first or second chimeric cysteine-rich region through another region.

[0279] In one embodiment, the DcR3 variant of the present invention comprises part or all of the region of wild-type DcR3 present between CRD4 and HBD as additional regions bound to the C-terminus of the first or second chimeric cysteine-rich region. In this embodiment, it is preferred that the additional regions are directly bound to the C-terminus of the first or second chimeric cysteine-rich region.

[0280] In other embodiments, the DcR3 variant of the present invention comprises part or all of a region of the C-terminus of the amino acid sequence adjacent to CRD4 in a TNF receptor superfamily (TNFRSF) molecule other than DcR3 as an additional region binding to the C-terminus of the first or second chimeric cysteine-rich region. In this embodiment, it is preferred that the additional region binds directly to the C-terminus of the first or second chimeric cysteine-rich region. The description of TNFRSF is the same as above. TNFRSF is preferably OPG.

[0281] In the first or second chimeric cysteine ​​enrichment region (e.g., the first chimeric cysteine ​​enrichment region containing the amino acid sequences (b) to (d) above), when the CRD4 of wild-type DcR3 is replaced by the CRD4 of OPG, the DcR3 variant of the present invention preferably includes multiple amino acid residues at the C-terminus of the amino acid sequence of OPG adjacent to the CRD4 as other regions bound to the C-terminus of the chimeric cysteine ​​enrichment region. That is, when the CR4 of wild-type DcR3 is replaced by the CRD4 of OPG, multiple amino acid residues at the C-terminus of the amino acid sequence of wild-type DcR3 adjacent to the CRD4 are preferably replaced by multiple amino acid residues at the C-terminus of the amino acid sequence of OPG adjacent to the CRD4. As multiple amino acid residues at the C-terminus of the amino acid sequence of OPG attached to CRD4, for example, amino acid residues at positions 186 to 194 of the amino acid sequence of OPG (Sequence No. 14) are preferred, but the number of amino acid residues substituting for multiple amino acid residues at the C-terminus of wild-type DcR3 attached to CRD4 can be suitably adjusted. The number of multiple amino acid residues at the C-terminus of the amino acid sequence of OPG attached to CRD4 is usually 1 to 12, preferably 1 to 10, more preferably 1 to 9, 1 to 6, or 1 to 3. In addition, when CRD4 is derived from wild-type DcR3 in the first or second chimeric cysteine ​​enrichment region (e.g., the first chimeric cysteine ​​enrichment region containing the amino acid sequences of (a) and (d) above), it is preferred that the C-terminus of the amino acid sequence of CRD4 in the first chimeric cysteine ​​enrichment region is combined with the C-terminus of the amino acid sequence of wild-type DcR3 attached to CRD4. Examples of such amino acid sequences include, for instance, amino acids at positions 194-195 of sequence number 2, but the number of amino acid residues constituting the C-terminal amino acid sequence of the CRD4 attached to wild-type DcR3 can be suitably adjusted. The number of amino acid residues constituting the C-terminal amino acid sequence of the CRD4 attached to wild-type DcR3 is typically 1 to 10, preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 to 2.

[0282] In other embodiments, the DcR3 variant of the present invention comprises a portion or all of the HBD of wild-type DcR3 as additional regions binding to the C-terminus of the first or second chimeric cysteine-rich region. In this embodiment, the additional regions may bind directly to the C-terminus of the first or second chimeric cysteine-rich region, or they may bind to the C-terminus of the first or second chimeric cysteine-rich region via a portion or all of a region of wild-type DcR3 present between CRD4 and the HBD, or via a portion or all of a region of a TNFRSF molecule other than DcR3 adjacent to the C-terminus of the amino acid sequence of CRD4.

[0283] For the DcR3 variant of the present invention, other regions that bind to the C-terminal side of the first or second chimeric cysteine ​​enrichment region preferably do not contain HBD and contain amino acid residues from position 186 to 194 of the amino acid sequence (serial number 14) containing OPG.

[0284] 3. DCR3 variants containing sugar chains

[0285] As a DcR3 variant of the present invention, it also includes DcR3 variants containing at least one glycan. Any DcR3 variant containing a glycan is included in the present invention as long as the cysteine-rich region or other amino acid residues contained in the above-mentioned DcR3 variant are bound to at least one glycan.

[0286] Glycoproteins have one or more glycans. When a glycoprotein has two or more glycans, the glycans can be of one type or two or more types. Examples of glycans in glycoproteins include glycans that are N-glycosidically bonded to amino acid residues of peptides or proteins (e.g., asparagine residues) and glycans that are O-glycosidically bonded to amino acid residues of peptides or proteins (e.g., serine residues, threonine residues). Examples of O-type glycans include Core1 and Core2, and examples of N-type glycans include high-mannose, hybrid, and complex glycans, with complex glycans being preferred.

[0287] 4. DCR3 variant containing the Fc region

[0288] As a variant of the DcR3 of the present invention, it also includes proteins of the same or different kind, which are directly or, if necessary, bound or fused to the N-terminal or C-terminal side of a chimeric cysteine-rich region (or a chimeric cysteine-rich region containing other regions) directly or, if necessary, via a suitable peptide linker. The number of amino acids constituting the peptide linker is not particularly limited, and examples include, for instance, 4, 5, 6, or 15.

[0289] Examples of polypeptides or proteins that bind to or are fused to chimeric cysteine-rich regions include, for example, the constant region or Fc region of immunoglobulins, peptides that bind FcRn (newborn Fc receptor), albumin, protein A, protein G, β-galactosidase, glutathione S-transferase (GST), maltose-binding protein, polyhistidine, FLAG peptide, etc., preferably the Fc region of immunoglobulins or its mutants (mutant Fc regions), and more preferably the Fc region of immunoglobulins derived from mammals or its mutants (mutant Fc regions).

[0290] When used in humans, the Fc region of an immunoglobulin (also referred to as an antibody) is preferably a human immunoglobulin. Examples of immunoglobulin types and subclasses include IgG, IgD, IgE, IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or IgA1, but these are not limited to these. When used in humans, the type and subclass of human immunoglobulin are preferred. Furthermore, when using the Fc region of an immunoglobulin as a polypeptide or protein that binds to or fuses to a chimeric cysteine-rich region, it is preferable that the Fc region of the immunoglobulin binds to or fuses to the C-terminal side of the chimeric cysteine-rich region.

[0291] Immunoglobulins are composed of polypeptides with heavy and light chains. The constant region of the heavy chain of human IgG, starting from the N-terminus, consists of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. The Fc region of IgG in this invention also includes a region formed by combining the CH2 and CH3 domains, and a region formed by combining part or all of the hinge domain with the CH2 and CH3 domains. The domains contained in the Fc region of IgG in this invention can be identified using EU index numbers. Specifically, the hinge domain is identified by EU index positions 216 to 230, the CH2 domain by positions 231 to 340, and the CH3 domain by positions 341 to 447.

[0292] Furthermore, peptides or proteins bound or fused to chimeric cysteine-rich regions also include modified peptides or proteins obtained by further substitution, deletion, insertion, or addition of one or more amino acids to alter the biological activity or properties of DcR3 variants, their in vivo kinetics such as blood half-life, or their physical or chemical properties such as protein stability. Modifications of peptides or proteins bound or fused to chimeric cysteine-rich regions include any natural mutations and artificial substitutions, deletions, insertions, or additions of amino acids. Examples of modified peptides or proteins include, for instance, mutant Fc regions containing amino acid sequences with deletions, substitutions, insertions, or additions of one or more amino acids in the Fc region of immunoglobulins. Examples of amino acid sequences that can be used as mutant Fc regions include amino acid sequences obtained by substituting, deleting, inserting, or adding one or more, preferably 2 to 30, more preferably 2 to 10, and particularly preferably 2 to 5 amino acids in the amino acid sequence of the Fc region of immunoglobulins; or amino acid sequences that have 80% or more, preferably 85% or more, and more preferably 90% or more identity with the amino acid sequence of the Fc region of immunoglobulins.

[0293] The addition or deletion of glycan addition sites mentioned above is also included in the modification of peptides or proteins that are bound to or fused to chimeric cysteine-rich regions. For example, glycan addition sites can be added by adding or inserting peptides containing N-type glycan addition sites. Specific peptide sequences include GGNGT or YGNGT composed of 5 amino acids [International Patent Publication No. 2014 / 153111].

[0294] As a substitute for human IgG1 that reduces or eliminates complement-dependent cytotoxic (CDC) activity, examples include substitutions of one or more amino acids selected from Leu (L234), Leu (L235), Asp (D265), Asp (D265), Lys (K322), Pro (P329), and Pro (P331) at position 234 (EU index), etc., with other amino acids. Specifically, examples include substitutions of one or more amino acids selected from L234, L235, D270, K322, P329, and P331 at position 329 with Ala, and substitutions of P331 with Ser or Gly, etc. [J. Immunol., 2000, 164: p.4178-4184, Cell.] Immunol., 2000, 200: p.16-26].

[0295] As a substitute for human IgG1 that reduces or eliminates effector activities such as antibody-dependent cell killing (ADCC) and antibody-dependent cell phagocytosis (ADCP), examples include those selected from Asn (N297) at position 297, Leu (L234) at position 234, Leu (L235) at position 235, Gly (G237) at position 237, Cys (C226) at position 226, Cys (C229) at position 229, Pro (P238) at position 238, Glu (E233) at position 233, and others at position 267. One or more amino acids in Ser (S267), Leu at position 328 (L328), and Pro at position 331 (P331) are substituted with other amino acids. Specific examples, based on the EU index, include substitutions such as N297 with Ala (N297A), N297 with Gln (N297Q), N297 with Gly (N297G), or L234 with Ala (L234A), L235 with Ala (L235A), and G237 with Ala reduction (G237A). It should be noted that " / " means "and" (the same applies below).

[0296] In addition, examples of modifications that increase the binding activity with FcγRIIb, which is an inhibitory receptor, include substitution of Gly with Asp at position 236 (G236D), substitution of Leu with Phe at position 328 (L328F), substitution of Ser with Asp at position 239 (S239D), and substitution of Ser with Glu at position 267 (S267E) according to the EU index [Curr. Opin. Cell. Biol., 2009, 20: p.685-691]. In addition, as modifications to enhance binding to FcRn (newborn Fc receptor) in the low pH environment of the endosome, prevent antibody loss, and prolong the blood half-life, examples include 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), and substitution of Thr at position 256 with Glu. Substitution (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. [J. Immunol., 2009, 182: p.7663-7671, MAbs, 2017, 9: p.844-853].

[0297] In a preferred embodiment, the mutant Fc region has the following substitution: in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass, Cys, indicated as position 220 according to the EU index, is replaced with Ser. Examples of mutant Fc regions involved in this method include: for instance, the Fc region of an immunoglobulin having the following amino acid sequence (hereinafter referred to as g1S, sequence number 72): after removing the CH1 domain and the Glu domain (position 216 according to the EU index) from the constant region (sequence number 153) of the heavy chain of human IgG1, the Cys domain (position 220 according to the EU index) involved in binding with the light chain is replaced by Ser (C220S); the Fc region of an immunoglobulin having the following amino acid sequence (hereinafter referred to as Eg1S, sequence number 156): after removing the CH1 domain from the constant region (sequence number 153) of the heavy chain of human IgG1, the Cys domain (position 216 to 447 according to the EU index) is replaced by Ser (C220S); and so on. When the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the first chimeric cysteine ​​enrichment region in the DcR3 variant of the present invention has the wild-type DcR3's CRD1 replaced by the OPG's CRD1 and the wild-type DcR3's CRD4 replaced by the OPG's CRD4. Furthermore, when the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, the second chimeric cysteine ​​enrichment region 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. It is preferred that the other amino acid replacing Glu at position 57 be selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and it is further preferred that it be selected from Lys, Leu, Arg, and Val. The other amino acid that replaces Arg at position 58 is preferably selected from Asp, Glu and Thr, and selection from Asp and Glu is even more preferred.When the substitution of Glu at position 57 with other amino acids is combined with the substitution of Arg at position 58 with other amino acids, it is preferable to combine the substitution of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with the substitution of Arg at position 58 with Asp, Glu, or Thr. It is further preferable to combine the substitution of Glu at position 57 with Lys, Leu, Arg, or Val with the substitution of Arg at position 58 with Asp or Glu. It is even more preferable to combine the substitution of Glu at position 57 with Lys or Arg with the substitution of Arg at position 58 with Asp or Glu.

[0298] In another preferred embodiment, the mutant Fc region has the following substitutions: in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG4 subclass, Ser at position 228 according to the EU index is replaced with Pro, Leu at position 235 according to the EU index is replaced with Glu, and Arg at position 409 according to the EU index is replaced with Lys. Examples of mutant Fc regions in this embodiment include the Fc of an immunoglobulin (hereinafter referred to as g4PEK, sequence number 74) having the following amino acid sequence: the CH1 domain is removed from the constant region (sequence number 154) of the heavy chain of human IgG4, and Ser at position 228 according to the EU index is replaced with Pro, Leu at position 235 is replaced with Glu, and Arg at position 409 is replaced with Lys, as described in International Patent Publication 2006 / 33386. When the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the first chimeric cysteine ​​enrichment region included in the DcR3 variant of the present invention has the wild-type DcR3's CRD1 replaced by the OPG's CRD1 and the wild-type DcR3's CRD4 replaced by the OPG's CRD4. Furthermore, when the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the second chimeric cysteine ​​enrichment region included in the DcR3 variant of the present invention 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. It is preferred that the other amino acid replacing Glu at position 57 be selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and it is further preferred that it be selected from Lys, Leu, Arg, and Val. The other amino acid replacing Arg at position 58 is preferably selected from Asp, Glu, and Thr, and selection from Asp and Glu is further preferred. When combining substitutions of Glu at position 57 with other amino acids with substitutions of Arg at position 58, it is preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with substitutions of Arg at position 58 with Asp, Glu, or Thr, further preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, or Val with substitutions of Arg at position 58 with Asp or Glu, and even more preferred to combine substitutions of Glu at position 57 with Lys or Arg with substitutions of Arg at position 58 with Asp or Glu.

[0299] In a further preferred embodiment, the mutant Fc region has the following substitutions: in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass, Leu at position 234, as indicated by the EU index, is replaced with Ala; Leu at position 235, as indicated by the EU index, is replaced with Ala; and Gly at position 237, as indicated by the EU index, is replaced with Ala. Examples of mutant Fc regions in this embodiment include the Fc regions of immunoglobulins having the following amino acid sequences (hereinafter referred to as g1SLALAGA or Eg1S LALAGA, sequence numbers 162 and 164): in the Fc regions of g1S (sequence number 72) or Eg1S (sequence number 156), Leu at position 234 is replaced with Ala; Leu at position 235 is replaced with Ala; Gly at position 237 is replaced with Ala; and so on. In the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the first chimeric cysteine ​​enrichment region in the DcR3 variant of the present invention has the wild-type DcR3 CRD1 replaced by the OPG CRD1 and the wild-type DcR3 CRD4 replaced by the OPG CRD4 as the first chimeric cysteine ​​enrichment region. Furthermore, in the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, the second chimeric cysteine ​​enrichment region 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. It is preferred that the other amino acid replacing Glu at position 57 be selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and it is further preferred that it be selected from Lys, Leu, Arg, and Val. The other amino acid replacing Arg at position 58 is preferably selected from Asp, Glu, and Thr, and selection from Asp and Glu is further preferred. When combining substitutions of Glu at position 57 with other amino acids with substitutions of Arg at position 58, it is preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with substitutions of Arg at position 58 with Asp, Glu, or Thr, further preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, or Val with substitutions of Arg at position 58 with Asp or Glu, and even more preferred to combine substitutions of Glu at position 57 with Lys or Arg with substitutions of Arg at position 58 with Asp or Glu.

[0300] In a further preferred embodiment, the mutant Fc region has the following substitution: in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass, Asn at position 434, as indicated by the EU index, is replaced by Ala. Examples of mutant Fc regions involved in this embodiment include: for example, the Fc region of an immunoglobulin having an amino acid sequence in which Asn at position 434 is replaced by Ala in the Fc region of g1S (serial number 72) or Eg1S (serial number 156) (hereinafter referred to as g1S N434A or Eg1S N434A, serial numbers 312, 160); the Fc region of an immunoglobulin having an amino acid sequence in which Asn at position 434 is replaced by Ala in the Fc region of g1S LALAGA (serial number 162) or Eg1S LALAGA (serial number 164) (hereinafter referred to as g1S LALAGANA or Eg1S LALAGANA, serial numbers 313, 166), etc. In the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the first chimeric cysteine ​​enrichment region in the DcR3 variant of the present invention has the wild-type DcR3 CRD1 replaced by the OPG CRD1 and the wild-type DcR3 CRD4 replaced by the OPG CRD4 as the first chimeric cysteine ​​enrichment region. Furthermore, in the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, the second chimeric cysteine ​​enrichment region 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. It is preferred that the other amino acid replacing Glu at position 57 be selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and it is further preferred that it be selected from Lys, Leu, Arg, and Val. The other amino acid replacing Arg at position 58 is preferably selected from Asp, Glu, and Thr, and selection from Asp and Glu is further preferred. When combining substitutions of Glu at position 57 with other amino acids with substitutions of Arg at position 58, it is preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with substitutions of Arg at position 58 with Asp, Glu, or Thr, further preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, or Val with substitutions of Arg at position 58 with Asp or Glu, and even more preferred to combine substitutions of Glu at position 57 with Lys or Arg with substitutions of Arg at position 58 with Asp or Glu.

[0301] In a further preferred embodiment, the mutant Fc region has the following substitutions: in the amino acid sequence of the heavy chain of an antibody belonging to the human IgG1 subclass, Met at position 252 according to the EU index is replaced with Tyr, Ser at position 254 according to the EU index is replaced with Thr, and Thr at position 256 according to the EU index is replaced with Glu. Examples of mutant Fc regions in this embodiment include the Fc regions of immunoglobulins having the following amino acid sequences (hereinafter referred to as g1SYTE or Eg1S YTE, sequence numbers 311 and 158): in the Fc region of g1S (sequence number 72) or Eg1S (sequence number 156), the amino acid sequence in which Met at position 252 is replaced with Tyr, Ser at position 254 is replaced with Thr, and Thr at position 256 is replaced with Glu. In the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, it is preferred that the first chimeric cysteine ​​enrichment region in the DcR3 variant of the present invention has the wild-type DcR3 CRD1 replaced by the OPG CRD1 and the wild-type DcR3 CRD4 replaced by the OPG CRD4 as the first chimeric cysteine ​​enrichment region. Furthermore, in the case where the DcR3 variant of the present invention includes the mutant Fc region involved in this manner, the second chimeric cysteine ​​enrichment region 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. It is preferred that the other amino acid replacing Glu at position 57 be selected from Lys, Leu, Arg, Val, Ala, Phe, His, Ile, and Met, and it is further preferred that it be selected from Lys, Leu, Arg, and Val. The other amino acid replacing Arg at position 58 is preferably selected from Asp, Glu, and Thr, and selection from Asp and Glu is further preferred. When combining substitutions of Glu at position 57 with other amino acids with substitutions of Arg at position 58, it is preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, Val, Ala, Phe, His, Ile, or Met with substitutions of Arg at position 58 with Asp, Glu, or Thr, further preferred to combine substitutions of Glu at position 57 with Lys, Leu, Arg, or Val with substitutions of Arg at position 58 with Asp or Glu, and even more preferred to combine substitutions of Glu at position 57 with Lys or Arg with substitutions of Arg at position 58 with Asp or Glu.

