WO2015199154A1 - IMPROVED Fc-BINDING PROTEIN, METHOD FOR PRODUCING SAID PROTEIN, ANTIBODY ADSORBENT USING SAID PROTEIN, AND METHOD FOR SEPARATING ANTIBODY USING SAID ADSORBENT - Google Patents

IMPROVED Fc-BINDING PROTEIN, METHOD FOR PRODUCING SAID PROTEIN, ANTIBODY ADSORBENT USING SAID PROTEIN, AND METHOD FOR SEPARATING ANTIBODY USING SAID ADSORBENT Download PDF

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WO2015199154A1
WO2015199154A1 PCT/JP2015/068259 JP2015068259W WO2015199154A1 WO 2015199154 A1 WO2015199154 A1 WO 2015199154A1 JP 2015068259 W JP2015068259 W JP 2015068259W WO 2015199154 A1 WO2015199154 A1 WO 2015199154A1
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seq
replaced
binding protein
amino acid
antibody
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PCT/JP2015/068259
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French (fr)
Japanese (ja)
Inventor
朝岡義晴
田中亨
寺尾陽介
山中直紀
木津奈都子
青木大
井出輝彦
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東ソー株式会社
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Priority claimed from JP2014147207A external-priority patent/JP6451119B2/en
Priority claimed from JP2014147206A external-priority patent/JP6451118B2/en
Priority claimed from JP2015115078A external-priority patent/JP6699096B2/en
Application filed by 東ソー株式会社 filed Critical 東ソー株式会社
Priority to CN201580045734.4A priority Critical patent/CN106574261B/en
Priority to EP15811514.7A priority patent/EP3162895B1/en
Priority to US15/321,916 priority patent/US10815289B2/en
Publication of WO2015199154A1 publication Critical patent/WO2015199154A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to an Fc binding protein having affinity for immunoglobulin. More specifically, the present invention relates to an Fc-binding protein having higher stability against heat and acid than wild type, a method for producing the protein, an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier, and the adsorbent. The present invention relates to a method for separating an antibody using.
  • Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Individual molecules recognize a single or the same group of immunoglobulin isotypes by a recognition domain on the Fc receptor by a recognition domain belonging to the immunoglobulin superfamily. This determines which accessory cells are driven in the immune response. Fc receptors can be further classified into several subtypes, such as Fc ⁇ receptors that are receptors for IgG (immunoglobulin G), Fc ⁇ receptors that bind to the Fc region of IgE, Fc ⁇ receptors that bind to the Fc region of IgA, etc. There is. Each receptor is further classified, and Fc ⁇ receptors have been reported to include Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, and Fc ⁇ RIIIb (Non-patent Document 1).
  • Fc ⁇ RIIIa is present on the surface of natural killer cells (NK cells) and macrophages and is an important receptor involved in ADCC (antibody-dependent cellular cytotoxicity) activity, which is important in the human immune system. It is.
  • the Fc ⁇ RIIIa and affinity for human IgG has been reported that binding constant indicates the strength of the bond (KA) is 10 7 M -1 or less (Non-Patent Document 2).
  • the amino acid sequence of human Fc ⁇ RIIIa (SEQ ID NO: 1) is published in public databases such as UniProt (Accession number: P08637).
  • FIG. 1 shows a schematic diagram of the structure of human Fc ⁇ RIIIa.
  • the number in FIG. 1 has shown the amino acid number, and the number respond
  • antibody drugs drugs containing antibodies
  • the antibody used for the antibody drug is purified to high purity using column chromatography after culturing cells capable of expressing the antibody (for example, Chinese hamster ovary (CHO) cells) obtained by genetic engineering techniques.
  • cells capable of expressing the antibody for example, Chinese hamster ovary (CHO) cells
  • CHO Chinese hamster ovary
  • Non-Patent Document 3 As a method for analyzing the molecular structure of an antibody used for an antibody drug, conventionally, LC-MS analysis (Non-Patent Document 3) including peptide mapping, analysis by two-dimensional electrophoresis, and glycan excision has been performed.
  • both methods involve very complicated operations.
  • chromatographic analysis can be mentioned. Specifically, it is possible to separate and quantify aggregates and degradation products by separating antibodies based on molecular weight using gel filtration chromatography. In addition, the difference in charge of antibody molecules can be separated by ion exchange chromatography.
  • the above-described chromatographic analysis has limited results because the minute structural changes of antibody molecules cannot be identified.
  • Patent Document 1 and Non-Patent Document 4 analysis by affinity chromatography among chromatographies can be performed based on the affinity between an affinity ligand immobilized on an insoluble carrier and an antibody. Therefore, minute structural changes of the antibody molecule can be identified (Patent Document 1 and Non-Patent Document 4).
  • Patent Document 1 and Non-Patent Document 4 it is practically difficult to separate antibody molecules on an industrial scale using the methods described in Patent Document 1 and Non-Patent Document 4, and improvements have been desired.
  • ADCC antibody-dependent cytotoxicity
  • ADCC activity In antibody medicine, the strength of ADCC activity possessed by an antibody is important.
  • antibody drugs are usually produced using genetic recombination techniques using animal cells as hosts, and because glycosylation in the host cannot be controlled, antibodies with a certain ADCC activity are expressed. It is difficult. In addition, it takes a lot of time and effort to separate antibodies from the expressed antibodies based on the strength of ADCC activity.
  • An object of the present invention is to provide an Fc-binding protein having improved stability to heat and acid, a method for producing the protein, and an antibody adsorbent using the protein.
