Cyclized cytokine and method for producing same

a cytokine and cyclization technology, applied in the direction of peptide/protein ingredients, drug compositions, extracellular fluid disorder, etc., can solve the problems of inconvenient patient care, inconvenient medication compliance, and undesirable metabolic half-life of bioactive proteins, and achieve high cyclization efficiency, increase thermostability and protease resistance, and small number of amino acids added

Inactive Publication Date: 2018-05-03
NAT INST OF ADVANCED IND SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0057]In the present invention, specifically developed is G-CSF(C177), a cyclized protein composed of an amino acid sequence in which only 2 amino acids are added to a linear control G-CSF. In addition, G-CSF(C163) is a cyclized protein composed of an amino acid sequence in which nonstructural regions at the N-terminus and the C-terminus are deleted and only 2 amino acids are added. Further, G-CSF(C166) or G-CSF(C170) is a cyclized protein composed of an amino acid sequence in which nonstructural regions at the N-terminus and the C-terminus are deleted and 5 or 9 amino acid residues, respectively, are added.
[0058]When compared with commercially available filgrastim and the linear control G-CSF, any of them can maintain intrinsic receptor-binding activity and have increased thermostability and protease resistance. Among them, G-CSF(C163), G-CSF(C166), and G-CSF(C170) were able to achieve high cyclization efficiency while the number of amino acids added is small.
[0059]Currently, G-CSF has been widely used in clinical practice including treatment of neutropenia caused by cancer chemotherapy. Development of biobetter drugs is in progress so as to enhance stability by using a variety of strategies such as a mutant G-CSF and a chemically-modified G-CSF. Among the strategies, a polypeptide chain cyclization technique aims at increasing stability while change in the amino acid sequence and change in the conformation of a protein can be minimized. An undesirable characteristic change such as an increase in immunogenicity can also be minimized. Thus, the technique seems to have a substantial advantage in the production of bioactive proteins for treatment of humans.
[0060]According to the present invention, it is possible to reduce, to minimum, addition of a sequence necessary for a cyclization reaction, which addition is the biggest disadvantage in increasing stability by cyclization in view of the above-described situations. Further, the present invention can resolve the problems of low cyclization reaction efficiency and a complicated purification step, thereby significantly contributing to technical advances in the art.

Problems solved by technology

When administered for treatment of humans, a bioactive protein often has an undesirable metabolic half-life.
This intrinsic metabolic half-life frequently causes suboptimal therapeutic efficacy, medication compliance problems, and dosing schedule and administration regimen that are inconvenient for patients.
Unfortunately, most such approaches are expensive and entail additional time-consuming processes during production.
In addition, PEG itself is immunogenic, so that anti-PEG antibodies are produced, resulting in a decrease in drug efficiency of a PEGylated protein and a decrease in safety, as disclosed in Non-Patent Literature 3.
In addition, when reaction efficiency of the cyclization reaction is insufficient, an additional purification step of separating the resulting cyclized protein from an unreacted linear protein is needed.

Method used

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  • Cyclized cytokine and method for producing same
  • Cyclized cytokine and method for producing same
  • Cyclized cytokine and method for producing same

Examples

Experimental program
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Effect test

example 1

To Design Improved G-CSFs

[0148](1) As used herein, a linear control G-CSF, which is a starting material for improvement, is defined as follows. Specifically, the linear control G-CSF is a linear polypeptide consisting of 175 amino acids set forth in SEQ ID NO: 1. When the amino acid sequence of the linear control G-CSF is compared with the amino acid sequence (SEQ ID NO: 14) of filgrastim, threonine at position 2 and cysteine at position 18 are replaced by alanine and serine, respectively. A previous report has demonstrated that these mutations do not affect the activity of G-CSF (Reference Document 3).