[0302] Examples of mutant Fc regions include, for example, mutant Fc regions containing the amino acid sequences described in sequence numbers 72, 74, 156, 158, 160, 162, 164, 166, 311, 312, or 313, or mutant Fc regions composed of such amino acid sequences, but are not limited to these.

[0303] The peptide linker is not limited to the addition of homologous or heterologous peptides, polypeptides or proteins to the N-terminus or C-terminus of the chimeric cysteine-rich region. Examples include peptide linkers such as the IEGRMD linker or the GS linker, as well as chemical linkers.

[0304] As a DcR3 variant of the present invention, a DcR3 variant comprising a first or second chimeric cysteine-rich region and an Fc region or a mutant Fc region may be optimally selected.

[0305] In one embodiment, the amino acid sequence included in the DcR3 variant of the present invention may be, for example, the amino acid sequences described in sequence numbers 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46, or an amino acid sequence obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids. In this embodiment, the DcR3 variant of the present invention may consist of or contain the above-described amino acid sequences, but is preferably composed of the above-described amino acid sequences. It should be noted that the above-described amino acid sequences contain HBD.

[0306] In other embodiments, examples of amino acid sequences included in the DcR3 variant of the present invention include, for example, the amino acid sequences described in sequence numbers 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 amino acid sequences obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids. In this embodiment, the DcR3 variant of the present invention may consist of or contain the above-described amino acid sequences, but is preferably composed of the above-described amino acid sequences. It should be noted that the above-described amino acid sequences do not contain HBD.

[0307] In other ways, examples of amino acid sequences included in the DcR3 variant of the present invention include, for example, amino acid sequences containing the following sequences: amino acid sequences described in sequence numbers 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 amino acid sequences obtained by deleting, substituting, inserting or adding 1 to 30 amino acids; and amino acid sequences described in sequence numbers 72, 74, 156, 158, 160, 162, 164, 166, 311, 312 or 313 as mutant Fc regions.

[0308] In other embodiments, examples of amino acid sequences included in the DcR3 variant of the present invention include, for example, sequence numbers 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, 24... The amino acid sequences described in 8, 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 amino acid sequences obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids. In this manner, the DcR3 variant of the present invention may consist of or contain the above-described amino acid sequences, but preferably consists of the above-described amino acid sequences. It should be noted that the above amino acid sequence includes the Fc region or its mutant (mutant Fc region).

[0309] It should be noted that sequence number 76 represents the amino acid sequence of chimeric B-Fc (IEGRMD g1S) (a DcR3 variant obtained by fusing chimeric B, the IEGRMD linker, and Fc (g1S)), sequence number 78 represents the amino acid sequence of chimeric C-Fc (IEGRMD g1S) (a DcR3 variant obtained by fusing chimeric C, the IEGRMD linker, and Fc (g1S)), and sequence number 80 represents the amino acid sequence of chimeric A-Fc (IEGRMD g1S). The amino acid sequence of g1S (a DcR3 variant obtained by fusing chimeric A, IEGRMD linkers with Fc(g1S)) is given by sequence number 82. Sequence number 82 represents the amino acid sequence of chimeric A-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A with Fc(g4PEK)). Sequence number 84 represents the amino acid sequence of 103-123OPG-Fc(g4PEK) (a DcR3 variant obtained by fusing 103-123OPG with Fc(g4PEK)). Sequence number 86 represents the amino acid sequence of N131S / N144S-Fc(g4PEK) (a DcR3 variant obtained by fusing N131S with Fc(g4PEK)). The amino acid sequences of the DcR3 variant obtained by fusing S / N144S with Fc(g4PEK) are as follows: sequence number 88 represents the amino acid sequence of T133A / S146A-Fc(g4PEK); sequence number 90 represents the amino acid sequence of N131S / N144S / N157S-Fc(g4PEK); and sequence number 92 represents the amino acid sequence of T133A / The amino acid sequence of S146A / T159A-Fc(g4PEK) (a DcR3 variant obtained by fusing T133A / S146A / T159A with Fc(g4PEK)), sequence number 94 represents the amino acid sequence of the chimeric A-E57K-Fc(g4PEK) (a DcR3 variant obtained by fusing A-E57K with Fc(g4PEK)), sequence number 96 represents the amino acid sequence of the chimeric A-E57L-Fc(g4PEK) (a DcR3 variant obtained by fusing A-E57L with Fc(g4PEK)), sequence number 9 8 represents the amino acid sequence of the chimeric A-R60K-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-R60K with Fc(g4PEK)), sequence number 150 represents the amino acid sequence of the chimeric A-Fc(g1S) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S)), sequence number 168 represents the amino acid sequence of the chimeric A-Fc(Eg1S) (a DcR3 variant obtained by fusing chimeric A with Fc(Eg1S)), and sequence number 170 represents the amino acid sequence of the chimeric A-Fc(Eg1S-YTE) (a DcR3 variant obtained by fusing chimeric A with Fc(Eg1S YTE)).Serial number 172 represents the amino acid sequence of chimeric A-Fc(Eg1S-N434A) (a DcR3 variant obtained by fusing chimeric A with Fc(Eg1S-N434A)), serial number 174 represents the amino acid sequence of chimeric A-Fc(g1S-LALAGA) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S-LALAGA)), serial number 176 represents the amino acid sequence of chimeric A-Fc(Eg1S-LALAGA) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S-LALAGA)), and serial number 178 represents the amino acid sequence of chimeric A-Fc(Eg1S-LALAGANA) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S-N434A)). The amino acid sequences of the DcR3 variant obtained by fusing 1S-LALAGANA are as follows: sequence number 190 represents the amino acid sequence of the chimeric A-E57R-Fc(g4PEK) variant (obtained by fusing A-E57R with Fc(g4PEK); sequence number 192 represents the amino acid sequence of the chimeric A-E57V-Fc(g4PEK) variant (obtained by fusing A-E57V with Fc(g4PEK); and sequence number 194 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc(g4PEK) variant (obtained by fusing A-E57K_R58D with Fc(g4PEK)). Serial number 196 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E with Fc(g4PEK)), serial number 198 represents the amino acid sequence of the chimeric A-E57R_R58D-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57R_R58D with Fc(g4PEK)), serial number 200 represents the amino acid sequence of the chimeric A-E57K-Fc(Eg1S) (a DcR3 variant obtained by fusing chimeric A-E57K with Fc(Eg1S)), and serial number 202 represents the amino acid sequence of the chimeric A-E57L-Fc(Eg1S). The amino acid sequences of the following are defined as follows: (A DcR3 variant obtained by fusing A-E57L with Fc(Eg1S)); sequence number 204 represents the amino acid sequence of the chimeric A-E57R-Fc(Eg1S); sequence number 206 represents the amino acid sequence of the chimeric A-E57V-Fc(Eg1S); and sequence number 208 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc(Eg1S).Serial number 210 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc(Eg1S) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E with Fc(Eg1S)); serial number 212 represents the amino acid sequence of the chimeric A-E57R_R58D-Fc(Eg1S) (a DcR3 variant obtained by fusing chimeric A-E57R_R58D with Fc(Eg1S); serial number 214 represents the amino acid sequence of the chimeric A-E57K-Fc(Eg1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57K with Fc(Eg1S YTE); and serial number 216 represents the amino acid sequence of the chimeric A-E57L-Fc(Eg1S YTE) (a DcR3 variant obtained by fusing chimeric A-E57L with Fc(Eg1S)). The amino acid sequences of the DcR3 variant obtained by fusing A-E57R-Fc (Eg1S YTE) are as follows: sequence number 218 represents the amino acid sequence of the chimeric A-E57R-Fc (Eg1S YTE); sequence number 220 represents the amino acid sequence of the chimeric A-E57V-Fc (Eg1S YTE); sequence number 222 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc (Eg1S YTE); and sequence number 224 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc (Eg1S YTE). The amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1S YTE) is given by sequence number 226. Sequence number 228 represents the amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1S YTE). Sequence number 230 represents the amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1S YTE). The amino acid sequences of the DcR3 variant obtained by fusing A-E57R-Fc (Eg1S N434A) and 234 represent the amino acid sequences of the DcR3 variant obtained by fusing A-E57R with Fc (Eg1SN434A).Serial number 236 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing A-E57K_R58D with Fc(Eg1S N434A)), serial number 238 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing A-E57K_R58E with Fc(Eg1S N434A)), and serial number 240 represents the amino acid sequence of the chimeric A-E57R_R58D-Fc(Eg1S N434A) (a DcR3 variant obtained by fusing A-E57K_R58E with Fc(Eg1S N434A)). The amino acid sequences of the DcR3 variant obtained by fusing A-E57K-Fc (Eg1S LALAGA) and Fc (Eg1S LALAGA) are as follows: sequence number 242 represents the amino acid sequence of the chimeric A-E57K-Fc (Eg1S LALAGA); sequence number 244 represents the amino acid sequence of the chimeric A-E57L-Fc (Eg1S LALAGA); sequence number 246 represents the amino acid sequence of the chimeric A-E57R-Fc (Eg1S LALAGA); and sequence number 248 represents the amino acid sequence of the chimeric A-E57V-Fc (Eg1S LALAGA). The amino acid sequence of LALAGA (a DcR3 variant obtained by fusing A-E57V with Fc (Eg1S LALAGA)) is given by sequence number 250. Sequence number 252 represents the amino acid sequence of A-E57K_R58E-Fc (Eg1S LALAGA) (a DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1S LALAGA)). Sequence number 254 represents the amino acid sequence of A-E57R_R58D-Fc (Eg1S LALAGA) (a DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1S LALAGA)). The amino acid sequences of the DcR3 variant obtained by fusing A-E57K with Fc (Eg1S LALAGANA) are shown. Sequence number 256 represents the amino acid sequence of the chimeric A-E57K-Fc (Eg1S LALAGANA) variant, and sequence number 258 represents the amino acid sequence of the chimeric A-E57L-Fc (Eg1S LALAGANA) variant.Serial number 260 represents the amino acid sequence of the chimeric A-E57R-Fc(Eg1S LALAGANA) (a DcR3 variant obtained by fusing chimeric A-E57R with Fc(Eg1S LALAGANA)), serial number 262 represents the amino acid sequence of the chimeric A-E57V-Fc(Eg1S LALAGANA) (a DcR3 variant obtained by fusing chimeric A-E57V with Fc(Eg1SLALAGANA)), serial number 264 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc(Eg1SLALAGANA) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D with Fc(Eg1S LALAGANA)), and serial number 266 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc(Eg1S LALAGANA) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D with Fc(Eg1S LALAGANA)). The amino acid sequence of LALAGANA (a DcR3 variant obtained by fusing A-E57K_R58E with Fc (Eg1SLALAGANA)) is given by sequence number 268, which represents the amino acid sequence of the chimeric A-E57R_R58D-Fc (Eg1SLALAGANA) (a DcR3 variant obtained by fusing A-E57R_R58D with Fc (Eg1S)). The amino acid sequences of the DcR3 variant obtained by fusing A-E57A with Fc(g4PEK) are as follows: sequence number 288 represents the amino acid sequence of the chimeric A-E57A-Fc(g4PEK); sequence number 290 represents the amino acid sequence of the chimeric A-E57F-Fc(g4PEK); sequence number 292 represents the amino acid sequence of the chimeric A-E57H-Fc(g4PEK); and sequence number 294 represents the amino acid sequence of the chimeric A-E57I-Fc(g4PEK). Serial number 296 represents the amino acid sequence of the chimeric A-E57M-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57M with Fc(g4PEK)), serial number 298 represents the amino acid sequence of the chimeric A-E57K_R58T-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57K_R58T with Fc(g4PEK), and serial number 30... 0 represents the amino acid sequence of the chimeric A-E57L_R58E-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57L_R58E with Fc(g4PEK), and sequence number 302 represents the amino acid sequence of the chimeric A-E57V_R58T-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57V_R58T with Fc(g4PEK)).Serial number 304 represents the amino acid sequence of chimeric A-E57V_R58E-Fc(g4PEK) (a DcR3 variant obtained by fusing chimeric A-E57V_R58E with Fc(g4PEK)), serial number 314 represents the amino acid sequence of chimeric A-Fc(g1S YTE) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S YTE)), serial number 315 represents the amino acid sequence of chimeric A-Fc(g1S N434A) (a DcR3 variant obtained by fusing chimeric A with Fc(g1S N434A)), serial number 316 represents the amino acid sequence of chimeric A-Fc(g1S LALAGANA) (a DcR3 variant obtained by fusing chimeric A with Fc(g1SLALAGANA)), and serial number 317 represents the amino acid sequence of chimeric A-E57K-Fc(g1S LALAGANA). The amino acid sequences of the following are defined: YTE (a DcR3 variant obtained by fusing A-E57K with Fc(g1S YTE)); sequence number 318 represents the amino acid sequence of A-E57L-Fc(g1S YTE); sequence number 319 represents the amino acid sequence of A-E57R-Fc(g1S YTE); sequence number 320 represents the amino acid sequence of A-E57V-Fc(g1S YTE); and sequence number 321 represents the amino acid sequence of A-E57K_R58D-Fc(g1S YTE). The amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58D with Fc(g1S YTE) is shown in sequence 322. Sequence number 323 represents the amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58D with Fc(g1S YTE). Sequence number 324 represents the amino acid sequence of the DcR3 variant obtained by fusing A-E57K_R58D with Fc(g1S YTE). The amino acid sequences of the DcR3 variant obtained by fusing A-E57L-Fc(g1S N434A) and Fc(g1S N434A) are shown. Serial number 325 represents the amino acid sequence of the chimeric A-E57L-Fc(g1S N434A) variant, and serial number 326 represents the amino acid sequence of the chimeric A-E57R-Fc(g1S N434A) variant.Serial number 327 represents the amino acid sequence of the chimeric A-E57V-Fc(g1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57V with Fc(g1S N434A)), serial number 328 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc(g1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57K_R58D with Fc(g1S N434A)), serial number 329 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc(g1S N434A) (a DcR3 variant obtained by fusing chimeric A-E57K_R58E with Fc(g1S N434A)), and serial number 330 represents the amino acid sequence of the chimeric A-E57R_R58D-Fc(g1S N434A) (g1S N434A). The amino acid sequence of N434A (a DcR3 variant obtained by fusing A-E57R_R58D with Fc(g1S N434A)) is given by sequence number 331. Sequence number 331 represents the amino acid sequence of A-E57K-Fc(g1S LALAGANA) (a DcR3 variant obtained by fusing A-E57K with Fc(g1S LALAGANA)). Sequence number 332 represents the amino acid sequence of A-E57L-Fc(g1S LALAGANA) (a DcR3 variant obtained by fusing A-E57L with Fc(g1SLALAGANA)). Sequence number 333 represents the amino acid sequence of A-E57R-Fc(g1SLALAGANA) (a DcR3 variant obtained by fusing A-E57R with Fc(g1S N434A)). The amino acid sequences of the DcR3 variant obtained by fusing A-E57V with Fc (g1S LALAGANA) are as follows: sequence number 334 represents the amino acid sequence of the chimeric A-E57V-Fc (g1S LALAGANA); sequence number 335 represents the amino acid sequence of the chimeric A-E57K_R58D-Fc (g1S LALAGANA); and sequence number 336 represents the amino acid sequence of the chimeric A-E57K_R58E-Fc (g1S LALAGANA). The amino acid sequence of the DcR3 variant obtained by fusing A-E57R_R58D with Fc(g1S LALAGANA) is given by sequence number 337.

[0310] In any of the above-described methods, any mutations (modifications) that can be added to the amino acid sequence include natural mutations and any substitutions, deletions, insertions, or additions of artificial amino acids. Furthermore, in any of the above-described methods, as a sequence obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids to the above-described amino acid sequence, examples include amino acid sequences obtained by substituting, deleting, inserting, or adding 1 or more, preferably 2 to 30, more preferably 2 to 10, particularly preferably 2 to 5 amino acids, 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 of the same identity as the amino acid sequence.

[0311] The DcR3 variants of the present invention can also be further chemically modified for the purpose of altering biological activity or its properties, in vivo kinetics such as blood half-life, or physical or chemical properties such as protein stability.

[0312] Examples of chemical modifications include polyethylene glycol (PEG) modification, acetylation, amidation, or phosphorylation, with PEG modification being particularly preferred. PEGylation involves attaching one or more PEG molecules to amino acid residues, such as those with an N-terminal amino group of a protein, or those with a Lys-containing ε-amino group, carboxyl group, thiol group, or hydroxyl group on their side chains.

[0313] The average molecular weight of PEG molecules can be used in the range of approximately 3,000 to approximately 50,000, but is not limited to this range.

[0314] One method for binding PEG molecules to DcR3 variants is to introduce active groups such as carboxyl, formyl (aldehyde), N-hydroxysuccinimide ester, amino, thiol, or maleimide groups into the terminal portion of the PEG, so that it reacts with functional groups such as amino, carboxyl, thiol, or hydroxyl groups present in the side chain of the DcR3 variant.

[0315] The signal peptide is not limited. When the N-terminal CRD domain of the DcR3 variant is derived from human DcR3, an example is a signal peptide of human DcR3 consisting of amino acid sequences from positions 1 to 29 of sequence number 2. Alternatively, when the N-terminal CRD domain of the DcR3 variant is derived from human OPG, an example is a signal peptide derived from human OPG consisting of amino acid sequences from positions 1 to 21 of sequence number 14. Furthermore, the signal peptide of the present invention may comprise any of artificial sequences, sequences derived from expression vectors, or sequences derived from other proteins suitable for expressing the DcR3 variant in a host cell. The DcR3 variant containing the signal peptide is an immature polypeptide; in one embodiment, the signal peptide is cleaved during maturation. The DcR3 variant of the present invention may also include variants with different N-termini obtained by cleaving at a position different from the predicted cleavage site of the signal peptide described above.

[0316] 5. Manufacturing method of DCR3 variant

[0317] The DcR3 variant of this invention can be used with Molecular Cloning: A Laboratory Manual, 3 rd Methods described in publications such as the edition of Cold Spring Harbor Laboratory Press (2001) and others, for example, to express and manufacture DNA encoding the DcR3 variant in a host cell.

[0318] The signal peptide is not limited. When the N-terminal CRD domain of the DcR3 variant is derived from human DcR3, an example is a signal peptide of human DcR3 composed of amino acid sequences from positions 1 to 29 of sequence number 2. Alternatively, when the N-terminal CRD domain of the DcR3 variant is derived from human OPG, an example is a signal peptide derived from human OPG composed of amino acid sequences from positions 1 to 21 of sequence number 14. Furthermore, any of the following can be used to manufacture the DcR3 variant of the present invention: artificial sequences, sequences derived from expression vectors, or sequences derived from other proteins of host cells suitable for expressing the DcR3 variant. The DcR3 variant of the present invention can also include variants with different N-termini obtained by cleaving at a position different from the predicted cleavage site of the signal peptide described above.

[0319] The DcR3 variants of the present invention can be obtained through artificial design 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 a mutation introduction method, site-specific mutation induction using primer-based PCR is preferred (Kunkel et al., Proc. Natl. Acad. Sci. USA, 1985, 82: 488-492). Other methods include: total synthesis of the mutated gene; PCR using primers containing the mutation, separating the fragments before and after the mutation site, ligating the two fragments by overlapping the mutated region, and inserting them into a vector using in-fusion cloning (Clontech).