  • Another object of the present invention is to provide a method for separating antibodies in a simple and highly efficient manner based on the difference in molecular structure in a method for separating antibodies using a carrier on which an affinity ligand is immobilized. is there.
  • Still another object of the present invention is to provide a method for separating antibodies based on the strength of antibody-dependent cytotoxic activity.
  • the present inventors have identified an amino acid residue involved in stability improvement in human Fc ⁇ RIIIa, and a mutant in which the amino acid residue is substituted with another amino acid residue, It has been found that it has excellent stability against heat and acid, and the present invention has been completed.
  • the present inventors add a certain concentration of chloride ion or sulfate ion to the equilibration solution of the column packed with the insoluble carrier on which the affinity ligand is immobilized. As a result, it was found that the resolution of the antibody was improved, and the present invention was completed.
  • the present inventors have used antibody-dependent cell cytotoxicity (ADCC) by using an adsorbent obtained by immobilizing an Fc-binding protein on an insoluble carrier.
  • ADCC antibody-dependent cell cytotoxicity
  • the amino acid sequence of SEQ ID NO: 37 comprises the amino acid residues from the 33rd to the 208th, and the amino acid residues from the 33rd to the 208th of the following (1) to (84) An Fc binding protein wherein at least one amino acid substitution has occurred.
  • the 117th glutamine in SEQ ID NO: 37 is replaced with leucine (44)
  • the 119th glutamic acid in SEQ ID NO: 37 is replaced with valine (45) 121 of SEQ ID NO: 37
  • the histidine is replaced with arginine (46)
  • the 130th proline of SEQ ID NO: 37 is replaced with leucine (47)
  • the 135th lysine of SEQ ID NO: 37 is replaced with tyrosine (48)
  • the 136th glutamic acid of SEQ ID NO: 37 is valine (49)
  • the 141st histidine of SEQ ID NO: 37 is replaced with glutamine (50)
  • the 146th serine of SEQ ID NO: 37 is replaced with threonine (51)
  • the 154th lysine of SEQ ID NO: 37 is replaced with arginine (52)
  • the 159th glutamine of SEQ ID NO: 37 was replaced with histidine (53)
  • SEQ ID NO: 3 The 163rd
  • (M) A polynucleotide encoding the Fc-binding protein according to any one of (A) to (D).
  • (R) a step of equilibrating the column by adding an equilibration solution to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized; and adding a solution containing an antibody to the equilibrated column,
  • An antibody separation method comprising a step of adsorbing on the carrier and a step of eluting the antibody adsorbed on the carrier using an eluent, wherein the equilibration solution contains chloride ions or sulfate ions of 30 mM or more. Including the separation method.
  • the Fc-binding protein of the present invention is a protein having a binding property to the Fc region of an antibody, and at least of the extracellular region (EC region of FIG. 1) of human Fc ⁇ RIIIa comprising the amino acid sequence set forth in SEQ ID NO: 1.
  • the extracellular region (C in FIG. 1) and the extracellular region (C in FIG. 1) may be included.
  • the amino acid substitution at the specific position is, in the amino acid sequence shown in SEQ ID NO: 1, Val27Glu (in this notation, the 27th valine of SEQ ID NO: 1 (43th in SEQ ID NO: 37) is substituted with glutamic acid.
  • mutants in which any one or more amino acid substitutions are known among Leu66His, Leu66Arg, Gly147Asp, Tyr158His, and Val176Phe are known.
  • An amino acid substitution may be included.
  • the amino acid residue at a specific position may be substituted with an amino acid other than those described above as long as it has antibody binding activity.
  • One example is a conservative substitution that substitutes between amino acids whose physical and / or chemical properties of both amino acids are similar. Conservative substitutions are not limited to Fc-binding proteins, and are generally known to those skilled in the art to maintain protein function between those with substitutions and those without substitutions. Examples of conservative substitutions include substitutions that occur between glycine and alanine, between aspartic acid and glutamic acid, between serine and proline, or between glutamic acid and alanine (protein structure and function, Medical Science International, 9 2005).
  • the number of amino acids to be substituted is not particularly limited.
  • Fc-binding proteins shown in the following (a) to (l) can be mentioned. These Fc-binding proteins are preferable in terms of improving stability against heat, acid or alkali.
  • A In the amino acid sequence shown in SEQ ID NO: 37, the amino acid residues from the 33rd to the 208th amino acid residues are included, and the 45th phenylalanine is the isoleucine in the 33rd to 208th amino acid residues.
  • Fc binding protein (Fc binding protein containing the amino acid sequence from the 33rd position to the 208th position in the amino acid sequence shown in SEQ ID NO: 39) wherein (B) including the 33rd to 208th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 37, and the 45th phenylalanine is the isoleucine in the 33rd to 208th amino acid residues.
  • Fc-binding protein in which valine is substituted with glutamic acid and 187th phenylalanine is substituted with serine (Fc-binding protein containing the amino acid sequence from the 33rd to the 208th of the amino acid sequence described in SEQ ID NO: 43).
  • the amino acid sequence of SEQ ID NO: 37 comprises the amino acid residues from the 33rd to the 208th, and the 37th glutamic acid is the glycine in the 33rd to 208th amino acid residues, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 133th valine Is a glutamic acid, 163rd glycine is replaced by valine, 187th phenylalanine is replaced by serine, and 194th serine is replaced by arginine (from the 33rd amino acid sequence of SEQ ID NO: 73) Amino acid sequence up to 208th Fc-binding protein that contains).