[0149](2) In order to make a polypeptide of interest cyclic by using the catalytic function of inteins, a residue of at least one of the N-terminus and the C-terminus should be serine or cysteine. In addition, if the residue at the C-terminus is proline, it is known that the reaction fails to proceed. Here, as a mutant modified so as to make the inteins reaction proceed by introducing...

example 2

To Synthesize Linear Control G-CSF-Expression Plasmid

[0154](1) A chemically synthesized nucleic acid sequence (SEQ ID NO: 25) encoding the amino acid sequence (SEQ ID NO: 1) of the linear control G-CSF was purchased.

[0155](2) As the chemically synthesized nucleic acid as a template, PCR amplification was carried out using two primers (SEQ ID NOs: 26 and 27). The resulting PCR product was purified and digested by restriction enzymes NcoI and BamHI. Next, an E. coli vector pET16b was digested by restriction enzymes NcoI and BamHI and a band at or near 5600 bp was excised and purified. The band was dephosphorylated using_E. coli alkaline phosphatase. The two samples purified were ligated using T7 DNA ligase.

[0156](3) E. coli DH5a was transformed with the resulting ligation product of (2) and was cultured on an LB plate medium containing 100 μg / m1 of ampicillin. The resulting transformants were subjected to colony PCR and DNA sequencing (GE Healthcare Bioscience, BigDye Terminator v3.1)...

example 3

To Construct Inteins Vector

[0157](1) A synthetic gene was purchased having a nucleotide sequence (SEQ ID NO: 28) encoding the inteins sequence (a sequence containing, in sequence, SEQ ID NO: 15 and SEQ ID NO: 16) derived from Nostoc punctiforme.

[0158](2) The synthetic gene of (1) was mixed with restriction enzymes NcoI and XhoI and was subjected to a cleavage reaction at 37° C. for 4 h. After gel electrophoresis, a band at or near 450 bp was excised and purified. Next, an E. coli vector pET16b was digested by NcoI and XhoI and a band at or near 5600 bp was excised and purified. The band was dephosphorylated using E. coli alkaline phosphatase. The two samples purified were ligated using T7 DNA ligase.

[0159](3) E. coli DH5a was transformed with the resulting ligation product and was cultured on an LB plate medium containing 100 μg / m1 of ampicillin. The resulting transformants were subjected to colony PCR and DNA sequencing (GE Healthcare Bioscience, BigDye Terminator v3.1) and were t...

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Abstract

The purpose of the present invention is to provide a method for producing a very stable, cyclized mutant protein such that high cyclization efficiency is achieved while the number of amino acids added is minimal and the biological properties of an original protein are maintained. In view of conformational information about the original protein, secondary structure-free regions at N/C terminal portions are deleted. Then, a protein database is screened for proteins with secondary structures similar to those of N/C terminal residues of a secondary structure-forming portion after the deletion. The screening results are used to determine the amino acid length of a loop structure through which the N-terminus and the C-terminus of the secondary structure-forming portion of the original protein are to be connected. A cyclized mutant protein is finally produced having a loop structure with the determined amino acid length.

Description

TECHNICAL FIELD[0001]The present invention relates to novel improved versions of a pharmaceutically bioactive protein, preferably a cytokine with a helix bundle conformation, and more preferably granulocyte colony-stimulating factor (G-CSF), which acts to promote production of granulocytes and to enhance functions of neutrophils, nucleic acids encoding the improved proteins, and a production method therefor.BACKGROUND ART(Protein Instability Problem When Used as Pharmaceutical)[0002]When administered for treatment of humans, a bioactive protein often has an undesirable metabolic half-life. This intrinsic metabolic half-life frequently causes suboptimal therapeutic efficacy, medication compliance problems, and dosing schedule and administration regimen that are inconvenient for patients.(Conventional Major Stability-improving Strategies and Their Problems)[0003]In the production of a bioactive protein for treatment of humans, its metabolic half-life has been extended by physical mean...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/535A61P7/00C12N15/70
CPCC07K14/535A61P7/00C12N15/70A61K38/00C12N2840/445C07K1/02C12P21/02C12N5/10A61K38/22C07K19/00
Inventor HONDA, SHINYAMIYAFUSA, TAKAMITSU
Owner NAT INST OF ADVANCED IND SCI & TECH
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