[0320] As a method for identifying mutations with a target binding mode, one can exemplify the following: preparing a library with randomly introduced mutations, displaying it on bacteriophages, yeast, etc., and screening based on binding activity to obtain mutants with the target binding mode. Alternatively, one can exemplify the following: expressing a vector containing a DNA mutation that replaces a specific amino acid with a different amino acid in a host cell and manufacturing it to obtain a mutant with the target binding mode. The DNA encoding the DcR3 variant of the present invention can be designed from the amino acid sequence of the DcR3 variant of the present invention to encode its base sequence and synthesized using a DNA synthesizer. Furthermore, it can be isolated by PCR using human cDNA as a template.

[0321] A recombinant vector is prepared by inserting the DNA encoding the DcR3 variant of the present invention obtained above downstream of the promoter of an appropriate expression vector, and the recombinant vector is introduced into a host cell suitable for the expression vector.

[0322] The base sequence of the DNA encoding the DcR3 variant can be substituted to create codons that are optimally suited for expression within the host cell, thereby increasing the yield of the target DcR3 variant. When preparing the DNA encoding this DcR3 variant, a signal peptide encoding a secretory protein is added to its 5' end. Using this DNA, a recombinant vector is prepared in the same manner as described above, and introduced into a host cell, allowing the peptide to be secreted into the culture medium for production. Examples of signal peptides include sequences derived from DcR3 or OPG, artificial sequences, sequences derived from expression vectors, or sequences derived from other proteins suitable for the host cell.

[0323] In one manner, the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant can be exemplified by, for example, the base sequences recorded in sequence numbers 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45.

[0324] In other ways, the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant can be exemplified by, for example, the base sequences recorded in sequence numbers 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.

[0325] In other ways, the base sequence of the DNA encoding the amino acid sequence of the DcR3 variant can be exemplified by, for example, sequence numbers 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, 2 The base sequences recorded at 13, 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.

[0326] Any vector can be used as an expression vector, as long as it can autonomously replicate or integrate into the chromosome in the host cell and contains an appropriate promoter at a location where the DNA encoding the polypeptide can be transcribed.

[0327] Any cell can be used as the host cell, such as yeast, insect cells, or animal cells, as long as it can express the gene encoding the DcR3 variant. Yeast, animal cells, or insect cells that can glycosylate proteins are preferred examples.

[0328] Examples of yeasts include microorganisms belonging to the genera *Saccharomyces*, *Schizosaccharomyces*, *Kluyveromyces*, *Trichosporon*, *Schwanniomyces*, *Pichia*, and *Candida*, such as *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Kluyveromyces Lactis*, *Trichosporon pullullans*, *Schwanniomyces alluvius*, and *Candida utilis*.

[0329] Examples of insect cells include Sf9 and Sf21 cells from the ovaries of the fall armyworm (Spodoptera frugiperda) [Baculovirus Expression Vectors, A Laboratory Manual, WHFreeman and Company, New York (1992)], High 5 cells from the ovaries of the white armyworm (Trichoplusia ni) (manufactured by Invitrogen), and S2 (Schneider 2) cells from the late embryos of the fruit fly (Drosophila melanogaster) (Thermo Scientific).

[0330] Examples of animal cells include Namalwa cells as human cells, COS cells as monkey cells, and CHO cells as 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 number: CCL-61), Freestyle CHO-S cells, CHO cells lacking the dihydrofolate reductase gene [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO cells lacking the 6-fucosetransferase gene (International Publication No. 2005 / 035586, International Publication No. 02 / 31140), 293 cells as human cells (ATCC number: CRL-1573), Freestyle 293F cells, Expi293 cells (Thermo Scientific), DUkXB11 (ATCC number: CCL-9096), Pro-5 (ATCC number: CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3, rat myeloma cells YB2 / 3HL.P2.G11.16Ag.20 (or also known as YB2 / 0), mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, or Syrian hamster cells BHK, HBT5637 (Japanese Patent Application Publication No. 63-299), etc.

[0331] When using yeast as the host cell, examples of expression vectors include YEP13 (ATCC number: 37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, or pHS15.

[0332] As a promoter, any promoter that can be expressed in yeast strains can be used, such as promoters of genes of glycolysis systems like hexokinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock peptide promoter, MFα1 promoter, or CUP1 promoter, etc.

[0333] As a method for introducing recombinant vectors, any method can be used as long as it introduces DNA into yeast. Examples include electroporation (Methods Enzymol., 194, 182 (1990)), spheroplast method (Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), lithium acetate method (J. Bacteriology, 153, 163 (1983)), or the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

[0334] When using insect cells as a host, peptides can be expressed using methods described in, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WHFreeman and Company, New York (1992), Bio / Technology, 6, 47 (1988).

[0335] That is, after introducing the recombinant gene into a vector and the genome of a deletion-type baculovirus into insect cells, and obtaining the recombinant virus in the insect cell culture supernatant, the recombinant virus can then infect the insect cells to induce peptide expression.

[0336] Examples of gene delivery vectors used in this method include pVL1392, pVL1393 (manufactured by Becton Dickinson), and pBlueBac4.5 (manufactured by Invitrogen).

[0337] As baculoviruses, for example, the alfalfa silver-striped noctuid moth nuclear polyhedrosis virus (Autographa californica nuclear polyhedrosis virus), which infects noctuid moths, can be used.

[0338] Methods for producing recombinant viruses by co-introducing the aforementioned recombinant gene vector and the aforementioned baculovirus into insect cells include, for example, the calcium phosphate method (Japanese Patent Application Publication No. 2-227075) or the liposome method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

[0339] When using S2 (Schneider 2) cells (Thermo Scientific) derived from late-stage embryos of Drosophila melanogaster as insect cells as a host, peptide expression can be achieved by introducing gene vectors such as pMTBiPV5-HisA (Thermo Scientific) into the host cells via the calcium phosphate method, for example, as described in Mol. Biotechnol., 2015, 10: 914-922.

[0340] When using animal cells as hosts, examples of expression vectors include pCI mammalian expression vector (Promega), pcDNA3.1(+) (Invitrogen), pcDNA I / Amp, pcDNA I, pcDM8 (Phenix), pAGE107 (Japanese Patent Application Publication No. 3-22979, Cytotechnology, 3, 133 (1990)), pAS3-3 (Japanese Patent Application Publication No. 2-227075), pCDM8 (Nature, 329, 840 (1987)), pREP4 (Invitrogen), pAGE103 (J. Biochem., 101, 1307 (1987)), pAGE210, pME18SFL3, or pKANTEX93 (International Publication No. 97 / 10354), etc.

[0341] As a promoter, any promoter that functions in animal cells can be used, such as the promoter of the IE (immediate early) gene of cytomegalovirus (CMV), the early promoter of SV40, the promoter of retroviruses, the metallothionein promoter, the heat shock promoter, or the SRα promoter. Alternatively, the enhancer of the IE gene of human CMV can be used in conjunction with the promoter.

[0342] As a method for introducing recombinant vectors into animal cells, any method that introduces DNA into animal cells can be used, such as electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (Japanese Patent Application Publication No. 2-227075), liposome method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), or the method described in Virology, 52, 456 (1973).

[0343] Temporary expression of the DcR3 variant can be performed using the expression vector of the DcR3 variant of the present invention obtained by the above method, or an expression vector obtained by modifying it.

[0344] For the host cells into which the expression vector is introduced, any host cell capable of expressing the DcR3 variant can be used, such as COS-7 cells (ATCC number: CRL1651) [Methods in Nucleic Acids Res., CRC Press, 283 (1991)]. For introducing the expression vector into COS-7 cells, methods such as the DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press, (1991)] or the liposome method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] can be used.

[0345] When using CHO-S cells or Expi293 cells (Thermo Scientific), the expression vector is introduced using liposomes [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], etc.

[0346] Using the expression vector of the DcR3 variant of the present invention obtained by the above method, or an expression vector obtained by modifying it, a transformant strain that stably expresses the DcR3 variant can be obtained.

[0347] After the expression vector was introduced, the transformants that stably expressed the recombinant antibody were cultured in an animal cell culture medium containing agents such as G418 sulfate, and then screened (Japanese Patent Application Publication No. 2-257891).

[0348] When the transformant is obtained by using eukaryotic organisms such as yeast as a host, any natural or synthetic culture medium can be used as the culture medium for culturing the transformant, as long as it contains carbon sources, nitrogen sources, and / or inorganic salts that the transformant can assimilate and can effectively carry out the culture of the transformant.

[0349] As a carbon source, any carbon source that the transformant can assimilate can be used, such as carbohydrates such as glucose, fructose, sucrose, molasses containing these compounds, starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, alcohols such as ethanol or propanol, etc.

[0350] As a nitrogen source, ammonium salts of inorganic 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 hydrolysate, soybean meal and soybean meal hydrolysate, various fermented microorganisms and their digests, etc., can be used.

[0351] As inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and / or calcium carbonate can be used.

[0352] The culture is preferably carried out under aerobic conditions such as shaking culture or deep aeration and stirring culture. The culture temperature is preferably 15~40°C, and the culture time is usually preferably 16 hours to 7 days. Maintaining the pH during culture at 3.0~9.0 is preferred. The pH can be adjusted using inorganic or organic acids, alkalis, urea, calcium carbonate, or ammonia.

[0353] In addition, antibiotics such as ampicillin or tetracycline can be added to the culture medium as needed during cultivation.

[0354] As a culture medium for culturing transformants obtained by using insect cells as hosts, commonly used TNM-FH medium (Becton Dickinson), Sf-900 II SFM medium (Invitrogen), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Graced insect medium (Nature, 195, 788 (1962)) or Schneider's Medium (Thermo Fisher) can be used.

[0355] For culturing transformants obtained using insect cells as a host, a pH of 6-7 and a temperature of 25-30°C are generally preferred, with a cultivation period of 1-5 days being ideal. Additionally, antibiotics such as gentamicin can be added to the culture medium as needed during the cultivation process.

[0356] Animal cell culture media include RPMI 1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical Co., Ltd.), EX-CELL 301 medium (manufactured by JRH), EX-CELL 325 PF CHO serum-free medium (Sigma-Aldlich), IMDM medium (manufactured by Invitrogen), hybridoma-SFM medium (manufactured by Invitrogen), Eagle's minimum essential medium (MEM) (Science, 122, 501 (1952)), Dulbecco modified Eagle medium (Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)), or media obtained by adding various additives such as FBS to these media.

[0357] Animal cell culture is generally preferably carried out in the presence of 5% CO2, with a pH of 6-8 and a temperature of 30-40°C, and a culture period of 1-7 days. Additionally, antibiotics such as kanamycin or penicillin can be added to the culture medium as needed during the culture process.

[0358] The DcR3 variant or its expression and accumulation in the culture supernatant were achieved by culturing the obtained transformants in a culture medium. Alternatively, the expression level of the DcR3 variant or its expression could be increased using a DHFR amplification system (Japanese Patent Application Laid-Open No. 2-257891).

[0359] The DcR3 variant involved in this invention is generated and accumulated in a culture in the manner described above, and collected from the culture, thereby enabling the production of the DcR3 variant involved in this invention.

[0360] To isolate and purify peptides produced by transformants, conventional protein isolation and purification methods can be used.

[0361] For example, in the case where the DcR3 variant of the present invention is secreted extracellularly, the DcR3 variant can be recovered from the culture supernatant. That is, by processing the culture using processes such as centrifugation, a culture supernatant is obtained, and the purified product can be obtained from the culture supernatant by using the following processes alone or in combination: conventional protein separation and purification methods, i.e., solvent extraction, salting out based on ammonium sulfate, desalting, precipitation based on organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)-agarose or DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography using resins such as S-Sepharose FF (GEHEALTHCARE Corporation), hydrophobic chromatography using resins such as butyl agarose or phenyl agarose, gel filtration using molecular sieves, affinity chromatography, focusing chromatography, or electrophoresis such as isoelectric point electrophoresis, etc.

[0362] For example, in cases where the DcR3 variant of the present invention has an Fc that can bind to immunoglobulins of protein G or protein A, protein G chromatography, which binds protein G or protein A as an affinity ligand to a support, or protein A chromatography, 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)]. Alternatively, methods for protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.

[0363] Alternatively, DcR3 variants that have undergone further chemical modification, such as peptides, sugar chains, or PEG, can be obtained from the DcR3 variants obtained via the above methods by using conventional chemical modification methods.

[0364] 6. Methods for evaluating the bioactivity, physical properties, and kinetics of DcR3 variants.

[0365] Preferred examples of 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 but having neutralizing activity against any of LIGHT and TL1A; and DcR3 variants having no neutralizing activity against FasL but having neutralizing activity against LIGHT and TL1A.

[0366] The DcR3 variant of the present invention can be used to determine its biological activities, physical properties, and in vivo kinetics, such as neutralizing activity, by using the following methods.

[0367] (1) Preparation of ligands

[0368] The sources of LIGHT, TL1A, and FasL used in this invention are not limited, and examples of LIGHT, TL1A, and FasL derived from eukaryotes can be cited. 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, can be cited.

[0369] Cells expressing LIGHT, TL1A, or FasL, or their ligands, can be obtained by introducing an expression vector containing cDNA encoding the full length or a portion thereof of LIGHT, TL1A, or FasL into suitable host cells such as E. coli, yeast, insect cells, or animal cells. Alternatively, the ligand can be obtained by purifying various human cultured cells or human tissues that express LIGHT, TL1A, or FasL in large quantities. The cultured cells or tissues can also be used directly as ligands. Furthermore, synthetic peptides containing partial sequences of LIGHT, TL1A, or FasL can be produced using chemical synthesis methods such as the Fmoc or tBoc methods.

[0370] In addition, LIGHT, TL1A and FasL can be manufactured by introducing the DNA encoding LIGHT, TL1A or FasL into a host cell and expressing it, for example, by methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).

[0371] For soluble LIGHT, TL1A, and FasL, they are generated after being expressed on the cell membrane as membrane-bound LIGHT, TL1A, and FasL, respectively, and then detached from the extracellular region by proteases. The cleavage sites have been identified for each ligand, but in preparing recombinants of soluble ligands, any sequence near the cleavage site can be included. For example, soluble LIGHT recombinants can be prepared from the region of positions 66-240, 74-240, and 83-240 from the N-terminus of the membrane-bound LIGHT amino acid sequence. Soluble TL1A recombinants can be prepared from the region of positions 72-251 from the N-terminus of the membrane-bound TL1A amino acid sequence. Soluble FasL can be prepared from the region of positions 130-281 and 134-281 from the N-terminus of the membrane-bound FasL amino acid sequence.

[0372] Recombinants with soluble ligands can be prepared by adding a His tag, FLAG tag, or similar tag to the N-terminus of the aforementioned region, and obtained through affinity purification. Examples of amino acid sequences that are soluble recombinants of LIGHT, TL1A, and FasL include those listed in sequence numbers 114, 116, 118, 120, 122, 124, 126, 128, 130, and 306. Examples of base sequences that are base sequences of DNA encoding the amino acid sequences of soluble recombinants of LIGHT, TL1A, and FasL include those listed in sequence numbers 113, 115, 117, 119, 121, 123, 125, 127, 129, and 305.

[0373] Functional LIGHT, TL1A, or FasL can form homotrimers. The molecular weight can be determined by SEC-MALS analysis of soluble LIGHT, TL1A, or FasL prepared using any of the above methods. SEC-MALS is an analytical method that calculates the molecular weight by 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 obtained by SEC (size exclusion chromatography)-HPLC separation. Examples of HPLC systems include Shimadzu's Prominence; examples of SEC columns include TOSOH's TSKgel; and examples of MALS detectors include Wyatt Technology's miniDAWN TREOS. Furthermore, methods for separating homotrimers from crude purified products that do not form trimers include SEC purification. Examples of SEC purification methods include using the AKTApurifier manufactured by GE HealthCare as an HPLC system and the Superdex 200 Increase 10 / 300 GL manufactured by GE HealthCare as an SEC column to fractionate the crude purified product based on molecular weight and recover only the molecular weight fraction of the homotrimer.

[0374] (2) Evaluation of the binding activity of DcR3 variants containing the human Fc region

[0375] Methods for determining the binding activity of DcR3 variants against soluble LIGHT, soluble TL1A, or soluble FasL include, for example, binding assays based on enzyme immunoassay (ELISA) and kinetic analyses based on Biacore. As ligands, gene-introduced cells obtained by introducing expression vectors containing DNA sequences encoding the extracellular domains of the respective ligands in LIGHT, TL1A, or FasL into E. coli, yeast, insect cells, or animal cells, or recombinant proteins, as well as serum, plasma, or culture supernatants containing ligands obtained from human tissues or human cells, are used.

[0376] In the case of ELISA, for example, anti-human IgG antibodies are immobilized on a 96-well plate, and after reacting with the DcR3 variant, each ligand is dispensed and bound. After washing, unlabeled anti-ligand antibodies or receptor-Fc fusions of each ligand are reacted, followed by reactions with anti-Fc antibodies labeled with biotin, enzymes, or chemiluminescent substances, or with labeled anti-ligand antibodies or receptor-Fc fusions of each ligand. Detection is performed corresponding to the labeled substance, and the binding of ligands to the DcR3 variant can be measured. Alternatively, in the case of Biacore, for example, using a Biacore T100 or Biacore T200, the kinetics of binding between the DcR3 variant and each ligand are measured, and the results are analyzed using the instrument's accompanying software. Specifically, after immobilizing anti-human IgG antibodies on a sensor chip CM5 via amino-coupling, the DcR3 variant is passed through and an appropriate amount is bound to the sensor chip. Binding and dissociation are then measured by passing multiple ligands of known concentrations through the chip. The obtained data are analyzed kinetically using a 1:1 binding model with the software provided with the instrument to obtain various parameters. Alternatively, after immobilizing each ligand onto the sensor chip using, for example, an amino-coupling method, binding and dissociation are measured by passing multiple DcR3 variants at known concentrations through the chip. The obtained data are then analyzed kinetically using a bivalent binding model with the software provided with the instrument to obtain various parameters. Alternatively, for a ProteinA sensor chip obtained by immobilizing the MabSelectSure ligand (as a variant of the ProteinA IgG binding domain) onto the sensor chip, DcR3 variants are passed through the chip to bind an appropriate amount, and binding and dissociation are measured by passing multiple ligands at known concentrations through it. The obtained data are then analyzed kinetically using a 1:1 binding model with the software provided with the instrument to obtain various parameters.

[0377] The binding activity of DcR3 variants against membrane-bound LIGHT, TL1A, or FasL can be determined as follows: For example, using gene-introduced cells obtained by introducing an expression vector containing the full-length DNA sequence encoding each ligand of LIGHT, TL1A, and FasL into animal cells, or using cells obtained by inducing expression of membrane-bound ligands through appropriate stimulation such as mitogen stimulation (e.g., PHA-L, PMA, or iomycin) on human cells like PBMCs or HUVECs, or stimulation with anti-CD3 antibodies and anti-CD28 antibodies, cytokines, IgG, or immune complexes, the activity can be measured by flow cytometry. Specifically, the DcR3 variant can react with cells expressing membrane-bound ligands, or with fluorescently labeled anti-Fc antibodies, or with DcR3 variants labeled with biotin or fluorescent dyes. After washing, the fluorescence intensity corresponding to the labeled substance is measured by flow cytometry.