  • the amino acid sequence shown in SEQ ID NO: 37 includes the 33rd to 208th amino acid residues, and in the 33rd to 208th amino acid residues, the 37th glutamic acid is glycine, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 133th valine Is glutamic acid, 156th threonine is isoleucine, 163rd glycine is valine, 174th tyrosine is histidine, 181st lysine is glutamic acid, 187th phenylalanine is serine, 194th serine is arginine In each Has been that Fc binding proteins (Fc-binding protein comprising an amino acid sequence from the 33 th of the amino acid sequence
  • (K) includes the 33rd to 208th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 37, and the 37th glutamic acid is the glycine in the 33rd to 208th amino acid residues, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 98th asparagine Acid is glutamic acid, 117th glutamine is leucine, 133th valine is glutamic acid, 156th threonine is isoleucine, 163rd glycine is valine, 174th tyrosine is histidine, 181st lysine is In glutamic acid, 18 Fc binding protein in which the phenylalanine at position No.
  • the 37th glutamic acid is the glycine
  • the 39th Leucine is methionine
  • 45th phenylalanine is isoleucine
  • 64th glutamine is arginine
  • 67th tyrosine is histidine
  • 70th glutamic acid is aspartic acid
  • 84th serine is proline
  • 98th aspartic acid is glutamic acid
  • 117th glutamine is leucine
  • 133th valine is glutamic acid
  • 156th threonine is isoleucine
  • 163rd glycine is valine
  • 174th tyrosine is 181st in histidine Fc-binding protein in which lysine is substituted with glutamic acid, 187th phenylalanine is substituted with serine, 194th serine with arginine, 201st threonine with alanine, and
  • the Fc-binding protein of the present invention may further be added with an oligopeptide useful for separation from a solution in the presence of a contaminant substance on the N-terminal side or C-terminal side.
  • the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid and the like.
  • an oligopeptide containing cysteine useful for immobilizing the Fc-binding protein of the present invention on a solid phase such as a support for chromatography is used as an N-terminal side or C-terminal side of the Fc-binding protein of the present invention. It may be further added to.
  • the length of the oligopeptide added to the N-terminal side or C-terminal side of the Fc binding protein is not particularly limited as long as the IgG binding property and stability of the Fc binding protein of the present invention are not impaired.
  • a polynucleotide encoding the oligopeptide is prepared and then genetically engineered using a method well known to those skilled in the art to the N-terminal side of the Fc-binding protein.
  • the chemically synthesized oligopeptide may be added by chemically binding to the N-terminal side or C-terminal side of the Fc-binding protein of the present invention.
  • a signal peptide for promoting efficient expression in the host may be added to the N-terminal side of the Fc binding protein of the present invention.
  • the signal peptide when the host is Escherichia coli include periplasm such as PelB (SEQ ID NO: 101), DsbA, MalE (the first to 26th region of the amino acid sequence described in UniProt No. P0AEX9), TorT, and the like.
  • An example is a signal peptide that secretes a protein (Japanese Patent Laid-Open No. 2011-097898).
  • the Fc binding protein of the present invention may or may not have a sugar chain.
  • animal cells, yeasts, insect cells and the like may be used as hosts.
  • an artificially synthesized sugar chain may be modified.
  • an Fc-binding protein having no sugar chain it may be used as a host in which sugar chain addition does not occur, such as Escherichia coli. Further, by performing an operation of removing a sugar chain from an Fc binding protein having a sugar chain, an Fc binding protein having no sugar chain can be obtained.
  • a method for producing the polynucleotide of the present invention (I) a method of artificially synthesizing a polynucleotide containing the nucleotide sequence by converting the amino acid sequence of the Fc-binding protein of the present invention into a nucleotide sequence, (II) A polynucleotide containing the whole or a partial sequence of an Fc binding protein is prepared artificially or from a cDNA of the Fc binding protein using a DNA amplification method such as PCR, and the prepared polynucleotide is appropriately used.
  • the method of connecting by various methods can be illustrated.
  • the conversion when converting from an amino acid sequence to a nucleotide sequence, the conversion is preferably performed in consideration of the frequency of codon usage in the host to be transformed.
  • the host is Escherichia coli
  • AGA / AGG / CGG / CGA is used for arginine (Arg)
  • ATA is used for isoleucine (Ile)
  • CTA is used for leucine (Leu)
  • GGA is used for glycine (Gly).
  • CCC is less frequently used in proline (Pro) (because it is a so-called rare codon)
  • it may be converted so as to avoid those codons.
  • Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute).
  • an error-prone PCR method can be used.
  • the reaction conditions in the error-prone PCR method are not particularly limited as long as a desired mutation can be introduced into a polynucleotide encoding an Fc binding protein.
  • four types of deoxynucleotides dATP / dTTP / dCTP
  • / DGTP deoxynucleotides
  • MnCl 2 is added to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to introduce a mutation into the polynucleotide. be able to.
  • the polynucleotide containing the entire or partial sequence of the Fc-binding protein is contacted / acted with a drug as a mutagen, or irradiated with ultraviolet rays, to obtain a polynucleotide.
  • a method of producing a mutation by introducing a mutation As a drug used as a mutagen in this method, a mutagenic drug commonly used by those skilled in the art such as hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfite, hydrazine may be used. .
