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EP-4056232-B1 - DCR3 VARIANT

EP4056232B1EP 4056232 B1EP4056232 B1EP 4056232B1EP-4056232-B1

Inventors

  • KANAI AKIKO
  • HOSOMI Hitomi
  • YONEZAWA Sakiko

Dates

Publication Date
20260506
Application Date
20200911

Claims (20)

  1. A DcR3 variant comprising a first chimeric cysteine-rich region or a second chimeric cysteine-rich region, wherein: the first chimeric cysteine-rich region consists of any one of the following amino acid sequences: (a) an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 26 or 50; (b) an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 28 or 52; (c) an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 30 or 54; and (d) an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 32 or 56; and the second chimeric cysteine-rich region consists of an amino acid sequence (e) obtained by introducing into the amino acid sequence of the first chimeric cysteine-rich region, deletion, substitution, insertion or addition of 1 to 5 amino acids.
  2. The DcR3 variant according to claim 1, wherein the DcR3 variant comprises one or more complex N-glycoside-linked glycans.
  3. The DcR3 variant according to claim 1 or 2, wherein the DcR3 variant has neutralizing activity to at least one or more of LIGHT, TL1A and FasL.
  4. The DcR3 variant according to any one of claims 1 to 3, wherein the DcR3 variant has neutralizing activity to all of LIGHT, TL1A and FasL.
  5. The DcR3 variant according to any one of claims 1 to 3, wherein the DcR3 variant has no neutralizing activity to FasL and has neutralizing activity to one or more of LIGHT and TL1A.
  6. The DcR3 variant according to any one of claims 1 to 3 and 5, wherein the DcR3 variant has no neutralizing activity to FasL and has neutralizing activity to both of LIGHT and TL1A.
  7. The DcR3 variant according to any one of claims 1 to 6, wherein the first cysteine rich region consists of an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 54.
  8. The DcR3 variant according to any one of claims 1 to 7, wherein the amino acid sequence (e) comprises one or two or more substitution selected from the group consisting of: substitution of Glu at position 57 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with another amino acid; substitution of Arg at position 58 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with another amino acid; and substitution of Arg at position 60 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with another amino acid.
  9. The DcR3 variant according to any one of claims 1 to 8, wherein the amino acid sequence (e) comprises substitution of Glu at position 57 and Arg at position 58 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with other amino acids.
  10. The DcR3 variant according to any one of claims 1 to 9, wherein the amino acid sequence (e) comprises one or two or more substitution selected from the group consisting of: substitution of Glu at position 57 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met; substitution of Arg at position 58 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Asp, Glu or Thr; and substitution of Arg at position 60 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Lys.
  11. The DcR3 variant according to any one of claims 1 to 10, wherein the amino acid sequence (e) comprises substitution of Glu at position 57 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Lys, Leu, Arg, Val, Ala, Phe, His, Ile or Met, and substitution of Arg at position 58 from the N-terminus of the amino acid sequence (a), (b), (c) or (d) with Asp, Glu or Thr.
  12. The DcR3 variant according to any one of claims 1 to 11, wherein the amino acid sequence (e) comprises substitution selected from the following (f) to (i): (f) substitution of Asn at positions 131 and 144 from the N-terminus of the amino acid sequence (b), (c) or (d) with other amino acids; (g) substitution of Asn at positions 131, 144 and 157 from the N-terminus of the amino acid sequence (b), (c) or (d) with other amino acids; (h) substitution of Thr at position 133 and Ser at position 146 from the N-terminus of the amino acid sequence (b), (c) or (d) with other amino acids; and (i) substitution of Thr at position 133, Ser at position 146 and Thr at position 159 from the N-terminus of the amino acid sequence (b), (c) or (d) with other amino acids.
  13. The DcR3 variant according to any one of claims 1 to 12, wherein the amino acid sequence (e) comprises substitution selected from the following (f') to (i'): (f') substitution of Asn at positions 131 and 144 from the N-terminus of the amino acid sequence (b), (c) or (d) with Ser; (g') substitution of Asn at positions 131, 144 and 157 from the N-terminus of the amino acid sequence (b), (c) or (d) with Ser; (h') substitution of Thr at position 133 and Ser at position 146 from the N-terminus of the amino acid sequence (b), (c) or (d) with Ala; and (i') substitution of Thr at position 133, Ser at position 146 and Thr at position 159 from the N-terminus of the amino acid sequence (b), (c) or (d) with Ala.
  14. The DcR3 variant according to any one of claims 1 to 13, wherein the amino acid sequence (e) is an amino acid sequence consisting of amino acids at positions 1 to 164 from the N-terminus of an amino acid sequence set forth in SEQ ID NO: 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286.
  15. The DcR3 variant according to any one of claims 1 to 14, wherein the DcR3 variant is a DcR3 variant comprising the first or second chimeric cysteine-rich region, and a part or a whole of a heparan sulfate-binding domain of the wild-type DcR3 bound to the C-terminal side of the first or second chimeric cysteine-rich region.
  16. The DcR3 variant according to any one of claims 1 to 14, wherein the DcR3 variant is a DcR3 variant comprising the first or second chimeric cysteine-rich region and not comprising a heparan sulfate-binding domain of the wild-type DcR3.
  17. The DcR3 variant according to claim 15 or 16, wherein the DcR3 variant comprises one of amino acid sequences selected from: (I) an amino acid sequence set forth in SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or an amino acid sequence having 99% or more identity to the abovementioned amino acid sequence; and (II) an amino acid sequence set forth in SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 180, 182, 184, 186, 188, 270, 272, 274, 276, 278, 280, 282, 284 or 286, or an amino acid sequence having 99% or more identity to the abovementioned amino acid sequence.
  18. The DcR3 variant according to any one of claims 1 to 17, wherein the DcR3 variant comprises an amino acid sequence set forth in SEQ ID NO: 66 or 186.
  19. The DcR3 variant according to any one of claims 1 to 18, wherein the DcR3 variant comprises an Fc region derived from a human IgG1, IgG2 or IgG4 antibody, or a mutated Fc region consisting of an amino acid sequence obtained by introducing into an amino acid sequence of the abovementioned Fc region, deletion, substitution, insertion or addition of one or several amino acids.
  20. The DcR3 variant according to claim 19, wherein the Fc region or the mutated Fc region is bound to the C-terminal side of the first or second chimeric cysteine-rich region via another region or a linker.