[0378] Examples of DcR3 variants of the present invention as determined by the above method include, for example, DcR3 variants that have binding activity against at least one of LIGHT, TL1A and FasL; DcR3 variants that have binding activity against all of LIGHT, TL1A and FasL; DcR3 variants that have no binding activity against FasL but have binding activity against either LIGHT or TL1A; or DcR3 variants that have no binding activity against FasL but have binding activity against both LIGHT and TL1A.

[0379] (3) Evaluation of neutralizing activity of DcR3 variants

[0380] Examples of methods for determining the neutralizing activity of DcR3 variants include, for instance, measuring the binding of receptors corresponding to LIGHT, TL1A, or FasL in a solution containing the DcR3 variant, or measuring cellular functions such as cytokine production and cell proliferation by adding the corresponding ligands to LIGHT, TL1A, or FasL receptor-expressing cells in a culture medium containing the DcR3 variant.

[0381] Inhibitory activity against the binding of human LIGHT to its corresponding receptor can be measured, for example, using the method described in US Patent 8,974,787 B2, by the following method: In a reaction solution containing the DcR3 variant, HVEM or LTβR labeled with biotin, fluorescent dye, etc., is reacted with cells expressing membrane-bound LIGHT. After washing, the fluorescence intensity corresponding to the labeled substance is measured by flow cytometry. Alternatively, in a reaction solution containing the DcR3 variant, LIGHT labeled with biotin, fluorescent dye, etc., is reacted with cells expressing HVEM or LTβR. After washing, the fluorescence intensity corresponding to the labeled substance is measured by flow cytometry. The inhibitory activity of the DcR3 variant against the binding of LIGHT to its corresponding receptor can be confirmed by the reduction in fluorescence intensity compared to the case without the DcR3 variant.

[0382] The inhibitory activity against the binding of TL1A or FasL to their corresponding receptors can also be determined using the same method described above.

[0383] Preferably, the DcR3 variants of the present invention are: DcR3 variants exhibiting inhibitory activity against the binding of at least one of LIGHT, TL1A, and FasL to their corresponding receptors; DcR3 variants exhibiting inhibitory activity against the binding of all of LIGHT, TL1A, and FasL to their corresponding receptors; DcR3 variants exhibiting no inhibitory activity against the binding of FasL to its corresponding receptor but exhibiting inhibitory activity against the binding of either LIGHT or TL1A to their corresponding receptors; or DcR3 variants exhibiting no inhibitory activity against the binding of FasL to its corresponding receptor but exhibiting inhibitory activity against the binding of LIGHT and TL1A to their corresponding receptors. In the present invention, the statement "no inhibitory activity against the binding of a ligand to its corresponding receptor" is used to mean that the inhibitory activity is significantly reduced compared to wild-type DcR3.

[0384] The inhibitory activity against cell function caused by the addition of LIGHT can be measured using, for example, the method described in US Patent 8,974,787 B2, in the following manner: In a culture medium supplemented with a DcR3 variant, using cells such as HT-29 (ATCC No.: HTB-38) that have been confirmed to express HVEM and LTβR as LIGHT receptors, the production of LIGHT-dependent chemokines such as IL-8, CCL5, or CCL20 is measured using ELISA, alphaLISA (Perkin Elmer), or CBA assay (BD Biosciences) on the culture supernatant. Alternatively, using cells such as IFN-γ-stimulated enteric fibroblasts (Lonza), the production of soluble or membrane-bound LIGHT-dependent chemokines such as CXCL-10 is measured using the same method. Alternatively, instead of soluble or membrane-bound light, light induced from PBMCs or T cells stimulated with anti-CD3 and anti-CD28 antibodies, or PMA and ionomycin, can be used and measured using the same method. The inhibitory activity of the DcR3 variant against the cellular function induced by the addition of soluble or membrane-bound light can be confirmed by comparing the reduction in the production of these chemokines with the absence of the DcR3 variant. In addition, in vivo, using methods such as those described in US Patent 8,974,787 B2, improvements in survival, body weight, disease severity, pathology, and human cell engraftment resulting from the administration of the DcR3 variant can be evaluated in an acute GVHD (Graft versus host disease) model obtained by xenografting human PBMCs into immunodeficient mice.

[0385] The inhibitory activity against TL1A-induced cellular function can be determined using methods such as those described in Mucosal Immunology, 2015, 8: pp. 545-558, as follows: In a culture medium supplemented with a DcR3 variant, cells are stimulated with a cocktail of cytokines, including blood, PBMCs, pan T cells, CD4-positive T cells, or memory CD4-positive T cells derived from primates such as humans or rodents such as mice, using IL-12, IL-18, and TL1A, or IL-12, IL-18, IL-15, and TL1A. The production of TL1A-dependent cytokines such as IFN-γ, IL-6, GM-CSF, TNF-α, IL-5, IL-13, IL-17, or IL-22 is measured using ELISA, alphaLISA, or CBA assays on the culture supernatant. Alternatively, using methods such as those described in Immunity, 2002, 16: pp. 479-492, in a culture medium supplemented with the DcR3 variant, panT 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 antibodies and anti-CD28 antibodies, and the production of TL1A-dependent IFN-γ and IL-2 is measured using the same method as described above. Alternatively, a membrane-bound TL1A-forcibly expressed strain can be used instead of soluble TL1A, and the measurement can be performed using the same method. Alternatively, TL1A induced from expression in PBMCs or monocytes stimulated with IgG, immune complexes, etc., can be used, and the measurement can be performed using the same method. The inhibitory activity of the DcR3 variant against TL1A-induced cellular function can be confirmed by the reduction of the cytokines or the inhibition of cell proliferation when compared with the case without the DcR3 variant. Furthermore, in vivo, the improvement in survival, body weight, disease severity, and pathology based on DcR3 variant administration can be evaluated in models such as TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced colitis described in Mucosal Immunology, 2011, 4: pp. 172-185, or DSS (sodium dextran sulfate)-induced colitis described in Mucosal Immunology, 2014, 7: pp. 1492-1503. The improvement in disease severity can also be evaluated in rodent models such as mice exhibiting TL1A-dependent inflammatory, allergic, or autoimmune disease.

[0386] The inhibitory activity against cellular function induced by the addition of FasL can be determined using methods such as those described in J. Rheumatol., 2013, 40: pp. 1316-1326, as follows: In a medium supplemented with the DcR3 variant, using Jurkat cells (DSMZ: ACC 282), apoptosis dependent on soluble FasL or antibody-crosslinked soluble FasL can be measured by Annexin V / Propium Iodide staining or by BrdU uptake and ATP levels in live cells. Alternatively, a membrane-formed FasL-forcibly expressed strain can be used instead of soluble FasL, and the measurement can be performed using the same method. Alternatively, stimulated PBMCs or FasL induced from T cells can also be used, and the measurement can be performed using the same method. The reduction in apoptosis compared to the case without the DcR3 variant confirms the inhibitory activity of the DcR3 variant against cellular function induced by the addition of FasL.

[0387] Examples of DcR3 variants of the present invention that can be measured by the above methods include, for example, DcR3 variants that have inhibitory activity against at least one of the cell functions induced by the addition of LIGHT, TL1A, and FasL; DcR3 variants that have inhibitory activity against all of the cell functions induced by the addition of LIGHT, TL1A, and FasL; DcR3 variants that have no inhibitory activity against cell functions induced by the addition of FasL but have inhibitory activity against one or more of the cell functions induced by the addition of LIGHT and TL1A; or DcR3 variants that have no inhibitory activity against cell functions induced by the addition of FasL but have inhibitory activity against cell functions induced by the addition of LIGHT and TL1A. In the present invention, the statement "no inhibitory activity against cell functions induced by the addition of a certain ligand" can be used to mean that the inhibitory activity is significantly reduced compared to wild-type DcR3.

[0388] (4) Evaluation of OPG ligand reactivity of DcR3 variants

[0389] One of the features of the DcR3 variant of the present invention is that any or all of the CRD1, CRD4, and CRD3 that do not participate in ligand binding are sequences derived from OPG.

[0390] The lack of binding activity of the DcR3 variant against RANKL and TRAIL, which are OPG ligands, can be evaluated by, for example, using the same method as described above for determining the binding activity against LIGHT, TL1A, and FasL.

[0391] One of the features of the DcR3 variant of the present invention is that it has no neutralizing activity against RANKL and TRAIL.

[0392] The neutralizing activity of DcR3 variants against RANKL can be evaluated using, for example, the TRAP (tartaric acid-resistant acid phosphatase) activity assay, which is used as an assay for differentiation of osteoclast precursor cells based on RANKL stimulation, as described in J. Immunol., 2012, 189: p.245-252.

[0393] In addition, regarding the neutralizing activity of DcR3 variants against TRAIL, apoptosis induction based on soluble TRAIL or cross-linked soluble TRAIL can be evaluated, for example, in human cancer cell lines expressing DR4 or DR5, using the same method as that used to determine the neutralizing activity against FasL described above.

[0394] (5) Kinetic evaluation of DcR3 variant

[0395] As a DcR3 variant of the present invention, a DcR3 variant that reduces or eliminates the binding of heparan sulfate contained in heparan sulfate proteoglycans (HSPGs) on the cell membrane is preferred, and one that does not have a heparan sulfate binding domain (HBD) is more preferred.

[0396] The presence or absence of binding of heparan sulfate to the cell membrane via HBD can be determined using methods such as those described in J. Immunol., 2006, 176: pp. 173-180, as described below. For any cell type, such as CHO cells, human cell lines, vascular endothelial cells, hepatocytes, or blood cells, wild-type DcR3 or a DcR3 variant can be reacted, as can a fluorescently labeled detection antibody, or wild-type DcR3 or a DcR3 variant labeled with biotin, fluorescent dye, etc. After washing, the fluorescence intensity corresponding to the labeled substance is detected by flow cytometry (FCM). For the reduction or disappearance of binding to the cell membrane caused by HBD lacking DcR3, the decrease in fluorescence intensity based on FCM can be investigated.

[0397] As a method for evaluating whether the above-mentioned binding on the cell membrane is mediated by the binding of DcR3 to HBD, examples can be given, such as the method described in J. Immunol., 2006, 176: pp. 173-180, in which heparin, heparan sulfate or other GAGs are added as inhibitors in the reaction of wild-type DcR3 or DcR3 variants, or the cells are first treated with enzymes such as heparinase or trypsin, and then wild-type DcR3 or DcR3 variants are reacted.

[0398] One of the features of the DcR3 variant of the present invention is that it improves in vivo kinetics compared to wild-type DcR3 by reducing the loss of heparan sulfate in vivo. In this invention, "improved in vivo kinetics" means a longer blood half-life or a higher area under the concentration-time curve (AUC) up to infinity compared to wild-type DcR3.

[0399] It should be noted that, as a comparison object, molecules containing wild-type DcR3 CRDs can also be used instead of wild-type DcR3. Examples include DcR3 FL-Fc (amino acid sequence: SEQ ID NO. 100, DNA base sequence: SEQ ID NO. 99), DcR3FL-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 a wild-type DcR3 CRD with an Fc, and R128Q-Fc [US Patents US6,835,814 B1, US6,965,01 B1] obtained by fusing an Fc to the C-terminus of a full-length DcR3 (amino acid sequence: SEQ ID NO. 112, DNA base sequence: SEQ ID NO. 111) with a single amino acid mutation within the HBD. When using molecules containing wild-type DcR3 as comparison objects, they are also recorded as wild-type DcR3 controls.

[0400] For the changes in blood concentration of DcR3 variants in rodents such as mice and non-human primates such as cynomolgus monkeys, any dose can be administered intravenously or subcutaneously, and blood samples can be collected at any time for measurement using a DcR3 or human Fc detection system. For kinetic parameters such as blood half-life and AUC, methods such as those described in Pharmacokinetics, 1999, 14: pp. 286-293 can be used to calculate these parameters based on changes in blood concentration using moment analysis.

[0401] Furthermore, it is known that a higher amount of sialic acid added to the ends of N-type glycans improves in vivo pharmacokinetics (J. Pharm. Sci., 2015, 104: pp. 1866-1884). Therefore, for the DcR3 variant of the present invention having N-type glycans, a higher amount of sialic acid is preferred. The amount of sialic acid added per molecule of protein can be calculated, for example, by separating labeled sialic acid using a sialic acid fluorescent labeling kit (Takara Corporation) using reverse-phase HPLC and comparing it with a standard curve of sialic acid.

[0402] (6) Physical property evaluation of DcR3 variants

[0403] A characteristic of the DcR3 variant of the present invention is that, when expressed, isolated or purified in mammalian cells, the content of the aggregate is lower than that of wild-type DcR3.

[0404] The presence of agglutination during protein expression and secretion can be determined using the following methods: For example, host cells and / or culture supernatants infused with a recombinant vector integrating a DcR3 variant are recovered. In the presence of anti-DcR3 antibodies or Fc fusion variants, His, FLAG, or other tagged variants, the approximate molecular weight is investigated under non-reducing conditions using Western blotting based on antibodies against them. In cases where protein agglutination occurs during protein expression and / or secretion, one or more bands larger than the expected molecular weight are detected.

[0405] For the molecular weight of proteins, methods that calculate it from the amino acid sequence can be cited. Alternatively, for more accurate molecular weights, SEC-MALS-based analytical methods can be used. The molecular weight can be calculated by using the maximum value of the scattering intensity detected by a multi-angle light scattering detector (MALS) for each peak separated by SEC (size exclusion chromatography)-HPLC and detected at a wavelength of 215 nm. Examples of HPLC systems include Shimadzu's Prominence, examples of SEC columns include TOSOH's TSKgel, and examples of MALS detectors include Wyatt Technology's miniDAWN TREOS.

[0406] To determine whether the isolated or purified protein has agglutinated, the protein can be detected by SDS-PAGE under non-reducing conditions, CBB staining, or by immunoblotting using the same methods described above. Furthermore, the content of the agglutinate can be calculated based on the area of ​​each peak detected at 215 nm using gel filtration chromatography (SEC) separated by HPLC. Examples of suitable SEC columns include the TOSOH TSKgel G3000SW and the Waters ACQUITY UPLC Protein BEH SEC. It should be noted that the preferred agglutinate content for analysis using the above methods is 0–60%, a more preferred agglutinate content is 0–40%, a further preferred agglutinate content is 0–30%, an even more preferred agglutinate content is 0–20%, and the most preferred agglutinate content is 0–10%.

[0407] In addition, the DcR3 variant of the present invention is characterized by lower hydrophobicity than wild-type DcR3.

[0408] The hydrophobicity of proteins can be determined using hydrophobic interaction chromatography (HIC) columns that interact with hydrophobic regions present on the protein surface. Examples of HIC columns include TOSOH's TSKgel Butyl-NPR.

[0409] Furthermore, the DcR3 variant of the present invention is characterized by improved thermal stability compared to wild-type DcR3. Examples of methods for determining protein thermal stability include calorimetry such as DSC (Differential Scanning Calorimetry), spectrophotometry to obtain autofluorescence or circular dichroism (CD) spectra during thermal or chemical denaturation, and DSF (Differential Scanning Fluorimetry) to detect hydrophobic regions exposed on the inner side of proteins as temperature increases using fluorescent dyes (such as Sypro Orange) [J Am Chem Soc, 2009, 131: p.3794-3795].

[0410] The DSF (Digital Substances Fraction) for evaluating protein thermal stability can be performed, for example, using the method described in J. Pharm. Sci., 2013, 102: pp. 2471-2483, by measuring the fluorescence intensity at various temperatures. Alternatively, software such as BioRad's CFX Manager can be used to plot melting curves and calculate the Tm (thermal unfolding transition midpoint) value. Similarly, the DSC (Digital Substances Fraction) for evaluating protein thermal stability can be performed, for example, using the method described in J. Pharm. Sci., 2012, 101: pp. 955-964, by calculating the heat capacity and Tm value at various temperatures.

[0411] 7. DcR3 variant compositions

[0412] Examples of DcR3 variant compositions of the present invention include compositions comprising DcR3 variant molecules. Examples of DcR3 variant compositions of the present invention include compositions comprising a plurality of DcR3 variant molecules having the same primary amino acid sequence as constituting the DcR3 variant molecule, which can be generated by post-translational modification such as oxidation / reduction reactions, glycan addition reactions, or 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-glycosidic complex glycans and DcR3 variants not having N-glycosidic complex glycans. Regarding the proportion of DcR3 variants having one or more N-glycosidic complex glycans, for example, relative to the total number of DcR3 variants of the present invention, 70-100% is preferred, more preferably 90-99%, and particularly preferably 95-98%.

[0413] 8. Pharmaceutical compositions containing DcR3 variants

[0414] One embodiment of the present invention is a composition containing an effective amount of the DcR3 variant of the present invention. The composition containing the DcR3 variant of the present invention can be used as an active ingredient in the prevention or treatment of autoimmune diseases, inflammatory diseases, or allergic diseases, including mucosal diseases. That is, autoimmune diseases, inflammatory diseases, or allergic diseases can be prevented or treated by administering a pharmaceutical composition containing the DcR3 variant of the present invention to a patient requiring prevention or treatment of autoimmune diseases, inflammatory diseases, or allergic diseases.

[0415] There are no limitations on the conditions or diseases for which the compositions of the present invention are used, but examples include, for instance, inflammatory bowel disease (IBD), systemic lupus erythematosus, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, ulcerative colitis, multiple sclerosis, celiac disease, idiopathic thrombotic thrombocytopenic purpura, myasthenia gravis, Sjögren's syndrome, scleroderma, asthma, uveitis, epidermal hyperplasia, alopecia areata, and Behçet's disease. Diseases, including arteritis, cartilage inflammation, osteolysis, arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Wright syndrome, SEA syndrome (seronegative, tendinopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, and juvenile systemic lupus erythematosus. Juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Rytter syndrome, dermatomyositis, psoriatic arthritis, vasculitis, myositis, polymyositis, dermatomyositis, deforming arthritis, 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 and other inflammatory diseases, autoimmune diseases, or allergic diseases, etc.

[0416] Pharmaceutical compositions containing the DcR3 variant of the present invention may contain the DcR3 variant, or a mixture thereof, as an active ingredient with any other therapeutic active ingredient. Alternatively, these pharmaceutical formulations may be manufactured by mixing the active ingredient with one or more pharmacologically permissible support carriers using any method generally known in the art of pharmaceutical formulation.

[0417] The content of the DcR3 variant of the present invention in the pharmaceutical composition varies depending on the dosage form, the pharmacologically permissible amount of the DcR3 variant of the present invention, etc., and is, for example, about 0.01 to 100% by weight. Furthermore, the content of the pharmacologically permissible carrier in the pharmaceutical formulation varies depending on the dosage form, the pharmacologically permissible amount of the DcR3 variant of the present invention, etc., and is, for example, 0 to 99.9% by weight.

[0418] Ideally, the most effective route of administration should be used, such as oral administration, intravenous administration, subcutaneous administration, intraoral administration, intratracheal administration, intrarectal administration, intramuscular administration, or intraperitoneal administration.

[0419] As a method of administration, it can be tablets, powders, granules, syrups, or injections.

[0420] As formulations suitable for oral administration, they can be manufactured using, for example, the following ingredients: for liquid formulations such as syrups, sugars such as water, 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 parabens; and flavorings such as strawberry or mint. Furthermore, tablets, powders, and granules can be manufactured 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, hydroxypropyl cellulose, or gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin.