  • the host that expresses the Fc-binding protein of the present invention is not particularly limited, and examples thereof include animal cells (CHO cells, HEK cells, Hela cells, COS cells, etc.), yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schomisacios). and japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, insect cells (Sf9, Sf21 etc.), E. coli (JM109 strain, BL21 (DE3) strain, W3110 strain etc.) and Bacillus subtilis.
  • Use of animal cells or Escherichia coli as a host is preferable in terms of productivity, and more preferable when Escherichia coli is used as a host.
  • the polynucleotide of the present invention When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but an expression vector (for example, bacteriophage, cosmid, or the like commonly used for transformation of prokaryotic cells or eukaryotic cells) may be used. It is more preferable to use a plasmid or the like in which the polynucleotide of the present invention is inserted at an appropriate position.
  • the expression vector is not particularly limited as long as it is stably present in the host to be transformed and can be replicated.
  • pET plasmid vector When Escherichia coli is used as a host, pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector A pBBR plasmid vector can be exemplified.
  • the appropriate position means a position where the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility are not destroyed.
  • promoter examples include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, ⁇ phage ⁇ PL promoter, ⁇ PR promoter when the host is Escherichia coli.
  • examples include SV40 promoter, CMV promoter, and CAG promoter.
  • the expression vector of the present invention In order to transform a host using the expression vector inserted with the polynucleotide of the present invention (hereinafter referred to as the expression vector of the present invention) produced by the above-described method, a person skilled in the art may carry out the method. For example, when a microorganism belonging to the genus Escherichia (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) is selected as a host, known literature (eg, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). And the like. When the host is an animal cell, electroporation or lipofection may be used. By transforming the transformant obtained by the above-described method with an appropriate method, a transformant capable of expressing the Fc-binding protein of the present invention (hereinafter referred to as the transformant of the present invention). ) Can be obtained.
  • the expression vector of the present invention may be extracted from the transformant of the present invention and prepared by a method suitable for the host used for transformation.
  • the host of the transformant of the present invention is Escherichia coli
  • it can be prepared from a culture obtained by culturing the transformant using an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (Qiagen). That's fine.
  • the Fc-binding protein of the present invention can be produced by culturing the transformant of the present invention and recovering the Fc-binding protein of the present invention from the obtained culture.
  • the culture includes not only the cultured cells of the transformant of the present invention itself but also a medium used for the culture.
  • the transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host.
  • an LB (Luria-Bertani) medium supplemented with necessary nutrient sources is preferable.
  • An example of the medium is given.
  • a drug corresponding to the drug resistance gene contained in the vector to the medium and culture.
  • kanamycin may be added to the medium.
  • an appropriate nutrient source may be added to the medium, and if desired, the medium is selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol.
  • One or more reducing agents may be included.
  • a reagent that promotes protein secretion from the transformant to the culture solution such as glycine may be added.
  • glycine is contained in the medium at 2% (w / v) or less. Addition is preferred.
  • the culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., more preferably around 25 ° C., but may be selected depending on the characteristics of the protein to be expressed.
  • the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0.
  • an inducible promoter when included in the vector of the present invention, it is preferable that the induction be performed under conditions that allow the Fc-binding protein of the present invention to be expressed well.
  • the inducer include IPTG (isopropyl- ⁇ -D-thiogalactopyranoside).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside.
  • the Fc-binding protein of the present invention may be recovered by separating / purifying from the culture. For example, when expressed in the culture supernatant, the cells are separated by centrifugation, and the Fc-binding protein of the present invention may be purified from the obtained culture supernatant.
  • the bacterial cells are collected by centrifugation, and then added with an enzyme treatment agent, a surfactant, etc., or by using ultrasonic waves, a French press, etc. What is necessary is just to refine
  • a method known in the art may be used, and an example is separation / purification using liquid chromatography.
  • Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. By performing a purification operation by combining these chromatography, the Fc binding property of the present invention can be obtained. Proteins can be prepared with high purity.
  • the binding activity to IgG is measured using the Enzyme-Linked ImmunoSorbent Assay (hereinafter referred to as ELISA) method or the surface plasmon resonance method.
  • ELISA Enzyme-Linked ImmunoSorbent Assay
  • the IgG used for the measurement of the binding activity is preferably human IgG, and human IgG1 and human IgG3 are particularly preferable.
  • the adsorbent of the present invention can be produced by binding the Fc binding protein of the present invention to an insoluble carrier.
  • the insoluble carrier is not particularly limited, and carriers made from polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, starch, polyvinyl alcohol, polymethacrylate, poly (2- Examples include carriers made of synthetic polymers such as hydroxyethyl methacrylate), polyurethane, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, and vinyl polymers, and carriers made of ceramics such as zirconia, zeolite, silica, and coated silica. it can.
  • insoluble carriers carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers.
  • the preferred carrier include polymethacrylate gel introduced with a hydroxyl group such as Toyopearl (manufactured by Tosoh Corporation), agarose gel such as Sepharose (manufactured by GE Healthcare), and cellulose gel such as Cellufine (manufactured by JNC).
  • the shape of the insoluble carrier is not particularly limited, and may be granular or non-particulate, porous or non-porous.
  • N-hydroxysuccinimide (NHS) activated ester group epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, halo What is necessary is just to fix
  • the carrier provided with the active group may be a commercially available carrier as it is, or may be prepared by introducing an active group on the surface of the carrier under appropriate reaction conditions.
  • examples of the method for introducing an active group on the surface of the carrier include a method in which one of compounds having two or more active sites reacts with a hydroxyl group, an epoxy group, a carboxyl group, an amino group, etc. present on the surface of the carrier. it can.
