Polypeptide having sialylated sugar chains attached thereto

A sialic acid sugar chain and sialylation technology, which is applied in the field of polypeptides with additional sialylated sugar chains, can solve problems such as inhomogeneity of sugar chains, and achieve the effects of improving antitumor activity, stable quality, and high blood retention

Active Publication Date: 2015-02-04
GLYTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it is known that when expressed in CHO cells, heterogeneity of sugar chains generally occurs

Method used

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  • Polypeptide having sialylated sugar chains attached thereto
  • Polypeptide having sialylated sugar chains attached thereto
  • Polypeptide having sialylated sugar chains attached thereto

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0277] (Embodiment 1) Synthesis of Thioester Fragment

Embodiment 1-1

[0278] (Example 1-1) Synthesis of IFN 1-78 (S1Thi-C30Acm-Q48C) Ethan (SEQ ID NO: 3)

[0279]Amino-PEGA resin (manufactured by Merck) (50 μmol) was added to the column for solid-phase synthesis, and 4-hydroxymethyl-3-methoxyphenoxybutyric acid (HMPB) (125 μmol), O-benzene Triazol-1-yl-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (125 μmol) and N-ethylmorpholine (125 μmol) were dissolved in dimethylformamide ( DMF) (1.25ml), stirred at room temperature for 4 hours. The resin was fully washed with DMF and dichloromethane (DCM). Then Fmoc-Trp(Boc)-OH (0.25mmol), 1-(mesysyl)-3-nitro-1,2,4-triazole (MSNT) (0.25mmol) and N-methyl Imidazole (0.187 mmol) was dissolved in DCM (1.25 ml), added to a column for solid-phase synthesis, and stirred for 4 hours.

[0280] The resin was washed with DCM and DMF, and the Fmoc group was treated with 20% piperidine / DMF solution (2 ml) for 15 minutes for deprotection. After washing with DMF, the peptide chain was elongated, and amino acids...

Embodiment 1-2

[0283] (Example 1-2) Synthesis of IFN 1-78 (S1Thi-C30Acm-Q48C-S75C) MESNA (SEQ ID NO: 4)

[0284] Amino-PEGA resin (manufactured by Merck) (50 μmol) was added to a column for solid-phase synthesis, and 3-Fmoc-4-diaminobenzoic acid (150 μmol), 1-[bis(dimethylamino)methylene Base]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) (150 μmol) and diisopropylethylamine (300 μmol) were dissolved in DMF (1.25ml), and in Stir at room temperature for 2 hours.

[0285] After stirring, the resin was washed with DMF, treated with 20% piperidine / DMF solution (2 ml) for 15 minutes, and the protective Fmoc group was removed, and then the resin was fully washed with DMF. In the subsequent elongation of the peptide chain, amino acids are sequentially condensed using the method shown below.

[0286] The amino acid protected with Fmoc or Boc group was dissolved in DMF, and this solution was added to a solid-phase synthesis column (0.25 mmol). Add 0.2 M 1-[bis(dimethylamino)methyl...

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Abstract

[Problem] To provide a polypeptide which has highly homogeneous sialylated sugar chains attached thereto and has an interferon β activity. [Solution] A sugar chain-containing polypeptide that is selected from the group consisting of (1) to (4): (1) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO:1; (2) a polypeptide which comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 by deletion, substitution or addition of one to several amino acids; (3) a polypeptide that is an analog of interferon β; and (4) a polypeptide that has an 80% or higher homology with the polypeptide comprising the amino acid sequence represented by SEQ ID NO:1, wherein amino acids at 4-6 positions are substituted with amino acids having sugar chains attached thereto, and all of non-reducing ends of these sugar chains are sialylated.

Description

technical field [0001] The present invention relates to polypeptides with added sialylated sugar chains. More specifically, it relates to a polypeptide having a highly uniform sialylated sugar chain added thereto and having interferon β activity. Background technique [0002] Natural human interferon beta (IFN-beta) is a glycoprotein composed of 166 amino acid residues. It is known that interferon β belongs to the cytokine family and is involved in immunoregulatory action, antiviral activity, and cell proliferation inhibitory action. In addition, human interferon β has three Cys at the 17th, 31st, and 141st positions in the natural amino acid sequence, and one complex N-linked oligosaccharide at the 80th Asn. In addition, it is known that there are disulfide bonds at the 31st and 141st Cys. [0003] Interferon β as a drug is produced using a cell expression system, and is classified as IFN-β-1a or IFN-β-1b depending on the host in which it is expressed. IFN-β-1a is expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/565A61K38/00A61P1/16A61P25/00A61P31/14A61P35/00
CPCA61K38/00C07K14/565A61P1/16A61P17/00A61P25/00A61P31/14A61P35/00
Inventor 大内政辉西原三佳手塚克成前田政敏梶原康宏坂本泉
Owner GLYTECH
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