[0421] As a formulation suitable for non-oral administration, it is preferable to form a sterile aqueous agent containing an active compound isotonic with the recipient's blood. For example, in the case of an injectable formulation, an injectable solution is prepared using a carrier formed from a saline solution, a glucose solution, or a mixture of saline and glucose solutions.

[0422] In addition, one or more auxiliary ingredients selected from diluents, preservatives, flavoring agents, excipients, disintegrants, lubricants, binders, surfactants, and plasticizers, as exemplified in oral formulations, may be added to these non-oral formulations.

[0423] Medicines containing the DcR3 variant of the present invention can be safely administered to mammals (e.g., humans, mice, rats, rabbits, dogs, cats, cattle, horses, pigs, or monkeys).

[0424] The dosage and frequency of administration of the DcR3 variant of the present invention vary depending on the route of administration, the patient's age, weight, disease, and the nature or severity of the symptoms to be treated. Generally, in the case of oral administration, adults are given 0.01 mg to 1 g once or several times daily, preferably 0.05 to 50 mg. In the case of non-oral administration, such as intravenous administration, adults are given 0.001 to 100 mg once or several times daily, preferably 0.01 to 10 mg. However, these dosages and frequencies vary depending on the aforementioned conditions.

[0425] [Example]

[0426] The following are embodiments of the present invention. However, the present invention is not limited to these embodiments.

[0427] [Example 1] Evaluation of the aggregation properties of wild-type DcR3 in mammalian cells

[0428] The following describes the preparation of a fusion protein (DcR3 FL-Fc) of wild-type DcR3 (also known as full-length DcR3) (Sequence No. 4), the adapter sequence IEGRMD (Sequence No. 106), and the human IgG1 Fc region (g1S) (Sequence No. 72). Figure 3 The fusion protein (S195-Fc) of human DcR3 (sequence number 100) with the HBD region missing, the adaptor sequence IEGRMD (sequence number 106) and the human IgG1 Fc region (sequence number 72) (row A, sequence number 100) Figure 3 The protein obtained by adding a FLAG tag (sequence number 110) to the full-length DcR3 (sequence number 4) (sequence number 104) (sequence number 102), and the protein obtained by adding a FLAG tag (sequence number 110) to the full-length DcR3 (sequence number 4).

[0429] For DcR3 FL-Fc, a synthetic signal peptide DNA fragment, a human DcR3 DNA fragment (Sequence No. 3), an adapter sequence IEGRMD DNA fragment (Sequence No. 105), and an Fc (g1S) DNA fragment (Sequence No. 71) (GENEWIZ or Sigma) were inserted into a pCIpuro vector (partially modified Promega pCI) obtained by digestion with restriction enzymes NheI and SalI (New England Biolabs) using the In-Fusion HD CloninG Kit (Clontech). The vector was then transformed into E. coli DH5α competent cells (TOYOBO) to obtain transformants containing the inserted DcR3 FL-Fc DNA fragment (Sequence No. 99). The same method was used for S195-Fc. A DNA fragment containing an artificially synthesized signal peptide sequence, a human DcR3 DNA fragment with HBD removed (Sequence No. 107), a DNA fragment containing the adapter sequence IEGRMD (Sequence No. 105), and a DNA fragment containing Fc(g1S) (Sequence No. 71) were used to obtain a transformant with an inserted S195-Fc DNA fragment (Sequence No. 101). For DcR3 FL-FLAG, a DNA fragment containing an artificially synthesized signal peptide sequence, a human DcR3 DNA fragment (Sequence No. 3), and a FLAG-tagged DNA fragment (Sequence No. 109) were used to obtain a transformant with an inserted DcR3 FL-FLAG DNA fragment (Sequence No. 103).

[0430] Each plasmid obtained from each transformant was introduced into any of the following host cells: Freestyle CHO-S cells, Freestyle 293F cells, and Expi293 cells (all from Thermo Scientific), to temporarily express the protein. Plasmid introduction was performed using any of the following: Freestyle MAX Reagent, 293Fectin Transfection Reagent, or ExpiFectamine 293 (all from Thermo Scientific).

[0431] Transfected cells were cultured for several days, and then the culture supernatant was collected. DcR3 FL-Fc and S195-Fc were affinity purified using MabSelect SuRe (GEHEALTHCARE), and DcR3 FL-FLAG was affinity purified using Anti-FLAG M2 affinity gel (Sigma). The culture supernatants of DcR3 FL-Fc and S195-Fc were passed through a resin-filled column, washed with PBS (Nacalai Tesque), dissolved in dissolution buffer (20 mM citrate, 50 mM NaCl, pH 3.4), and quickly neutralized with neutralization buffer (1 M Na phosphate, pH 7.0). The culture supernatant of DcR3 FL-FLAG was similarly packed into resin, washed with PBS, dissolved in dissolution buffer (100 mM glycine-HCl, pH 3.5), and quickly neutralized with neutralization buffer (1 M Tris-HCl, pH 8.0). The absorbance (A280) of each dissolution fraction at 280 nm was measured, and the fractions with the highest absorbance values ​​were recovered. The buffer for the recovered fractions was replaced with PBS using a NAP column (GE HealthCare), and the material passing through a 0.22 μm filter was used for protein purification. The concentrations of DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG at 280 nm were calculated based on absorbance coefficients of 1.03, 1.17, and 0.77, respectively. SDS-PAGE was performed under non-reducing and reducing conditions with 100 mM DTT, followed by gel Coomassie staining (Nacalai Tesque) to determine the molecular weight. The estimated molecular weights predicted from the amino acid sequences of DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG were approximately 56.4 kDa, 44.7 kDa, and 31.0 kDa, respectively. Under non-reducing conditions, DcR3FL-Fc and S195-Fc, as Fc fusion bodies, exist as dimers, and their estimated molecular weights are approximately 112.8 kDa and 89.4 kDa, respectively.

[0432] The results of SDS-PAGE under non-reducing conditions showed that most of the transiently expressed DcR3 FL-Fc, S195-Fc, and DcR3 FL-FLAG in mammalian cells remained in the sample wells, and the migratory portions also became smeared or ladder-like, indicating that all recombinants were highly aggregated. Figure 1 (Small Figure A).

[0433] Furthermore, for the commercially available full-length DcR3-Fc (Abcam) prepared using HEK293 cells as the host cell, under non-reducing conditions, immunoblotting was performed using rabbit anti-human IgG Fc polyclonal antibody as the primary antibody and goat anti-rabbit IgG antibody (Dako) as the secondary antibody. Similar to the purified product mentioned above, most of it existed as a clump. Figure 1 Small image B).

[0434] On the other hand, almost no agglutination was observed in the commercially available DcR3 FL-Fc (R&D Corporation) expressed in insect Sf21 cells, confirming that the agglutination properties varied depending on the type of host cell that produced wild-type DcR3. Figure 1 (Figure C). Additionally, using insect cells S2 as host cells, DcR3 FL-Fc (Sequence No. 100), R218Q-Fc with an R218Q mutation (replacing Arg at position 218 of human DcR3 (Sequence No. 2) with Gln (US Patents US6835814B1, US6965012B1, Sequence No. 340), and S195-Fc (Sequence No. 102) lacking HBD were prepared, and their agglutination properties were evaluated. S2 cell lines stably expressing DcR3 FL-Fc, R218Q-Fc, and S195-Fc were obtained using pMTBiPV5-HisA (Thermo Scientific) and the Drosophila Expression System (Thermo Scientific), along with a synthetic human DcR3 DNA fragment (Sequence No. 3), a DNA fragment obtained by introducing the R218Q mutation into human DcR3 (Sequence No. 111), or a human DcR3 DNA fragment with HBD removed (Sequence No. 107), the adapter sequence IEGRMD (Sequence No. 105), and the Fc(g1S) DNA fragment (Sequence No. 71). These cells were purified using MabSelect SuRe affinity chromatography from the culture supernatant of the stable expression lines.

[0435] As a result, DcR3 FL-Fc, R218Q-Fc, and S195-Fc produced by S2 cells showed almost no aggregation ( Figure 1 (Figure C). Quantitative evaluation of the aggregate content was performed using HPLC (Shimadzu Corporation) gel filtration chromatography (SEC) (TSK gel G3000 SWXL 7.8 mm x 300 mm) (TOSOH Corporation). Analysis of Expi 293 and S195-Fc derived from S2, and the percentages (%) of monomers, aggregates, and decomposition products obtained by calculating peak areas are shown in Table 1.

[0436] [Table 1]

[0437]

[0438] The results above show that the expression of wild-type DcR3 in mammalian cells increases the amount of condensates generated.

[0439] [Example 2] Preparation of DcR3 variants that do not aggregate in mammalian cell expression lines

[0440] To date, no human DcR3 variant that does not condense in mammalian cell expression lines is known. Therefore, an attempt was made to prepare a DcR3 variant that maintains DcR3 activity while reducing the amount of condensate formation. DcR3 is a soluble molecule composed of 300 residues, with a signal peptide at the N-terminus followed by four cysteine-rich domains characteristic of the TNF receptor superfamily (TNFRSF) (CRD1, CRD2, CRD3, CRD4), and a heparan sulfate binding domain (HBD) containing a heparan sulfate binding motif and rich in basic amino acids at the C-terminus. Figure 2 , Figure 3 (A). LIGHT, TL1A, and FasL, all ligands of DcR3, bind via CRD2 and CRD3 of DcR3. Therefore, by keeping the regions containing CRD2 and CRD3 of DcR3 unchanged, and replacing CRD1 and / or CRD4 of DcR3 with soluble decoylet receptor osteoprotegerin (OPG), which is a similar TNFRSF molecule, a DcR3 variant was prepared.

[0441] Various DcR3 variants with the following structures were prepared by means of: chimeric A (serial number 54) obtained by fusing with an Fc sequence (IEGRMD g1S (serial number 339) or g4PEK (serial number 74) of the heavy chain of human IgG4 according to EU index, where Ser at position 228 is replaced with Pro, Leu at position 235 is replaced with Glu, and Arg at position 409 is replaced with Lys) to obtain chimeric A-Fc. Figure 3 The C line, sequence number 80 or 82); to make CRD1 in human DcR3 the amino acid sequence of human OPG, CRD2, CRD3 and CRD4 the amino acid sequence of human DcR3, and the chimeric B (sequence number 50) obtained by removing the amino acid sequence of HBD, fused with the Fc sequence (IEGRMD g1S) to obtain chimeric B-Fc ( Figure 3The G line, sequence number 76); to make CRD1, CRD2, and CRD3 in human DcR3 the amino acid sequence of human DcR3, CRD4 the amino acid sequence of human OPG, and the chimeric C (sequence number 52) obtained by removing the amino acid sequence of HBD, fused with the Fc sequence (IEGRMD g1S) to obtain the chimeric C-Fc ( Figure 3 H line, sequence number 78); to replace the amino acid sequence of chimeric A's CRD3 containing positions 18 to 36 and the two amino acids on its C-terminus with human OPG to obtain 103-123OPG (sequence number 56), and fuse it with the Fc sequence (g4PEK) to obtain 103-123OPG-Fc(g4PEK) ( Figure 3 (D line, sequence number 84). It should be noted that for the DcR3 variant of CRD4 derived from DcR3, a TS sequence is added to the C-terminus of CRD4 as the amino acid sequence of DcR3 (sequence number 2) from position 194 to position 195. For the DcR3 variant of CRD4 derived from OPG, an SGNSESTQK sequence is added to the C-terminus of CRD4 as the amino acid sequence of OPG (sequence number 14) from position 186 to position 194.

[0442] DNA fragments encoding signal peptide sequences, chimeric A, chimeric B, chimeric C, or 103-123OPG, and DNA fragments with HBD sequences removed (chimeric A: sequence number 53, chimeric B: sequence number 49, chimeric C: sequence number 51, 103-123OPG: sequence number 55) were artificially synthesized and linked with DNA fragments encoding Fc sequences (IEGRMD g1S or g4PEK) (sequence numbers 338 and 73). These fragments were then inserted into the pCIpuro vector using the same method as in Example 1 to obtain plasmids containing DNA fragments encoding various DcR3 variants (chimeric A-Fc: sequence number 79 or 81, chimeric B-Fc: sequence number 75, chimeric C-Fc: sequence number 77, 103-123OPG-Fc: sequence number 83). The obtained plasmid was introduced into any one of the host cells, namely Freestyle CHO-S cells, Freestyle 293F cells, or Expi293 cells, in the same manner as in Example 1, to temporarily express the protein, and then the protein was purified from the culture supernatant using MabSelectSuRe affinity purification.

[0443] The percentages (%) of monomers, aggregates and decomposition products of the various DcR3 variants prepared were calculated by SEC-HPLC using the same method as in Example 1, or by using an ACQUITY UPLC Protein BEH SEC 4.6 mm x 150 mm SEC (Waters Corporation) (Table 2).

[0444] [Table 2]

[0445]

[0446] The results showed that any of the DcR3 variants, including chimeric A-Fc, chimeric B-Fc, chimeric C-Fc, and 103-123OPG-Fc, exhibited a reduction in aggregation compared to the wild-type DcR3. The chimeric A-Fc variant showed the highest reduction in aggregation, followed by chimeric B-Fc and then chimeric C-Fc. Therefore, it was demonstrated that substitution of CRD1 and CRD4 derived from OPG has a reducing effect on aggregation, and that substitution of both CRD1 and CRD4 in combination further reduces aggregation.

[0447] Chimeric A-Fc, chimeric B-Fc, and chimeric C-Fc (all IEGRMD g1S) were subjected to SDS-PAGE under both non-reducing and reducing conditions with 100 mM DTT. The results showed that, even when mammalian cells were used as the host cell, almost no tailing or ladder-like banding was observed in chimeric A-Fc and chimeric B-Fc. Figure 4 ).

[0448] To calculate the absolute molecular weight, analyses were performed using HPLC (Prominence, Shimadzu Corporation) based on SEC (TSKgel G3000 SWXL 7.8mm x 300mm) (TOSOH Corporation) on the chimeric A-Fc (IEGRMD g1S or g4PEK) and 103-123OPG-Fc (g4PEK). Separation was performed using 50 mmol / L phosphate buffer (pH 7.0, 500 mmol / L NaCl) at a flow rate of 0.75 mL / min. For each peak detected at 215 nm, the absolute molecular weight (% is uncertain) was calculated using the intensity of scattered light detected by a multi-angle light scattering detector (MALS) (miniDAWN TREOS, Wyatt Technology Corporation). The results are shown in Table 3.

[0449] [Table 3]

[0450]

[0451] 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, thus confirming that each DcR3 variant exists as a dimer.

[0452] [Example 3] Analysis of the addition rate of N-type sugar chains of chimeric A-Fc (g4PEK) and evaluation of its effect on aggregation.

[0453] For the Asn in chimeric A-Fc(g4PEK) (serial number 82) predicted to have N-type glycan addition based on the amino acid sequence, there are 3 sites (N131, N144, N157) in the CRD4 region derived from OPG and 1 site (N260) in the Fc region. Therefore, the presence or absence of N-type glycan addition in chimeric A-Fc(g4PEK) was evaluated using the following method. Sample preparation involved cleaving the N-type glycan of the reduced and alkylated chimeric A-Fc(g4PEK) using PNGase F, followed by protein digestion with trypsin, endopeptidase 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 using a MAGIC2000 HPLC (Michrom Bioresources) equipped with a C18 reverse-phase column (0.2 mm x 150 mm) (GL Science) and an LTQ Orbitrap XL ion-hydrazine-Orbitrap Hybrid mass spectrometer (Thermo Scientific), via gradient dissolution with 5-65% (v / v) acetonitrile / 0.1% formic acid. The obtained peptide fragments were identified by MASCOT analysis (Matrix Science) of the amino acid sequence of the chimeric A-Fc (g4PEK). For N-type glycans with added Asn, the peak shift in the MS spectrum and the glycan addition site within the peptide were identified using PNGaseF treatment to convert to Asp and a mass increase of 0.984 Da as an indicator. The results showed that N-type glycans can be added to Asn residues in any of the N131, N144, N157, and N260 glycans. N157 and N260 had glycans added to almost all of their peptide fragments, while N131 and N144 were both found to be fragments without added glycans.

[0454] Next, to evaluate the effect of the N-type glycans added to the three Asn (N131, N144, N157) sites of CRD4 derived from OPG on aggregation, amino acid substitutes for removing the glycans at two sites (N131 and N144) or three sites (N131, N144, and N157) were prepared. As two-site glycan-removing substitutes, N131S / N144S-Fc (g4PEK) (ser) (serium 86) or T133A / S146A-Fc (g4PEK) (serium 88) (Ala) were prepared, in which N131 and N144 were replaced with Ser, respectively. As 3-site glycan ablation variants, N131S / N144S / N157S-Fc (g4PEK) (Sequence No. 90) was prepared by replacing N131, N144, and N157 with Ser, respectively, or T133A / S146A / T159A-Fc (g4PEK) (Sequence No. 92) was prepared by replacing T133, S146, and T159 with Ala, respectively. The synthetic signal peptide DNA fragments and the DNA fragments encoding each glycan ablation variant (Sequence Nos. 85, 87, 89, and 91) were inserted into the pCIpuro vector using the same method as in Example 1 and introduced into Expi293 cells. After transient expression of each glycan ablation variant, the culture supernatant was subjected to affinity purification based on MabSelectSuRe. The prepared glycan-removed products were analyzed by SEC-UPLC (apparatus: ACQUITY UPLC, column: ACQUITY UPLC Protein BEH SEC 200 Å, 1.7 μm, 4.6 x 150 mm) (Waters Corporation). The percentages (%) of monomers, aggregates, and decomposition products calculated from peak areas are shown in Table 4.

[0455] [Table 4]

[0456]

[0457] Regarding the condensate content of the 2-site glycan removal variants, compared to chimeric A-Fc (Table 2), the condensate content was slightly increased in each amino acid substitution variant, while for the 3-site glycan removal variants, a further increase in condensate content was observed in each amino acid substitution variant. This indicates that the addition of the N-type glycans at the three sites (N131, N144, N157) in CRD4 derived from OPG, especially the glycan added at N157, helps to reduce the condensate content of chimeric A. On the other hand, even after removing all three N-type glycans, the proportion of condensate content was lower than that of DcR3 FL-Fc and S195-Fc (Table 1). This shows that the N-type glycans and the OPG sequence have the effect of reducing the condensate content of wild-type DcR3, and combining the two has a further effect of reducing the condensate content.

[0458] [Example 4] Physical property evaluation of various DcR3 variants

[0459] To analyze the reasons for the reduced aggregation effect of the various DcR3 variants prepared in Examples 2 and 3, the following physicochemical analyses were performed. Hydrophobic interaction chromatography (HIC) is an analytical method where the higher the hydrophobicity of the protein surface, the longer the dissolution time from the column. Using a hydrophobic chromatography column (TSKgel Butyl-NPR 4.6 mm x 35 mm) (TOSOH), 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) was used as the mobile phase. After separation of 8 μg of sample at a flow rate of 0.5 mL / min, the dissolution time (in minutes) detected at a wavelength of 215 nm is shown in Table 5.