  • examples of the compound that introduces an epoxy group into the hydroxyl group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.
  • Examples of the compound that introduces an epoxy group on the carrier surface with the compound and then introduces a carboxyl group on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3- Examples thereof include aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.
  • Examples of the compound that introduces a maleimide group into the hydroxyl group, epoxy group, carboxyl group or amino group present on the surface of the carrier include N- ( ⁇ -maleimidocaproic acid) hydrazide, N- ( ⁇ -maleimidopropionic acid) hydrazide, 4- [ 4-N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [ ⁇ -maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N-hydroxys
  • Compounds that introduce a haloacetyl group into the hydroxyl group or amino group present on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated.
  • An example is a method in which ⁇ -alkenyl alkanglycidyl ether is reacted with a hydroxyl group or amino group present on the surface of the carrier, and then the ⁇ -alkenyl moiety is halogenated with a halogenating agent to activate.
  • ⁇ -alkenyl alkanglycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether.
  • halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.
  • the method for introducing an active group on the surface of the carrier there is a method for introducing an activating group into the carboxyl group present on the surface of the carrier using a condensing agent and an additive.
  • the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole.
  • the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.
  • examples of the compound that introduces an active group on the surface of the carrier include tresyl chloride (forms a tresyl group as an activating group) and vinyl bromide (forms a vinyl group as an activating group).
  • Examples of the buffer used when the Fc-binding protein of the present invention is immobilized on an insoluble carrier include acetate buffer, phosphate buffer, MES buffer, HEPES buffer, Tris buffer, and borate buffer.
  • the reaction temperature at the time of immobilization may be appropriately set in consideration of the reactivity of the active group and the stability of the Fc-binding protein of the present invention from the temperature range from 5 ° C. to 50 ° C., preferably It is in the range of 10 ° C to 35 ° C.
  • the separation method of the present invention comprises a step of equilibrating a column by adding an equilibration solution to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized, and a solution containing an antibody is added to the equilibrated column.
  • the step of adsorbing the antibody to the carrier and the step of eluting the antibody adsorbed to the carrier using an eluate the equilibration solution is 30 mM or more of chloride ions or sulfuric acid. It is characterized by containing ions.
  • the degree of separation of antibody components can be improved from 1.1 times to 1.8 times in terms of Rs value.
  • the concentration of chloride ion or sulfate ion contained in the equilibration solution may be 30 mM or more, preferably 30 mM or more and 1500 mM or less, more preferably 30 mM or more and 1000 mM or less, and 30 mM or more and 500 mM or less. And more preferably 50 mM or more and 500 mM or less.
  • the adsorbed antibody using the equilibration solution it may be eluted using an elution solution that weakens the affinity between the antibody and the Fc-binding protein.
  • an elution solution that weakens the affinity between the antibody and the Fc-binding protein.
  • a weakly acidic buffer solution having a pH of 5.0 to 6.9 containing 30 mM or more of chloride ions or sulfate ions was used as an equilibration solution, and an acidic buffer solution having a pH of 2.5 to 4.5 was used as an eluent.
  • a gradient elution method is mentioned.
  • the buffer may be appropriately selected from known buffers based on the pH of the buffer to be prepared.
  • buffer examples include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinosulfonic acid), and MOPS (3-Morpholinosulfonic sulfonic acid). acid), citric acid, succinic acid, glycine, and piperazine.
  • the separation method of the present invention can be separated if it is an antibody having at least the Fc region of an antibody to which a sugar chain is added and has an affinity for an Fc binding protein.
  • Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substitutions thereof that are generally used as antibodies used in antibody pharmaceuticals.
  • bispecific antibodies bispecific antibodies
  • fusion antibodies of Fc regions of antibodies with added sugar chains and other proteins complexes of Fc regions of drugs with added sugar chains and drugs (ADC), etc. Even an antibody whose structure has been artificially modified can be separated by the separation method of the present invention.
  • the separation method of the present invention includes, for example, adding a buffer solution containing an antibody to a column packed with an adsorbent obtained by immobilizing an Fc binding protein on an insoluble carrier using a liquid delivery means such as a pump. After the antibody is specifically adsorbed to the adsorbent, an appropriate eluate is added to the column, whereby the adsorbed antibody can be separated based on the strength of ADCC activity. Note that it is preferable to equilibrate the column with an appropriate buffer before adding the buffer containing the antibody to the column because the antibody can be separated with higher purity.
  • the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution.
  • the pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8.
  • the eluate for eluting the antibody adsorbed on the adsorbent based on the strength of ADCC activity is a buffer solution on the acidic side of the solution used when the antibody is adsorbed on the adsorbent.
  • the buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side.
  • the pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.
  • a column packed with the adsorbent of the present invention is loaded with sugar.
  • a buffer solution containing an antibody having a chain is added using a liquid delivery means such as a pump to specifically adsorb the antibody having a sugar chain to the adsorbent of the present invention, and then an appropriate eluate is added.
  • the antibody having a sugar chain may be eluted.
  • the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution.
  • the pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8.
  • the eluate for eluting the antibody having a sugar chain adsorbed to the adsorbent of the present invention it is more acidic than the solution used for adsorbing the antibody having a sugar chain to the adsorbent of the present invention.
  • Side buffer examples of the buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side.
  • the pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.
  • the elution position (elution fraction) of the antibody varies depending on the sugar chain structure of the antibody. Therefore, by separating the antibody using the adsorbent of the present invention, the difference in the sugar chain structure of the antibody can be identified.