Description

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a DcR3 variant as claimed, which is a variant of wild-type DcR3. More particularly, the present invention relates to a DcR3 variant as claimed that has binding activity (preferably neutralizing activity) to a ligand of DcR3, and that results in a decreased amount of aggregates as compared to wild-type DcR3 when produced using a cell derived from a mammal as a host, and/or that exhibits improved in vivo kinetics. Background Art A tumor necrosis factor (TNF) superfamily (TNFSF) and a TNF receptor superfamily (TNFRSF) each form 18 ligands and 29 receptor families having similar structures. Antibodies and Fc fusion proteins against many molecules included in these families have been developed and launched, and have exhibited therapeutic effects in treatment of various autoimmune diseases (Non-Patent Document 1). Although many TNFRSFs are expressed on a cell membrane and transmit a signal downstream by ligand binding, some molecules are decoy receptors (DcRs) not involved in signaling. As the decoy receptors, four types of DcR1, DcR2, DcR3 and osteoprotegerin (OPG) have been identified. OPG is a soluble decoy receptor for RANKL and TRAIL, and inhibits signaling by competing with the binding to ligands of a RANKL receptor and a TRAIL receptor. Meanwhile, DcR1 and DcR2 are decoy receptors for TRAIL, and DcR3 is a decoy receptor for three molecules of LIGHT, TL1A and FasL, and all of the decoy receptors neutralize ligands by competitively inhibiting the binding of the ligands to a signaling receptor (Non-Patent Document 2). DcR3 is a soluble molecule consisting of 300 amino acid residues. The N-terminal side has a signal peptide, followed by four cysteine-rich domains (CRDs) (CRD1, CRD2, CRD3 and CRD4), which are characteristic of TNFRSF, and the C-terminal side has a heparan sulfate-binding domain (HBD), which includes a heparan sulfate-binding motif and is rich in basic amino acids. All of LIGHT, TL1A and FasL bind via CRD2 and CRD3 of DcR3 (Non-Patent Documents 3, 4 and 5). DcR3 has, in addition to a function as a decoy receptor by ligand neutralization, a function as an immunomodulatory molecule based on the activity of HBD. For example, it has been reported that DcR3 directly binds via HBD to glycosaminoglycan (GAG) including heparan sulfate on the cell membrane of monocytes, macrophages or dendritic cells, thus initiating various immunosuppressive and immunostimulatory effects such as Th2 induction by differentiation of dendritic cells, induction of M2 macrophages, enhancement of adhesion of monocytes, osteoclast differentiation, or decreased expression of MHC class II molecules (Non-Patent Documents 6 and 12). DcR3 ligands have been reported to be involved in autoimmune diseases, inflammatory diseases, allergy, cancer, infection or other various inflammation reactions. For example, all of LIGHT, TL1A and FasL are included in the susceptibility locus in inflammatory bowel disease (IBD), and particularly, regarding TL1A, existence of a plurality of gene polymorphisms associated with pathological conditions has been known. There have also been reports of increased expression of DcR3 ligands in the blood or tissue of IBD patients, and improvement in pathological conditions by inhibition of DcR3 ligands in a mouse enteritis model (Non-Patent Documents 6 to 9). Although expression of DcR3 in human normal tissues is at an extremely low level, the expression is induced by infection or tissue damage. Furthermore, it has been known that the level of DcR3 in blood is increased in various autoimmune diseases or inflammatory diseases such as IBD, systemic lupus erythematosus (SLE), atopic dermatitis (AD) or rheumatoid arthritis (RA). Although no DcR3 homolog has been identified in mice, improvement in pathological conditions in human DcR3 transgenic mice and drug efficacy by administration of plasmids or recombinant DcR3 have been confirmed in mouse disease models such as a type I diabetes mellitus model, a multiple sclerosis model or a nephritis model (Non-Patent Documents 6 and 10). Genentech Inc. cloned the human DcR3 gene, and showed that a fusion of DcR3 and the Fc region of human IgG1 binds to soluble human FasL and inhibits human FasL-dependent apoptosis in vitro (Patent Document 1). Eli Lilly and Company obtained FLINT corresponding to a protease-resistant DcR3 mutant obtained by introducing one-amino-acid mutation (R218Q) into wild-type DcR3, and reported that the in vivo kinetics thereof is more improved in mice and monkeys than that of wild-type DcR3. However, the half-lives in blood when wild-type DcR3 and FLINT were administered at 0.5 mg/kg by intravenous injection to cynomolgus monkeys were extremely short, with values of 9 hours or 12.3 hours, respectively (Patent Documents 2 and 3, Non-Patent Document 11). Prior Art Document [Patent Document] Patent Document 1: JP 4303883 B2Patent Document 2: US 6,835,814 B1Patent documen