[0460] [Table 5]

[0461]

[0462] Compared to the full-length DcR3 monoamino acid mutant R218Q-Fc prepared from insect S2 cells, the dissolution times of any of the following mutants—S195-Fc derived from S2, chimeric A-Fc (IEGRMD g1S, g4PEK) derived from mammalian cells, 103-123OPG-Fc (g4PEK), and the 2-site glycan ablation variant—were all shortened. This can be attributed to improved physical properties due to reduced hydrophobicity, resulting in a decrease in globule size. Furthermore, the chimeric C and 3-site glycan ablation variants, which showed a slightly weaker reduction in globule size, possessed hydrophobicity equal to or greater than that of R218Q-Fc, indicating a correlation between the proportion of globule size and protein hydrophobicity.

[0463] Then, the thermal stability of the protein was evaluated by differential scanning calorimetry (DSF). In 96-well white microplates (BioRad), 9.5 μg of various DcR3 variants were mixed with 1 μL of SYPRO Orange Protein Gel Stain (Invitrogen) diluted 50-fold with water in a 20 μL mixture. The temperature was increased from 20°C to 95°C using a C1000 thermal cycler (BioRad) in increments of 0.5°C for 10 seconds. Fluorescence at each temperature was detected using the FRET channel, and melting curves were calculated using CFX Manager software (BioRad). Figure 5 ) and melting temperature (Tm value) (°C) (Table 6).

[0464] [Table 6]

[0465]

[0466] Compared to the full-length DcR3 single-amino acid mutant R218Q-Fc prepared from insect S2 cells, the Tm value of S195-Fc derived from S2 was significantly increased, and its thermal stability was improved, thus teaching that the HBD region contributes to thermal instability. Furthermore, the Tm value of the chimeric A-Fc (IEGRMD g1S) derived from mammalian cells was higher than that of S195-Fc. Therefore, it is evident that DcR3 variants obtained by replacing a portion of the CRD of DcR3 with a portion of the CRD of OPG not only exhibit reduced aggregation but also improved protein thermal stability (Table 6). Figure 5 ).

[0467] [Example 5] Evaluation of the responsiveness of the DcR3 variant with the heparin sulfate binding domain (HBD) removed to normal human cells and CHO cells.

[0468] The in vivo kinetics of wild-type DcR3 and its mutant FLINT (R218Q mutation) in mice and cynomolgus monkeys were reported to be extremely poor (Drug Metabolism and Disposition, 2003, 31: p. 502-507). One of the contributing factors is that the heparan sulfate-binding domain (HBD) present in wild-type DcR3 allows DcR3 to directly bind to heparan sulfate proteoglycans on the cell membrane (J. Immunol., 2006, 176: 173-180).

[0469] Therefore, flow cytometry (FCM) was used to analyze the reactivity of HBD-removed DcR3 variants S195-Fc, chimeric A-Fc (IEGRMD g1S, g4PEK), and 103-123OPG-Fc (g4PEK) to CHO cells, which were used as human primary cells and production cells. As a positive control, DcR3 FL-Fc containing the HBD region was used, and as a negative control, K194-Fc and anti-DNP antibody (g4PEK) were used. K194-Fc (amino acid sequence: sequence number 152, DNA base sequence: sequence number 151) is a protein obtained by fusing the amino acid sequence of OPG (sequence number 14) from Met at position 1 to Lys at position 194 with the IEGRMD linker (sequence number 106) and the amino acid sequence of Fc (g1S) (sequence number 72). It was transiently expressed in Expi293 cells using the method described in Example 1 and purified from the culture supernatant using Mabselect SuRe (GE HealthCARE). The anti-DNP antibody (g4PEK) was obtained using the following antibody: the variable region of the anti-2,4-dinitrophenol (DNP) antibody described in Clin. Cancer Res., 2005, 11(8), pp. 3126-3135 was inserted into a vector encoding the Fc sequence (g4PEK), introduced into CHO cells and expressed, and the antibody was purified from the culture supernatant using Protein A. HUVECs (Lonza) and male human hepatocytes (Bioreclamation IVT) were used as primary human cells. Primary human cells were cultured using the culture media specified for each cell type as described in the appendix, on collagen-coated adhesion plates (IWAKI). CHO cells were cultured in suspension using EX-CELL 325 PF CHO Serum-Free Medium (Sigma-Aldrich).

[0470] HUVECs and hepatocytes were dissected using 0.02% EDTA solution and a cell scraper, and passed through a cell filter (40 μm). The dissected HUVECs, hepatocytes, and CHO cells separated from the suspension culture medium were washed with FCM buffer (PBS containing 1% BSA, 1 mmol / L EDTA, and 0.05% NaN3), and then resuspended in FCM buffer.

[0471] Next, to become 1 x 10 5Cells were seeded in 96-well U-plates (Falcon) using a per-well method. The prepared Fc fusion proteins were added at a concentration of 10 μg / mL, and the cells were incubated on ice for 1 hour. After washing with FCM buffer, the cells were suspended in an IVE / 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 stained on ice for 1 hour. For HUVECs and hepatocytes, Human FcR Blocking Reagent (Miltenyi Biotech) was added. After washing with FCM buffer, the fluorescence intensity was analyzed using a flow cytometer (FACS Fortessa, BDBioscience).

[0472] Analysis of PE staining intensity in live cell fractions negative for the LIVE / DEAD Fixable Aqua Dead Cell Stain Kit revealed that DcR3 FL-Fc (IgG1) (R&D Corporation) showed significant binding to any of HUVECs, hepatocytes, and CHO cells. Conversely, the HBD-removed DcR3 variant did not bind to any of them. Figure 6 ).

[0473] [Example 6] Evaluation of the in vivo kinetics of the DcR3 variant in mice

[0474] [Table 7]

[0475]

[0476] Mouse kinetic studies were performed on S195-Fc and each DcR3 variant as shown in Table 7. For chimeric A-Fc (g4PEK) and 103-123OPG-Fc (g4PEK), each DcR3 variant was prepared by stable expression in CHO cells using the method described below. DNA fragments containing synthetic signal peptide sequences and DNA fragments encoding each DcR3 variant (sequence numbers 81 and 83) were inserted into recombinant vectors prepared using the process described in International Publication No. 2012 / 081628, using the same method as in Example 1. These vectors were then introduced into CHO cells via electroporation. Culture and drug screening were performed using conventional methods, and cells with a viable cell percentage of approximately 98% were designated as stable expression lines. These stable expression lines were cultured in media such as EX-CELL 325 PF CHOSerum-Free Medium (Sigma-Aldrich) for a certain period, and then the culture supernatant was recovered. Each DcR3 variant was purified using the method described in Example 1.

[0477] BALB / c mice aged 5-6 weeks ( A single intravenous administration of 10 mg / kg of S195-Fc and each DcR3 variant (n = 2 or 3) was administered. Blood samples were collected from the tail vein at any time after administration, and the concentrations of S195-Fc and each DcR3 variant in the serum were determined using the following method: Biotinylated monkey anti-human IgG polyclonal antibody was reacted with streptavidin-immobilized beads, and the conjugated serum concentrations of S195-Fc and each DcR3 variant were detected using AlexaFluor 647-labeled monkey anti-human IgG polyclonal antibody. Assays were performed using a Gyrolab xP workstation (Gyros AB), and kinetic parameters were calculated using moment analysis. The same standard material used to prepare the calibration curve was used as the analyte administered to the animals. The conversion of serum concentrations of S195-Fc produced from S2 cells to chimeric A-Fc (IEGRMD g1S) produced from CHO-S cells is shown in the figure. Figure 7 In addition, for S195-Fc and each DcR3 variant shown in Table 7, the blood half-life (h) of the elimination phase after a single administration and the area under the concentration-time curve up to infinity were calculated. They are shown in Table 8.

[0478] [Table 8]

[0479]

[0480] Compared to S195-Fc, which served as a wild-type DcR3 control, the DcR3 variant exhibited a significantly improved blood concentration shift. Figure 7 The half-lives are almost identical, but... Improved by more than 10 times (Table 8).

[0481] It has been reported that the blood half-lives of wild-type DcR3 and FLINT (R218Q mutation) at a single intravenous administration of 0.5 mg / kg to CD-1 mice are 1.2 hours and 3.1 hours, respectively. Additionally, The values ​​were 0.48 and 0.36, respectively (Drug Metabolism and Disposition, 2003, 31: pp. 502-507). Compared to the concentration shift obtained by multiplying the dosage ratio under the assumption of linearity, the DcR3 variant showed higher exposure relative to wild-type DcR3 and FLINT.

[0482] [Example 7] Evaluation of the binding activity of DcR3 ligands

[0483] (1) Preparation of DcR3 ligands

[0484] Prepare soluble recombinant forms of DcR3 ligands (LIGHT, TL1A, FasL) for human, cynomolgus monkey, or mouse (amino acid sequence: sequence number 114, 116, 118, 120, 122, 124, 126, 128, 130; DNA base sequence: sequence number 113, 115, 117, 119, 121, 123, 125, 127, 129).

[0485] For soluble recombinant LIGHT from humans, cynomolgus monkeys, or mice, the N-terminus is used to add a His tag (His10) and a GS adapter (GGGSGGGSGGGSIEGR), with the extracellular region of LIGHT (human LIGHT: Asp74-Val240 (accession number 132), cynomolgus monkey LIGHT: Asp74-Val240 (accession number 134), mouse LIGHT: Asp72-Val239 (accession number 136)) downstream.

[0486] For soluble recombinant TL1A from humans, cynomolgus monkeys, or mice, the N-terminus is used to add a His tag (His6) and a GS adapter (GGGSGGGSGGGS), with the extracellular region of TL1A (human TL1A: Leu72-Leu251 (accession number 138), cynomolgus monkey TL1A: Leu72-Leu251 (accession number 140), mouse TL1A: Ile94-Leu270 (accession number 142)) attached downstream.

[0487] For soluble recombinant FasL from humans, cynomolgus monkeys, or mice, the sequence used is the one with a His tag (His6) added to the N-terminus, and the extracellular region of FasL (human FasL: Pro134-Leu281 (accession number 144), cynomolgus monkey FasL: Pro133-Leu280 (accession number 146), mouse FasL: Pro132-Leu279 (accession number 148)) linked downstream.

[0488] A DNA fragment containing a synthetic signal peptide sequence and a DNA sequence containing a tagged soluble DcR3 ligand (GENEWIZ) were inserted downstream of the CMV promoter in the pCI-Hygro2.01 vector (partially modified Promega pCI) using the In-Fusion HD CloninG Kit (Clontech) and transformed into E. coli DH5α competent cells (TOYOBO).

[0489] The obtained plasmid was introduced into Expi293 cells (Thermo Scientific) to induce temporary protein expression. The plasmid was introduced using ExpiFectamine 293 (Thermo Scientific), and after culturing for 3 days, the culture supernatant was recovered.

[0490] For human FasL and cynomolgus monkey FasL, the protein was temporarily expressed by introducing it into Freestyle CHO-S cells (Thermo Scientific). The plasmid was introduced using Freestyle MAX Reagent (Thermo Scientific), and the culture supernatant was recovered after 3 days of culture.

[0491] 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 resin-packed column, washed with washing buffer (60 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4), and then dissolved with dissolution buffer (250 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4).

[0492] 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 resin-packed column, washed with washing buffer (2 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4), and then dissolved with dissolution buffer (250 mmol / L imidazole, 20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 7.4).

[0493] Each dissolution fraction was replaced with PBS using a NAP column (GE Healthcare) and sterilized through a 0.22 μm filter. The purity of the purified protein was confirmed by SDS-PAGE. Polymer formation was analyzed by SEC-UPLC (Apparatus: ACQUITY UPLC, Column: ACQUITY UPLC Protein BEH SEC 200Å, 1.7 μm, 4.6 x 150 mm) (Waters). Peaks corresponding to the molecular weight of trimers were confirmed in almost all recombinant soluble DcR3 ligands, while no trimer peak was detected in mouse light itself, indicating it was a monomer.

[0494] (2) Binding activity assay using BIAcore

[0495] Binding activity against DcR3 ligands (LIGHT, TL1A, FasL) was analyzed using the SPR method with a BIAcore T-100 (GEHEALTHCARE) for the various wild-type DcR3 controls and DcR3 variants shown in Table 9. HBS-EP + Buffer was used as the buffer.

[0496] After immobilizing 10,000 RU of anti-human antibody onto a Series S Sensor Chip CM5 using the Human Antibody Capture Kit (GE HealthCARE), various wild-type DcR3 and DcR3 variants were captured by flow-through at 10 μL / min for 30 seconds. Meanwhile, a protein-free buffer was flow-through the reference flow cell. Subsequently, as the analyte, human, cynomolgus monkey, or mouse DcR3 ligands diluted to 0.08–80 nmol / L were flow-through at 10 μL / min for 2 minutes, with binding monitored, followed by flow-through of buffer for 3 minutes, with dissociation monitored. Finally, a regeneration reaction was performed by flow-through of 3 mol / L magnesium chloride at 20 μL / min for 1 minute. Using BIAcore T-100 evaluation software and a 1:1 binding model, with each DcR3 ligand 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), the kinetic constants (kDa) were calculated. a k d K D (Table 10-12). The results showed that, except for mouse LIGHT which could not produce trimers, various wild-type DcR3 and DcR3 variants were confirmed to bind to various DcR3 ligands in humans, cynomolgus monkeys, and mice.

[0497] [Table 9]

[0498]

[0499] [Table 10]

[0500]

[0501] [Table 11]

[0502]

[0503] [Table 12]

[0504]

[0505] [Example 8] Evaluation of binding activity of OPG ligands (RANKL, TRAIL)

[0506] RANKL is known to bind to RANK and be associated with bone resorption, while TRAIL binds to the TRAIL receptor and is associated with cell death. The binding activity of various prepared human DcR3 recombinants to RANKL and TRAIL was analyzed by ELISA. As positive controls for binding OPG ligands, the C-terminal deletion variants of OPG K194-Fc, RANK-Fc (Enzo LifeScience), and TRAIL R1-Fc (R&D Systems) prepared in Example 5 were used. As negative controls, anti-DNP antibody (IgG1) was used (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 the vector encoding the Fc of IgG1, introduced into CHO cells for expression, and purified from protein A).

[0507] Anti-human IgG1 (prepared to 10 μg / mL) with PBS was aliquoted into 96-well plates (MAXISORP NUNC-IMMUNO PLATE, Thermo Scientific) at 50 μL / well, and incubated overnight at 4°C to allow adsorption. After removing the immobilization solution, 1 g of Block Ace powder (DS Pharma Bio Medical) was dissolved in 100 mL of water, and the prepared 1% Block Ace was aliquoted into 100 μL / well. The plates were incubated at room temperature for 1 hour for blocking, and then washed three times with PBS containing 0.1% Tween (hereinafter referred to as PBST). Next, various human DcR3 recombinants or DcR3 variants, and RANK-Fc were aliquoted into plate 1 at 50 μL / well using 1% BSA-PBS to achieve a concentration of 1 μg / mL. Similarly, various wild-type DcR3 or DcR3 variants, and TRAIL R1-Fc were aliquoted into plate 2 at 50 μL / well using 1% BSA-PBS to achieve a concentration of 1 μg / mL. The plates were then incubated at room temperature for 1 hour.

[0508] After washing each plate three times with PBST, dispense 50 μL / well of RANKL (Peprotech) diluted with 1% BSA-PBS to 0.64-50000 pg / mL into plate 1, and dispense 50 μL / well of TRAIL (Peprotech) diluted with 1% BSA-PBS to 0.64-50000 pg / mL into plate 2. Let stand at room temperature for 1 hour.

[0509] After washing the plates three times with PBST, dispense 50 μL / well of biotinylated anti-RANKL antibody (Peprotech) diluted to 0.4 μg / mL with 1% BSA-PBS into plate 1, and dispense 50 μL / well of biotinylated anti-TRAIL antibody (R&D) diluted to 0.4 μg / mL with 1% BSA-PBS into plate 2. Incubate at room temperature for 1 hour.

[0510] After washing each plate three times with PBST, dispense 50 μL / well of streptavidin-HRP diluted 10,000 times with 0.1% Block Ace and let stand at room temperature for 1 hour.

[0511] After washing each plate three times with PBST, dispense 50 μL of TMB + Substrate Chromogen (Dako) into each well and allow it to stand at room temperature for 1 minute. Then, dispense 50 μL of 0.5 mol / L sulfuric acid solution into each well to terminate the colorimetric reaction. Measure the absorbance at 450 nm (sample wavelength) and 570 nm (reference wavelength) using a microplate reader.

[0512] The results showed that K194-Fc bound to RANKL and TRAIL as OPG ligands, while RANK-Fc and TRAIL R1-Fc bound to RANKL and TRAIL, respectively. However, DcR3 FL-Fc (R&D Corporation), S195-Fc, chimeric A-Fc (IEGRMD g1S), and chimeric B-Fc (IEGRMD g1S) did not bind to any ligand. Figure 8 Combined with the results of Example 7, it was confirmed that the DcR3 variant prepared by replacing a portion of the CRD of DcR3 with a portion of the CRD of OPG showed equivalent binding activity to DcR3 ligands as wild-type DcR3, but did not bind to OPG ligands.

[0513] [Example 9] Determination of neutralizing activity of DcR3 ligand

[0514] The neutralizing activities of various wild-type DcR3 controls and DcR3 variants against LIGHT, TL1A, and FasL were determined according to the methods described below. DNP antibodies were prepared using the methods described in Examples 5 and 8.

[0515] (1) LIGHT neutralization activity assay

[0516] Using the human colorectal cancer cell line HT-29 (ATCC number: HTB-38), the neutralizing activity of various wild-type DcR3 and various DcR3 variants was determined, with LIGHT-added IL-8 production as the indicator. Cell culture and neutralizing activity evaluation were performed using McCoy's 5A medium (Gibco) supplemented with 10% FBS (Gibco) and penicillin / streptomycin (Nacalai Tesque).

[0517] HT-29 cell line was used at 2 x 10 4 Cells were seeded in 96-well adhesion culture plates (Sumitomo Bakelite) and various wild-type DcR3 or DcR3 variants were added at final concentrations of 0.1, 1, or 10 μg / mL. Subsequently, human LIGHT (Gly66-Val240) with an N-terminal FLAG tag (DYKDDDDK) (described in JP2013153749) was added at a final concentration of 0.1 μg / mL, with a total culture volume of 200 μL / well. Cells were incubated at 37°C for 3 days in a 5% CO2 incubator. The culture supernatant was then collected, and the IL-8 concentration in the supernatant was determined using the AlphaLISA IL-8 Immunoassay Research Kit (Perkin Elmer).

[0518] The results confirmed that, across various human DcR3 variants, IL-8 production decreased in a concentration-dependent manner, demonstrating LIGHT neutralizing activity. Figure 9 ).

[0519] (2) TL1A neutralizing activity assay

[0520] Using human T cells, the neutralizing activity of various wild-type DcR3 and various DcR3 variants was measured, with TL1A-based IFN-γ production as the indicator. Cell culture and neutralizing activity evaluation were performed using X-VIVO15 medium (Lonza).