  • the structure of the glycans There are no particular limitations on the structure of the glycans that can be identified.
  • the adsorbent of the present invention can be separated based on the difference in the sugar chain structure of the antibody, it can also be used for separation of the sugar chain itself.
  • the adsorbent of the present invention can identify antibody separation, antibody separation based on the strength of ADCC activity, and the difference in the sugar chain structure of the antibody. Even when the Fc receptors (Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa, Fc ⁇ RIIIb, FcRn) are used, the difference in the sugar chain structure can be similarly identified.
  • the Fc-binding protein of the present invention is a protein in which an amino acid residue at a specific position in the extracellular region of human Fc ⁇ RIIIa is substituted with another amino acid residue.
  • the Fc-binding protein of the present invention has improved heat and acid stability compared to wild-type human Fc ⁇ RIIIa. Therefore, the Fc binding protein of the present invention is useful as a ligand of an adsorbent for separating immunoglobulin.
  • the separation method of the present invention can easily and accurately separate an antibody based on a medicinal effect or a molecule containing an Fc region of an antibody using chromatography. Therefore, according to the present invention, manufacturing process management and quality control of antibody drugs can be performed with higher accuracy.
  • the separation method of the present invention uses an adsorbent obtained by immobilizing an Fc-binding protein (for example, human Fc ⁇ RIIIa to which a sugar chain is not added) on an insoluble carrier, so that the antibody is antibody-dependent cytotoxic activity (ADCC). ) Can be separated on the basis of strength.
  • an Fc-binding protein for example, human Fc ⁇ RIIIa to which a sugar chain is not added
  • ADCC antibody-dependent cytotoxic activity
  • FIG. 1 is a schematic diagram of human Fc ⁇ RIIIa.
  • the numbers in the figure indicate the amino acid sequence numbers described in SEQ ID NO: 1.
  • S represents a signal sequence
  • EC represents an extracellular region
  • TM represents a transmembrane region
  • C represents an intracellular region.
  • FrA and FrB in the figure indicate the positions of fraction A and fraction B, respectively.
  • N1 to N6 in the figure correspond to N1 to N6 in Table 10
  • M1, M2 and D1 correspond to M1, M2 and D1 in Table 11, respectively.
  • FrA, FrB, and FrC in the figure indicate the positions of fraction A, fraction B, and fraction C, respectively.
  • Example 1 Preparation of Fc-binding protein expression vector (1) Based on the amino acid sequence from the 17th glycine (Gly) to the 192nd glutamine (Gln) of the human Fc ⁇ RIIIa amino acid sequence described in SEQ ID NO: 1, Using the DNAworks method (Nucleic Acids Res., 30, e43, 2002), a nucleotide sequence in which a codon was converted from a human type to an E. coli type was designed. The designed nucleotide sequence is shown in SEQ ID NO: 2.
  • the DNA mix in Table 1 means a solution obtained by sampling a predetermined amount of each of 18 types of oligonucleotides having the sequences described in SEQ ID NOs: 3 to 20 and mixing them.
  • the second-stage PCR uses FcRp1 synthesized in (2-1) as a template, SEQ ID NO: 21 (5′-TAGCCATGGGCATGCCGTACGAGAATCTGCCCGAAAGCTCTGTGGATGTCCCTGTGGGTAATCTGTG An oligonucleotide consisting of the sequence described in 1 was used as a PCR primer.
  • a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, the second step at 62 ° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at ° C. for 30 cycles.
  • the polynucleotide obtained in (2) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII.
  • Escherichia coli BL21 strain (DE3) was transformed with the ligation product.
  • the obtained transformant was cultured in an LB medium containing 50 ⁇ g / mL kanamycin, and then the expression vector pET-eFcR was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen).
  • the amino acid sequence of the polypeptide expressed by the expression vector pET-eFcR is shown in SEQ ID NO: 25, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26, respectively.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • Example 2 Mutation Introduction to Fc Binding Protein and Library Preparation Of the Fc binding protein expression vector pET-eFcR prepared in Example 1, the polynucleotide portion encoding the Fc binding protein was subjected to error-prone PCR. Mutation was randomly introduced. (1) Error prone PCR was performed using pET-eFcR prepared in Example 1 as a template. In error-prone PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution is heat-treated at 95 ° C. for 2 minutes, the first step at 95 ° C. for 30 seconds, the second step at 60 ° C.
  • the PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
  • the reaction solution was introduced into E. coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin (18 hours at 37 ° C.), and then placed on the plate. The formed colonies were used as a random mutant library.
  • Example 3 Screening of heat-stabilized Fc binding protein (1)
  • the random mutant library (transformant) prepared in Example 2 was subjected to 2YT liquid medium containing 50 ⁇ g / mL kanamycin (peptone 16 g / L, yeast).
  • (Extract 10 g / L, sodium chloride 5 g / L) was inoculated into 200 ⁇ L, and cultured with shaking at 30 ° C. overnight using a 96-well deep well plate.
  • Blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing SKIM MILK (manufactured by BD) and 150 mM sodium chloride. (4-2) Fc for evaluating antibody binding activity after washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20, 150 mM NaCl) A solution containing a binding protein was added to react the Fc binding protein with the immobilized gamma globulin (1 hour at 30 ° C.).
  • Table 4 summarizes the amino acid substitution position and the remaining activity (%) after heat treatment of the Fc binding protein expressed by the transformant selected in (5) with respect to the wild type (without amino acid substitution) Fc binding protein.