[0521] Frozen healthy human PBMCs (AllCells) were thawed in a 37°C water bath and suspended in DNase I (STEMCELL) culture medium warmed to 37°C. The medium was incubated at 37°C with low-speed shaking for 2 hours, and then T cells were isolated using the EasySep Human T cell Enrichment Kit (STEMCELL). The isolated T cells were then cultured at 1 x 10⁻⁶ cells / mL. 5Cells were seeded in 96-well suspension plates (Sumitomo Bakelite) and various wild-type DcR3 controls or DcR3 variants were added at final concentrations of 0.1, 1, and 10 μg / mL. Recombinant His10 human TL1A prepared in Example 6 was then added at a final concentration of 0.1 μg / mL. Human IL-12 (Miltenyi Biotech) and recombinant human IL-18 (MBL) at a final concentration of 2 ng / mL and 50 ng / mL, respectively, were added, with a total culture volume of 200 μL / well. Cells were incubated at 37°C for 3 days in a 5% CO2 incubator. The culture supernatant was then recovered, and the IFN-γ concentration in the supernatant was determined using the AlphaLISA IFN-γ Immunoassay Research Kit (Perkin Elmer).

[0522] The results confirmed that, across all DcR3 variants, IFN-γ production decreased in a concentration-dependent manner, demonstrating TL1A neutralizing activity. Figure 10 ).

[0523] (3) Assay of FasL neutralizing activity

[0524] Using the Jurkat T-cell leukemia cell line (DSMZ: ACC 282), the neutralizing activity of various wild-type DcR3 controls and various DcR3 variants was determined, with apoptosis induced by FasL supplementation as an indicator. Cell culture and neutralizing activity evaluation were performed using RPMI 1640 medium (Nacalai Tesque) supplemented with 10% FBS and penicillin / streptomycin.

[0525] Jurkat cell line at 5 x 10 4 Cells were seeded in 96-well suspension plates, and various wild-type DcR3 or DcR3 variants were added at final concentrations of 0.01, 0.1, and 1 μg / mL. Recombinant human His6 FaS Ligand (CST Japan) was then added at a final concentration of 0.1 μg / mL, with a total culture volume of 100 μL / well. Cells were incubated overnight at 37°C in a 5% CO2 incubator. CellTiter-Glo (Promega) was then added to Jurkat plates at 100 μL / well, and the number of viable cells, measured as an indicator of ATP production, was determined using a lux meter (Veritas, Promega).

[0526] The results confirmed a concentration-dependent increase in ATP production derived from living cells across various DcR3 variants, demonstrating FasL neutralizing activity. Figure 11 ).

[0527] [Example 10] Ligand neutralizing and binding activities of FasL-reduced variants

[0528] Based on the chimeric A-Fc(g4PEK), monoamino acid substitutes with binding and neutralizing activities for TL1A and LIGHT in the DcR3 ligand, but with reduced binding and neutralizing activities for FasL, were prepared and evaluated. Variants of CRD2 and CRD3 shown in Table 13, obtained by substituting any amino acid into Ala or other amino acids, were prepared, and their neutralizing activity against the DcR3 ligand was determined using the same method as in Example 9. As a result, for the following single amino acid substitutes, the neutralizing activity is selectively reduced for FasL (Table 13): chimeric A-E57K-Fc (g4PEK) obtained by replacing Glu at position 57 from the N-terminus with Lys or Leu (amino acid sequence: SEQ ID NO: 94, DNA base sequence: SEQ ID NO: 93), 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) obtained by replacing Arg at position 60 with Lys (amino acid sequence: SEQ ID NO: 98, DNA base sequence: SEQ ID NO: 97).

[0529] For the chimeric variants of A-E57K-Fc(g4PEK) (serial number 94), A-E57L-Fc(g4PEK) (serial number 96), and A-R60K-Fc(g4PEK) (serial number 98) whose neutralizing activity selectively decreases for FasL, the binding activity to the DcR3 ligand was determined using the same method as in Example 7. The results showed that the chimeric variants of A-E57K-Fc(g4PEK), A-E57L-Fc(g4PEK), and A-R60K-Fc(g4PEK) all exhibited significantly reduced binding only to FasL, while binding to TL1A and LIGHT was maintained. Figure 12A , Figure 12B , Figure 12C ).

[0530] [Table 13]

[0531]

[0532] [Example 11] Evaluation of the agglutination properties of commercially available wild-type DcR3 in mammalian cells

[0533] Using the same method as in Example 1, electrophoresis was performed on 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 a commercially available Fc fusion polymer (Enzo) with approximately half of the DcR3 molecule missing from the C-terminus of HBD, prepared using HEK293 cells as host cells, under reducing and non-reducing conditions. Based on the mobility under reducing conditions, the expected molecular weight of the monomer was approximately 50 kDa to 60 kDa. The mobility under non-reducing conditions was much greater than the expected molecular weight of the dimer; all commercially available products were predominantly present as aggregates. Figure 13 ).

[0534] [Example 12] Preparation of FasL-reduced binding variant

[0535] In order to select variants that bind to TL1A and LIGHT in the DcR3 ligand and whose binding selectivity for FasL is reduced, a monoamino acid substitute (E57X; X is any amino acid other than Glu) was prepared by replacing Glu (E57) at position 57 from the N-terminus of the chimeric A-Fc(g4PEK) (serial number 82) with an amino acid other than Glu. In addition, for E57K, E57L, E57R, and E57V obtained by replacing E57 with any one of Lys, Leu, Arg, or Val, the following fusion compounds were prepared: Any one of the adjacent amino acids in the chimeric A-Fc (g4PEK) (serial number 82), namely Trp (W53) at position 53, Asn (N54) at position 54, Tyr (Y55) at position 55, Leu (L56) at position 56, or Arg (R58) at position 58, was further replaced with a specific amino acid Z (Z being any one of Asp, Glu, Asn, Gln, Pro, Thr, or Gly) to obtain 2-amino acid substitutes (E57X_W53Z, E57X_N54Z, E57X_Y55Z, E57X_L56Z, E57X_R58Z); each 2-amino acid substitute was labeled. Figure 14A The variant number shown is a fusion of Fc (g4PEK).

[0536] Specifically, for chimeric A-E57K (amino acid sequence: sequence number 66, DNA base sequence: sequence number 65), chimeric A-E57R (amino acid sequence: sequence number 180, DNA base sequence: sequence number 179), chimeric A-E57V (amino acid sequence: sequence number 182, DNA base sequence: sequence number 181), chimeric A-E57K_R58D (variant number: 45-10, amino acid sequence: sequence number 184, DNA base sequence: sequence number 183), chimeric A-E57K_R58E (variant number: 45-18, amino acid sequence: sequence number 186, DNA base sequence: sequence number 185), and chimeric A, a portion of the Fc sequence corresponding to the various mutations introduced as shown in Table 14 was prepared. Fusion complex (base sequence: sequence numbers 213, 217, 219, 221, 223, 227, 231, 233, 235, 237, 255, 259, 261, 263, 265, 149, 167, 169, 171, 173, 175, 177; amino acid sequence: sequence numbers 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 represents Glu at position 216 of the human IgG1 heavy chain according to the EU index. The mutation introduction sites C220S, M252Y, S254T, T256E, N434A, L234A, L235A, and G237A represent, respectively, the following substitutions according to the EU index: Cys at position 220 is replaced by Ser, Met at position 252 is replaced by Tyr, Ser at position 254 is replaced by Thr, Thr at position 256 is replaced by Glu, Asn at position 434 is replaced by Ala, Leu at position 234 is replaced by Ala, Leu at position 235 is replaced by Ala, and Gly at position 237 is replaced by Ala.

[0537] [Table 14]

[0538]

[0539] Chimeric A-Fc (g1S) (base sequence: SEQ ID NO: 149, amino acid sequence: SEQ ID NO: 150) was prepared as follows: The IEGRMD sequence was deleted from the chimeric A-Fc (IEGRMD g1S) (SEQ ID NO: 80) containing the linker sequence IEGRMD (SEQ ID NO: 106) prepared in Example 2, and the result was stably expressed in CHO cells using the method described in Example 6. Chimeric A-Fc (Eg1S) (base sequence: SEQ ID NO: 167, amino acid sequence: SEQ ID NO: 168) was prepared as follows: The sequence obtained by linking chimeric A (SEQ ID NO: 54) to Eg1S was stably expressed in CHO cells using the method described in Example 6.

[0540] The chimeric A-Fc (Eg1S YTE, Eg1S N434A, Eg1S LALAGA, Eg1S LALAGANA) (base sequences: sequence numbers 169, 171, 175, 177; amino acid sequences: sequence numbers 170, 172, 176, 178) were obtained by fusing each Fc to the C-terminus of chimeric A (sequence number 54) and then temporarily expressing the resulting sequences in CHO-S cells.

[0541] The chimeric A-Fc (g1S YTE, g1S N434A, g1S LALAGA, g1S LALAGANA) (amino acid sequences: sequence numbers 314, 315, 174, 316) were obtained by fusing each Fc to the C-terminus of chimeric A (sequence number 54) and then temporarily expressing the results in Expi293 cells.

[0542] The plasmids expressing the fusion of each amino acid substitute with Fc (g4PEK) were prepared as follows: using the DNA sequence of either chimeric A-Fc (g4PEK) or E57X-Fc (g4PEK; X is K, R, or V) as a template, PCR primers designed to contain the mutation site were used to amplify two regions: from the NheI site to the site with the introduced amino acid substitution, and from the site with the introduced amino acid substitution to the SalI site. The resulting products were inserted below the CMV promoter of the pCIpuro vector using the same method as in Example 1.

[0543] For fusions of each amino acid substitute with each Fc of Eg1S YTE, Eg1S N434A, or Eg1S LALAGANA, plasmids are prepared as follows: using the DNA sequence of any of the chimeric A-Fcs with their respective mutant Fc sequences as templates, PCR primers designed to contain the mutant sites are used to amplify two regions: from the EcoRI site to the site with the introduced amino acid substitution, and from the site with the introduced amino acid substitution to the Bsu36I site. The resulting products are then inserted into the EcoRI and Bsu36I sites of the chimeric A-various mutant Fc vectors that serve as templates.

[0544] For fusions of each amino acid substitute with each Fc of g1S YTE, g1S N434A, or g1S LALAGANA, plasmids are prepared as follows: using the DNA sequence of any of the above amino acid substitutes-Fc as a template, PCR primers designed to remove E216 are used to amplify two regions: from the EcoRI site to the deleted Glu site, and from the deleted Glu site to the Bsu36I site. The resulting products are then inserted into the EcoRI and Bsu36I sites of the amino acid substitute-Fc vector used as a template.

[0545] Each plasmid was introduced into Expi293 cells to induce transient expression, and affinity purification based on MabSelectSuRe was performed from the culture supernatant. The contents of the prepared chimeric A-Fc, monomers of each single or 2-amino acid substituted Fc, aggregates, and decomposition products were calculated based on peak areas analyzed by SEC-UPLC (ACQUITY UPLC ProteinBEH SEC 4.6 mm x 150 mm) (Waters) or SEC-HPLC (TSKgel SuperSW3000 4.0 μm, 4.6 mm x 300 mm) (TOSOH) using the same method as in Example 2.

[0546] As a result, most of the prepared chimeric A-Fc, mono- and 2-amino acid substituted Fcs with various mutant Fcs maintained a lower condensate content than S195-Fc. Figure 14A , Figure 14B , Figure 14C ).

[0547] [Example 13] Evaluation of the binding activity of DcR3 soluble trimer ligands

[0548] For various chimeric A-Fc with different DcR3-Fc, S195-Fc, and Fc sequences, as well as various FasL binding-reduced variants prepared by Example 12, the method described in Example 7 was partially modified to evaluate the binding activity of soluble human LIGHT trimer, soluble human TL1A trimer, and soluble human FasL trimer.

[0549] (1) Preparation of DcR3 soluble trimer ligands

[0550] For the human soluble recombinant LIGHT, the sequence used is FLAG-LIGHT (base sequence: SEQ ID NO: 305, amino acid sequence: SEQ ID NO: 306), which involves adding a FLAG tag (DYKDDDDK) to the N-terminus and linking the extracellular region of LIGHT (Asp74-Val240) (SEQ ID NO: 132) downstream of it. For the human soluble recombinant TL1A, the sequence used is His6-TL1A (base sequence: SEQ ID NO: 115, amino acid sequence: SEQ ID NO: 116), which involves adding a His tag (His6) and a GS linker (GGGSGGGSGGGS) to the N-terminus and linking the extracellular region of TL1A (Leu72-Leu251) (SEQ ID NO: 138) downstream of it. Each plasmid was prepared using the same method as in Example 7 and transiently expressed on Expi293 cells.

[0551] FLAG-LIGHT was purified using ANTI-FLAG M2 Affinity Gel (Sigma). The culture supernatant was passed through a resin-packed column, washed with washing buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.4), and then dissolved with dissolution buffer (0.1 M glycine hydrochloride, pH 3.5).

[0552] Purification of His6-TL1A was performed as follows: Culture supernatant was passed through a column packed with Complete His-Tag Purification Resin (Roche), washed with wash buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl), and then dissolved with dissolution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). Dissolution buffer obtained by replacing PBS with a NAP column (GE HEALTHCARE) was passed through a column packed with Ni Sepharose Fast Flow resin (GE HEALTHCARE), washed with wash buffer prepared using the His Buffer Kit (GE HEALTHCARE) (60 mM imidazole, 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4), and then dissolved with dissolution buffer (250 mM imidazole, 20 mM sodium phosphate, 0.5 M NaCl, pH 7.4).

[0553] The dissolution fractions of FLAG-LIGHT and His6-TL1A were replaced with PBS using a NAP column (GE HEALTHCARE), passed through a 0.22 μm filter, and sterilized. The resulting purified protein was separated into trimer fractions using HPLC (Shimadzu Corporation) gel filtration chromatography (SEC) (TSKgel G3000 SWXL 7.8 mm x 300 mm) (TOSOH Corporation).

[0554] Human soluble recombinant FasL was prepared by adding a His tag (His6) to the N-terminus and attaching it downstream to the extracellular region of FasL (Pro134-Leu281) (serial number 144) to obtain Human His6 FaS Ligand / TNFSF6 (Cell Signaling Technology). It was confirmed to be a trimer by performing SEC-MALS using the same method as in Example 2.

[0555] (2) Binding activity assay using BIAcore

[0556] DcR3-Fc (R&D Company) and S195-Fc were evaluated according to the contents described in Example 7. Chimeric A-g4PEK (serial number 82) was prepared according to Example 6. Chimeric A-Fc (g1S, Eg1S) was prepared according to Example 12. Fc (g4PEK) fusions prepared according to Example 9 or Example 12 were evaluated as various FasL-reducing variants.

[0557] Binding activity to the human DcR3 trimer ligand was analyzed using the SPR method. For the determination of binding activity to human LIGHT and human TL1A, BIAcore T-100 (GE HEALTHCARE) or BIAcore T-200 (GE HEALTHCARE) was used to determine the binding activity to human FasL, respectively. HBS-EP + Buffer was used as the buffer solution.

[0558] 10,000 RU of anti-human antibody was immobilized on a Series S Sensor Chip CM5 using a Human Antibody Capture Kit (both from GE HealthCARE). Various wild-type DcR3 and DcR3 variants were then flown at 10 μL / min for 30 seconds to capture them. Conversely, a protein-free buffer was flowed through a reference flow cell. Then, as the analyte, each human DcR3 trimer ligand diluted to 0.02–80 nmol / L was flowed at 30 μL / min for 2 minutes to monitor binding, followed by flow through buffer for 3 minutes to monitor dissociation. Next, 3 mol / L magnesium chloride was flowed at 30 μL / min for 1 minute to perform a regeneration reaction. Binding activity against human LIGHT and human TL1A was assessed using BIAcore T-100 evaluation software, a 1:1 binding model, with the ligands set as trimers (human LIGHT trimer: 62.4 kDa, human TL1A trimer: 66.2 kDa), and the kinetic constants (kDa) were calculated. a k d K D In the determination of binding activity of human FasL, the BIAcore T-100 evaluation software and 1:1 binding model or the BIAcore T-200 evaluation software and 1:1 binding model were used, with the ligand set as a monomer (human FasL monomer: 19.8 kDa). Various kinetic constants (kDa) were calculated. a k d K D ).

[0559] As a result, chimeric A-Fc (g1S), chimeric A-Fc (Eg1S), and chimeric A-Fc (g4PEK) all confirmed binding to each DcR3 trimer ligand ( Figure 15A ).

[0560] Among the FasL-binding reduced variants (g4PEK) prepared in Example 12, the following single-amino acid substitution chimeric variants were used: 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"), 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"), 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"), and A-E57V-Fc. Fc (g4PEK) (amino acid sequence: sequence number 192, DNA base sequence: sequence number 191) (hereinafter sometimes referred to as "E57V-Fc"), chimeric A-E57A-Fc (g4PEK) (amino acid sequence: sequence number 288, DNA base sequence: sequence number 287) (hereinafter sometimes referred to as "E57A-Fc"), chimeric A-E57F-Fc (g4PEK) (amino acid sequence: sequence number 290, DNA base sequence: sequence number 289) (hereinafter sometimes referred to as "E57F-Fc"), chimeric A-E57H-Fc (g4PEK) (amino acid sequence: sequence number 292, DNA base sequence: sequence number 291) (hereinafter sometimes referred to as "E57V-Fc") The following are sometimes referred to as "E57H-Fc": chimeric A-E57I-Fc (g4PEK) (amino acid sequence: sequence number 294, DNA base sequence: sequence number 293) (hereinafter sometimes referred to as "E57I-Fc"), chimeric A-E57M-Fc (g4PEK) (amino acid sequence: sequence number 296, DNA base sequence: sequence number 295) (hereinafter sometimes referred to as "E57M-Fc"), and the diamino acid substituted chimeric A-E57K_R58D-Fc (g4PEK) (variant number: 45-10, amino acid sequence: sequence number 194, DNA base sequence: sequence number 193) (hereinafter sometimes referred to as "45-10-Fc"), chimeric A-E 57K_R58T-Fc (g4PEK) (variant number: 45-11, amino acid sequence: sequence number 298, DNA base sequence: sequence number 297) (hereinafter sometimes referred to as "45-11-Fc"), chimeric A-E57K_R58E-Fc (g4PEK) (variant number: 45-18, amino acid sequence: sequence number 196, DNA base sequence: sequence number 195) (hereinafter sometimes referred to as "45-18-Fc"), chimeric A-E57L_R58E-Fc (g4PEK) (variant number: 46-4, amino acid sequence: sequence number 300, DNA base sequence: sequence number 299) (hereinafter sometimes referred to as "46-4-Fc")Chimeric A-E57R_R58D-Fc (g4PEK) (Variant number: 82-5, amino acid sequence: sequence number 198, DNA base sequence: sequence number 197) (hereinafter sometimes referred to as "82-5-Fc"), Chimeric A-E57V_R58T-Fc (g4PEK) (Variant number: 85-6, amino acid sequence: sequence number 302, DNA base sequence: sequence number 301) (hereinafter sometimes referred to as "85-6-Fc"), Chimeric A-E57V_R For 58E-Fc (g4PEK) (variant number: 85-8, amino acid sequence: sequence number 304, DNA base sequence: sequence number 303) (hereinafter sometimes referred to as "85-8-Fc"), compared with chimeric A-Fc (g4PEK), the KD values ​​of either are less than 3-fold for both the LIGHT trimer and TL1A trimer, and the KD values ​​for the FasL trimer are greater than 3-fold or the Rmax value is reduced to less than 5, confirming that the binding selectivity for FasL is reduced. Figure 16A , Figure 16B ).

[0561] E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (each with Fc of g4PEK) were purified to over 95% monomer purity by SEC-HPLC (column; TSKgel G3000 SWXL 7.8mm x 300mm, TOSOH Corporation; HPLC; Shimadzu Corporation). The kinetic constants for BIAcore determination are shown in the figure. Figure 15B middle.

[0562] [Example 14] Evaluation of the neutralizing activity of DcR3 soluble ligands

[0563] The method described in Example 9 was modified to evaluate the neutralizing activity of various DcR3 variants against soluble human LIGHT, soluble human TL1A, and soluble human FasL.

[0564] (1) Preparation of various chimeric A-Fc with different Fc sequences

[0565] Chimeric A-Fc (g1S, Eg1S) used the substances prepared according to Example 12. For each chimeric A-Fc prepared according to Example 12, which is obtained by fusing each of the Fcs of Eg1S YTE, Eg1S N434A, Eg1S LALAGA or Eg1SLALAGANA shown in Table 14 to the C-terminus of chimeric A, the substances were all prepared by transient expression in CHO-S cells and purified to more than 95% monomer purity using AKTApurifier (GE HEALTHCARE) SEC (Superdex 200 Increase 10 / 300 GL) (GE HEALTHCARE).

[0566] (2) Neutralizing activity of soluble human light

[0567] The inhibitory activity of light-dependent CXCL10 production from IFN-γ-stimulated enteric fibroblasts (Lonza) against chimeric A-Fc (g1S, Eg1S, Eg1S YTE, Eg1S N434A, Eg1S LALAGA, Eg1S LALAGANA, g4PEK) was evaluated. Enteric fibroblasts were cultured in type I collagen-coated culture flasks (BD) using SmGM-2 Bullet Kit (Lonza) medium. Cells were sputtered at 1 x 10⁻⁶ cells / mL. 4 Cells / wells were seeded in type I collagen-coated 96-well plates (BD Biosciences) and then added at final concentrations of IFN-γ (10 ng / mL), the trimer FLAG-LIGHT prepared in Example 13 (20 ng / mL), and various DcR3 variants (19.5, 78.1, 313, 1250, 5000, 20000 ng / mL, or 4.88, 19.5, 78.1, 313, 1250, 5000 ng / mL, respectively), and cultured for 3 days. The culture supernatant was recovered, and the CXCL10 concentration was determined using the CXCL10 / IP-10 (human) AlphaLisa Detection Kit (PerkinElmer). The results showed that a concentration-dependent inhibition of CXCL10 production was confirmed for chimeric A-Fc cells with any Fc sequence, demonstrating neutralizing activity against soluble LIGHT. Figure 17A , Figure 17B ).

[0568] For the FasL-depleted variants E57K-Fc, E57L-Fc, E57R-Fc, and E57V-Fc (all Fc being g4PEK), the inhibitory activity against LIGHT-dependent IL-8 production from HT-29 cells was evaluated using the same method as in Example 9. The results showed that for each variant, concentration-dependent inhibition of IL-8 production was confirmed, exhibiting neutralizing activity against soluble LIGHT. Figure 17C ).

[0569] For the FasL-degraded variants 45-10-Fc, 45-18-Fc, and 82-5-Fc (all Fcs are g4PEK), similar to the chimeric A-Fc, the number of enterofibroblasts per pore was 2 x 10-1. 4 The inhibitory activity of the analyte on LIGHT-dependent CXCL10 production from IFN-γ-stimulated enteric myofibroblasts was evaluated at final concentrations of 19.5, 78.1, 313, 1250, 5000, and 20000 ng / mL. The results showed that for each variant, concentration-dependent inhibition of CXCL10 production was confirmed, demonstrating neutralizing activity against soluble LIGHT. Figure 17D ).

[0570] (3) Neutralizing activity of soluble human TL1A

[0571] Using the same method as in Example 9, the inhibitory activity of various DcR3 variants against TL1A-dependent IFN-γ production from IL-12 and IL-18-stimulated human T cells was evaluated. The results showed that chimeric A-Fc cells with either Fc sequence were confirmed to inhibit IFN-γ production in a concentration-dependent manner and possess neutralizing activity against soluble TL1A. Figure 18A , Figure 18B Furthermore, neutralizing activity against soluble TL1A was also confirmed in the FasL-reduced binding variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (all Fcs are g4PEK). Figure 18C , Figure 18D ).

[0572] (4) Neutralizing activity of soluble human FasL

[0573] Using the same method as in Example 9, the inhibitory activity of various DcR3 variants against FasL-dependent cell death in Jurkat cells or A3 cells as a Jurkat subclone was evaluated. The results confirmed that chimeric A-Fc cells possessing either Fc sequence inhibited A3 cell death in a concentration-dependent manner and exhibited neutralizing activity against soluble FasL. Figure 19A , Figure 19B On the other hand, for the FasL-binding reduced variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (all Fcs are g4PEK), it was confirmed that any one of them significantly reduced the inhibitory activity against Jurkat cell death and selectively reduced the neutralizing activity against soluble FasL. Figure 19C , Figure 19D ).

[0574] [Example 15] Evaluation of the binding activity of DcR3 membrane-type ligands

[0575] The binding activities of various DcR3 variants to human LIGHT, human TL1A, and human FasL were evaluated by flow cytometry using forced expression lines of membrane ligands. HEK293 cells for forced expression of human LIGHT were used, as described in US8974787. For TL1A and FasL, PCR amplification was performed using commercially available ORF clones (Origene) with Met residues and a FLAG tag (DYKDDDDK) added to the N-terminus (base sequence: SEQ ID NO. 307, 309; amino acid sequence: SEQ ID NO. 308, 310). The amplified molecules were inserted downstream of the CMV promoter of the pCIpuro vector using the In-Fusion HD CloninG Kit (Clontech) and transformed into *E. coli* DH5α competent cells (TOYOBO).

[0576] The obtained plasmids were introduced into CHO-K1 cells (ECACC) using Nucleofector and Cell Line Nucleofector Kit T (both from Lonza) for drug screening with 10 μg / mL puromycin (Thermo Fisher Scientific). Drug-resistant cells were stained with DyLight488-labeled anti-human TL1A antibody (Novus Biologics) or APC-labeled anti-human FasL antibody (BD Pharmingen), and high-expression fractions were sorted using a cell sorter (Sony). After expansion culture, cells were stained with PE-labeled anti-human TL1A antibody (Novus Biologics) or PE-labeled anti-human FasL antibody (BioLegend), and then sorted again to obtain membrane-type human TL1A or membrane-type human FasL high-expression cell lines.

[0577] Using the same method as in Example 5, the following conditions were modified to evaluate the binding activity of various DcR3 variants to membrane-bound ligand-forced expression strains and host cells. For HEK293 and membrane-bound LIGHT forced expression strains, the reaction was carried out at 1 μg / mL of each protein and 10 ng / mL of secondary antibody (goat F(ab')2 anti-human IgG R-phycoerythrin Conjugate, Southern Biotech). For membrane-bound TL1A and membrane-bound FasL forced expression strains, the reaction was carried out at 1 or 10 μg / mL of each protein and 0.1 or 1 μg / mL of secondary antibody.

[0578] As a result, the chimeric A-Fc (g1S, Eg1S, g4PEK) did not react with the 293 or CHO-K1 cells used as host cells, but reacted specifically with the membrane-type ligand-forced expression strains. Therefore, it was confirmed that various chimeric A-Fcs with different Fc sequences all possessed binding activity to the membrane-type DcR3 ligand. Figure 20A , Figure 20B Furthermore, the binding activity of the FasL-binding variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (all Fcs are g4PEK) was similarly evaluated. The results confirmed that they maintained binding activity to membrane-bound human LIGHT and membrane-bound human TL1A. On the other hand, except for E57L, the binding activity to membrane-bound human FasL was significantly reduced. Figure 21A , Figure 21B , Figure 21C ).

[0579] [Example 16] Evaluation of the binding activity of the DcR3 primary ligand

[0580] The following methods were used to evaluate the binding activity of various DcR3 variants to different DcR3 ligands derived from primary cells.

[0581] (1) Primary light binding activity

[0582] The induction of membrane-bound light expression in activated human T cells is known (The Journal of Immunology, 2004, 173: p. 502-507.). Frozen healthy human PBMCs (AllCells) were thawed and stimulated overnight with a final concentration of 50 ng / mL PMA (Sigma) and 1 μg / mL iomycin (Sigma). The binding of chimeric A-Fc (Eg1S) to membrane-bound light induced by expression in CD3-positive T cells was then evaluated using the following methods. As a negative control, the anti-DNP antibody (IgG1) described in Example 8 was used. Stimulated PBMCs were recovered and reacted with Human FcR Blocking Reagent (Miltenyi Biotech). Then, chimeric A-Fc or anti-DNP antibodies labeled with BV421-labeled CD3 antibody (BD Pharmingen), 7-AAD Staining Solution (BD Pharmingen), and Alexa Fluor488 Antibody Labeling Kit (Thermo Science) were added to a final concentration of 0.4 μg / mL. After reaction, the cells were washed, and the fluorescence intensity of Alexa Fluor488 in live-cell fractions of CD3-positive T cells was analyzed by flow cytometry. To confirm the expression of membrane-bound light in induced-stimulated PBMCs, Human FcRBlocking Reagent (Miltenyi Biotech) was used to react with induced-stimulated PBMCs. 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) were then added, respectively. After the reaction, the cells were washed, and the fluorescence intensity of PE in live-cell fractions of CD3-positive T cells was analyzed by flow cytometry.

[0583] The results confirmed that LIGHT expression was induced on CD3-positive human T cells in induced-stimulated PBMCs, and that Alexa Fluor488-labeled chimeric A-Fc bound to membrane-bound LIGHT on activated CD3-positive human T cells. Figure 22 Small picture A, Figure 22 Small image B).

[0584] (2) Primary TL1A binding activity

[0585] HUVEC cells (Lonza) cultured using the same method as in Example 5 were treated for 24 hours with a final concentration of 10 ng / mL recombinant human IL-1 alpha (R&D) and a final concentration of 20 μM TACE inhibitor TAPI-1 (Calbiochem). The binding of chimeric A-Fc (IEGRMDg1S) to induced membrane-bound TL1A was evaluated by a competition assay with TL1A antibody 1D1 1.31 (US2015 / 0132311). TL1A antibody 1D1 1.31 was prepared by linking the amino acid sequences of VL and VH described in US2015 / 0132311 to the constant region of human IgG1, transiently expressing it in Expi293 cells using the method described in Example 1, and purifying the antibody from the culture supernatant using Mabselect SuRe (GE HealthCARE). As a negative control, the anti-DNP antibody (IgG1) described in Example 8 was used. After reacting the recovered cells in Human FcR Blocking Reagent (Miltenyi Biotech), they were then added to a final concentration of 8.3 μg / mL with any of the following: anti-DNP antibody, chimeric A-Fc, or TL1A antibody labeled using the Zenon Alexa Fluor 647 Human IgG Labeling Kit (Molecular Probes). Following the reaction, the cells were washed, and the fluorescence intensity of Alexa Fluor 647 was analyzed by flow cytometry. Alternatively, under competitive conditions, the cells could be pre-reacted with any of the following: unlabeled DNP antibody, TL1A antibody, or chimeric A-Fc at a final concentration of 50 μg / mL.

[0586] As a result, the binding of labeled chimeric A-Fc competes with unlabeled TL1A antibody, and the binding of labeled TL1A antibody competes with unlabeled chimeric A-Fc. Therefore, it was confirmed that chimeric A binds to membrane-bound TL1A on stimulated HUVEC cells. Figure 23 ).

[0587] (3) Primary FasL binding activity

[0588] The binding of chimeric A-Fc (IEGRMD g1S) to primary soluble FasL produced from human T cells induced with activation-induced cell death (AICD) was evaluated using the following method. Primary soluble FasL was prepared by using human T cells isolated from frozen healthy human PBMCs using the same method as in Example 9, at a ratio of 2 x 10-1. 4Cells were seeded in 96-well U-plates (BD Biosciences) and cultured for 24 days with 1 μg / mL PHA-L (eBiosciences). Then, 1 μg / mL IL-2 (Peptrotech) was added and the cells were cultured for 5 days. After 5 days, the cells were harvested and seeded in 96-well U-plates immobilized with 5 μg / mL anti-CD3 antibody OKT3 (BioLegend). AICD was induced, and the cells were cultured overnight. The culture supernatant was then collected and concentrated to one-tenth of its original volume using an Amicon ultra-15 (Millipore) fraction with a molecular weight of 10 kDa, pre-washed with sterile water.

[0589] 96-well immunoassay plates (ThermoScientific) immobilized with 10 μg / mL anti-human IgG antibody (American Qualex) were blocked with 1% Block Ace (DS Pharma Bio Medical) and then captured with 20 μg / mL chimeric A-Fc (IEGRMD g1S) or Fas-Fc (R&D). After washing, the plates were reacted with recombinant FasL (abcam) or AICD culture supernatant concentrated 10 times as a standard. After washing, the plates were reacted with biotinylated anti-FasL antibody (abcam). After washing, the plates were reacted with streptavidin-HRP (PIERCE). After washing again, TMB solution (abcam) was added to develop the color, and the colorimetric reaction was stopped by 2N sulfuric acid solution. The absorbance at 450 nm was measured.

[0590] Results of capturing the board with matte A-Fc (IEGRMD g1S) are shown in Figure 24 In small figure A, the results of capturing the Fas-Fc plate are shown. Figure 24 In small figure B, 10 x AICD supernatant (sup.) refers to culture supernatant containing T cells induced with AICD under stimulation with anti-CD3 antibody OKT3, while "none" refers to culture supernatant containing T cells not induced with AICD. For plates capturing either chimeric A-Fc or Fas-Fc, FasL was detected only in the culture supernatant of AICD-induced T cells. This confirms that chimeric A-Fc binds to soluble FasL generated from AICD-induced T cells. Figure 24 Small picture A, Figure 24 Small image B).

[0591] [Example 17] Physical property evaluation of DcR3 variant

[0592] For various chimeric A-Fc (g1S, Eg1S, Eg1S YTE, Eg1S N434A, Eg1S LALAGANA, Eg1S LALAGA, g4PEK) and FasL-reduced binding variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, 82-5-Fc (all g4PEK), the dissolution time (minutes) based on hydrophobic interaction chromatography (HIC) and the Tm value (°C) based on differential scanning calorimetry (DSF) were calculated using the same method as in Example 4.

[0593] The results confirmed that introducing multiple amino acid mutations into the IgG1 Fc sequence had no significant effect on hydrophobicity. The dissolution time of the FasL-binding-reduced variants was not significantly different from that of the chimeric A-g4PEK variant, thus confirming that substitutions of one or two amino acids did not affect hydrophobicity. Figure 25 ).

[0594] Regarding the Tm values ​​based on DSF, the effects of amino acid substitutions or insertions in the IgG1 Fc sequence and the introduction of YTE mutations on thermal stability were observed. The Tm values ​​of the FasL-binding variants were not significantly different from those of the chimeric A-g4PEK, thus confirming that the introduction of one or two amino acid substitutions into the CRD region of DcR3 did not affect thermal stability. Figure 26 ).

[0595] [Example 18] Evaluation of the in vivo kinetics of the DcR3 variant in mice

[0596] Chimeric A-Fc (Eg1S) and FasL-deprived variants E57K-Fc, E57L-Fc, E57R-Fc, E57V-Fc, 45-10-Fc, 45-18-Fc, and 82-5-Fc (all Fcs are g4PEK) were evaluated in vivo in mice using the same methods described in Example 6. Chimeric A-Fc (Eg1S) was prepared by stable expression on CHO-K1 cells, and each FasL-deprived variant was prepared by transient expression on CHO-S cells. All were purified to over 95% monomer purity using AKTApurifier (GE HEALTHCARE) SEC (Superdex 200 Increase 10 / 300 GL).

[0597] Calculate the plasma half-life (h) of the elimination phase following a single intravenous administration of each DcR3 variant at 10 mg / kg (n = 2 or 3) to 5–6 week old BALB / c mice (♀), and the AUC0-∞ (μg * h / mL) as the area under the concentration-time curve to infinity. Figure 27 The standard substances used to determine the concentration of DcR3 variants in the serum of mice administered E57K-Fc, E57R-Fc, and E57V-Fc were the respective DcR3 variants prepared by transient expression in Expi293 cells.

[0598] As a result, the AUC of any DcR3 variant was significantly improved compared to S195-Fc (IEGRMD g1S) as wild-type DcR3 described in Example 6.

[0599] [Example 19] In vivo efficacy evaluation of DcR3 variant

[0600] The in vivo efficacy of chimeric A-Fc (g4PEK) was evaluated using a mouse model of acute interspecific graft-versus-host disease (GVHD).

[0601] (1) Preparation of an acute xenogeneic GVHD model in mice

[0602] An acute xenogeneic GVHD model in mice was established using the same method described in JP5209625. Six-week-old female mice with severe combined immunodeficiency (SCID) were intraperitoneally injected with 100 μg of rat anti-mouse IL2 receptor-β (IL2Rβ) chain antibody TMβ1 (Bio X Cell) on days -2 and 5 to deplete endogenous mouse natural killer cells. On day -1, mice were irradiated with a lethal dose below 1.7 Gy using a CellRad X-ray irradiation device (Faxitron). On day 0, 3 x 10 6 Personal PBMCs (AllCells) were transferred into the peritoneal cavity. Following this, 300 μg of chimeric A-Fc or DNP antibody (both g4PEK) prepared in 100 μL of PBS was injected intraperitoneally. For the chimeric A-Fc administration group, additional 300 μg of chimeric A-Fc was administered on days 4 and 8. Twelve days later, disease scores based on GVHD response were determined visually, and spleens were collected from euthanized mice to evaluate the number of human cells in the spleen.

[0603] (2) Evaluation of GVHD severity score

[0604] For the gross observations on day 12, scores ...

Claims

1. A variant of DcR3, which is a variant of wild-type trap receptor 3 (hereinafter referred to as DcR3), the DcR3 variant exhibiting improved in vivo kinetics compared to the wild-type DcR3.

2. The DcR3 variant as described in claim 1, having one or more N-glycosidic bonds in a complex glycan chain.

3. The DcR3 variant as described in claim 1, which has neutralizing activity against at least one of LIGHT, TL1A and FasL.

4. The DcR3 variant as described in claim 1, which has neutralizing activity against LIGHT, TL1A and FasL.

5. The DcR3 variant as claimed in claim 1, which has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A or more.

6. The DcR3 variant as claimed in claim 1, which has no neutralizing activity against FasL and has neutralizing activity against LIGHT and TL1A.

7. A DcR3 variant comprising a first chimeric cysteine-rich region or a second chimeric cysteine-rich region, wherein the first chimeric cysteine-rich region comprises an amino acid sequence obtained by replacing at least a portion of the cysteine-rich domain of wild-type DcR3 (hereinafter referred to as CRD) with at least a portion of the cysteine-rich domain of a TNF receptor superfamily molecule other than DcR3, and the second chimeric cysteine-rich region comprises an amino acid sequence obtained by deleting, substituting, inserting, or adding 1 to 30 amino acids in the amino acid sequence of the first chimeric cysteine-rich region.

8. A DcR3 variant composition comprising the DcR3 variant as described in claim 1 or 7.

9. The composition of claim 8, comprising a DcR3 variant having one or more N-glycosidic complex glycans and a DcR3 variant not having an N-glycosidic complex glycan.

10. DNA encoding the DcR3 variant as described in claim 1 or 7.

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