  • the amino acid residues from the 17th glycine to the 192nd glutamine and the 17th to 192nd amino acid residues are Met18Arg (this notation is SEQ ID NO: 1 represents that 18th methionine of 1 is substituted with arginine, the same applies below), Val27Glu, Phe29Leu, Phe29Ser, Leu30Gln, Tyr35Asn, Tyr35Asp, Tyr35Sr, Tyr35His, Lys46Thr, Lys46Thr, Lys46Thr, , Glu54Asp, Glu54Gly, Asn56Thr, Gln59Arg, Phe61Tyr, Glu64Asp, Ser6 Arg, Ala71Asp, Phe75Le
  • FcR2 the Fc-binding protein with the highest residual activity, in which the amino acid substitution of Val27Glu and Tyr35Asn occurred, was named FcR2 and contained a polynucleotide encoding FcR2.
  • the vector was named pET-FcR2.
  • the amino acid sequence of FcR2 is shown in SEQ ID NO: 27, and the sequence of the polynucleotide encoding FcR2 is shown in SEQ ID NO: 28.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR2 corresponding to the 17th to 192nd region of SEQ ID NO: 1
  • the 209th to 210th glycine (Gly) Is a linker sequence
  • histidines (His) from 211 to 216 are tag sequences.
  • glutamic acid of Val27Glu is present at the 43rd position
  • asparagine of Tyr35Asn is present at the 51st position.
  • Example 4 Production of Amino Acid-Substituted Fc Binding Protein The stability was further improved by accumulating amino acid substitutions that were found in Example 3 and involved in improving the thermal stability of the Fc binding protein. Accumulation of substituted amino acids was mainly performed using PCR, and three types of Fc-binding proteins shown in (a) to (c) below were prepared.
  • B FcR4 obtained by further performing amino acid substitution of Phe75Leu and Glu121Gly on FcR2.
  • C FcR5a in which Asn92Ser amino acid substitution was further performed on FcR4
  • Asn92Ser amino acid substitution was further performed on FcR4
  • FcR3 Val27Glu, Tyr35Asn, and Phe75Leu were selected from among the amino acid substitutions involved in improving thermal stability, which were revealed in Example 3, and FcR3 in which these substitutions were accumulated in a wild-type Fc-binding protein was prepared. Specifically, FcR3 was produced by introducing a mutation causing Phe75Leu into a polynucleotide encoding FcR2. (A-1) PCR was performed using pET-FcR2 obtained in Example 3 as a template.
  • oligonucleotides having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29 were used as primers for the PCR.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction in which the third step for 1 minute was set to 1 cycle were performed by heat treatment at 72 ° C. for 7 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m3F.
  • A-2) Except that pET-FcR2 obtained in Example 3 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 30 (5′-CCACCGTCGCCCGCATCAATAAGGTTAGCTGC-3 ′) was used as a PCR primer. , (A-1).
  • the purified PCR product was designated as m3R.
  • A-3) Two types of PCR products (m3F and m3R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed, and a PCR product m3p in which m3F and m3R were linked was obtained.
  • (A-4) PCR was performed using the PCR product m3p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding FcR3 in which an amino acid substitution at one site was introduced into FcR2 was prepared.
  • the amino acid sequence of FcR3 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 31, and the sequence of the polynucleotide encoding the FcR3 is shown in SEQ ID NO: 32.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR3 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences.
  • glutamic acid of Val27Glu is present at the 43rd position, asparagine of Tyr35Asn is present at the 51st position, and leucine of Phe75Leu is present at the 91st position.
  • FcR4 Val27Glu, Tyr35Asn, Phe75Leu and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, and these substitutions were accumulated in the wild-type Fc binding protein.
  • FcR4 was produced. Specifically, FcR4 was produced by introducing a mutation that causes Phe75Leu and Glu121Gly to the polynucleotide encoding FcR2.
  • oligonucleotide comprising the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29, which was obtained in Example 3, using the plasmid expressing Fc-binding protein (Table 4) containing amino acid substitutions of Ala71Asp, Phe75Leu and Glu121Gly as a template.
  • PCR product m4R was obtained by PCR in the same manner as in (a-1) using as a PCR primer.
  • B-2) After mixing the two types of PCR products (m3R, m4R) obtained in (b-1), PCR was performed in the same manner as in (a-3) to link m3R and m4R.
  • the obtained PCR product was designated as m4p.
  • (B-3) A method similar to (a-4), using the PCR product m4p obtained in (b-2) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR4.
  • (B-4) After purification of the polynucleotide obtained in (b-3), digestion with restriction enzymes NcoI and HindIII and expression vector pETmalE previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • the amino acid sequence of FcR4 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 33, and the sequence of a polynucleotide encoding the FcR4 is shown in SEQ ID NO: 34.
  • SEQ ID NO: 33 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • FcR5a Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser, and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, and these substitutions were changed to the wild type Fc binding protein. Accumulated FcR5a was produced. Specifically, FcR5a was prepared by introducing a mutation that caused Asn92Ser to the polynucleotide encoding FcR4 prepared in (b).
  • C-2 except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 36 (5′-GATCGCTCCAGGGTGCTCAGGCTGGTTCTGGC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m5aR.
  • C-3 After mixing the two kinds of PCR products (m5aF, m5aR) obtained in (c-1) and (c-2), PCR was performed in the same manner as in (a-3), and m5aF And m5aR were ligated.
  • the obtained PCR product was designated as m5ap.
  • C-4 A method similar to (a-4), using the PCR product m5ap obtained in (c-3) as a template and an oligonucleotide comprising the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR5a.
  • C-5 After purifying the polynucleotide obtained in (c-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No.
  • the amino acid sequence of FcR5a added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 37, and the sequence of the polynucleotide encoding the FcR5a is shown in SEQ ID NO: 38.
  • SEQ ID NO: 37 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • Example 5 Mutation Introduction to FcR5a and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR5a prepared in Example 4 (c) by error-prone PCR.
  • (1) Error-prone PCR was performed using the expression vector pET-FcR5a prepared in Example 4 (c) as a template.
  • the reaction solution was heat-treated at 95 ° C. for 2 minutes, and the first step at 95 ° C. for 30 seconds. The reaction was carried out by performing 35 cycles of a second step of 60 ° C.
  • the PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
  • reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
  • Example 6 Screening of Heat Stabilized Fc Binding Protein (1) By culturing the random mutation library prepared in Example 5 by the method described in Examples 3 (1) to (2), Fc binding protein was obtained. Expressed. (2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, and further 20-fold with 0.1 M sodium carbonate buffer (pH 10.0). Dilute to Thereafter, the diluted solution was heat-treated at 40 ° C. for 15 minutes, and the pH was returned to near neutral with 1M Tris buffer (pH 7.0). (3) Example 3 (4) shows the antibody binding activity of the Fc binding protein when the heat treatment of (2) is performed and the antibody binding activity of the Fc binding protein when the heat treatment of (2) is not performed.
  • the residual activity was measured by the ELISA method described in 1. Calculated. (4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing Fc-binding proteins with improved thermal stability compared to FcR5a were selected. The selected transformant was cultured in a 2YT liquid medium containing 50 ⁇ g / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen). (5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
  • Table 8 shows a summary of amino acid substitution positions for FcR5a and the residual activity (%) after heat treatment of the Fc-binding protein expressed by the transformant selected in (4).
  • Phe29Ile this notation is SEQ ID NO: 1 represents that phenylalanine at position 29 (position 45 in SEQ ID NO: 37) is substituted with isoleucine, and so on
  • Phe29Leu Glu39Gly, Gln48Arg, Tyr51Ser, Phe61Tyr, Asp77Gly, Asp82Glu, Gln90Alg, L Of Lys119Glu, Thr140Ile, Leu142Gln, Phe171Ser, Leu175Arg, Asn180Ser and Ile188Val Fc binding protein amino acid substitutions Zureka has at least one
  • FcR7a Fc binding protein in which Phe29Ile and Val117Glu amino acid substitutions were named FcR7a
  • FcR7a an expression vector containing a polynucleotide encoding FcR7a was designated as pET -It
  • the amino acid sequence of FcR7a is shown in SEQ ID NO: 39
  • the sequence of the polynucleotide encoding FcR7a is shown in SEQ ID NO: 40.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR7a corresponding to the 17th to 192nd region of SEQ ID NO: 1
  • histidines (His) from 211 to 216 are tag sequences.
  • isoleucine of Phe29Ile is present at the 45th position and glutamic acid of Val117Glu is present at the 133rd position.
  • Example 7 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 6 involved in improving the thermal stability of Fc binding protein in FcR7a. Accumulation of substituted amino acids was mainly performed using PCR, and four types of improved Fc binding proteins shown in (a) to (d) below were prepared.
  • FcR8 obtained by further performing amino acid substitution of Phe171Ser on FcR7a
  • FcR9 obtained by further substituting Gln48Arg amino acid for FcR8
  • FcR10 obtained by further performing amino acid substitution of Gln48Arg and Tyr51Ser on FcR8
  • D FcR11 obtained by further substituting amino acid substitution of Gln90Arg for FcR10
  • FcR8 Phe29Ile, Val117Glu, and Phe171Ser were selected from the amino acid substitutions involved in improving thermal stability, which were revealed in Example 6, and FcR8 in which these substitutions were accumulated in FcR5a (Example 4 (c)) was prepared. did. Specifically, FcR8 was produced by introducing a mutation that causes Phe171Ser into a polynucleotide encoding FcR7a. (A-1) PCR was performed using pET-FcR7a obtained in Example 6 as a template.
  • an oligonucleotide having a sequence described in SEQ ID NO: 23 and SEQ ID NO: 41 was used as a primer in the PCR.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction in which the third step for 1 minute was set to 1 cycle were performed by heat treatment at 72 ° C. for 5 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m8F.
  • A-2 Except that pET-FcR7a obtained in Example 6 was used as a template, and an oligonucleotide consisting of SEQ ID NO: 42 (5′-GACACGCGCAGCATTTCCTGCCCTGGGGCTG-3 ′) and the sequence shown in SEQ ID NO: 24 was used as a PCR primer. , (A-1).
  • the purified PCR product was designated as m8R.
  • reaction solution having the composition shown in Table 6.
  • the reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed, and a PCR product m8p in which m8F and m8R were linked was obtained.
  • (A-4) PCR was performed using the PCR product m8p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding FcR8 in which an amino acid substitution at one site was introduced into FcR7a was prepared.
  • FcR8 which is a polypeptide in which amino acids are substituted at 3 positions on FcR5a (8 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected microbial cells (transformants) Plasmid pET-FcR8 containing was obtained.
  • A-7) The nucleotide sequence of pET-FcR8 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR8 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 43, and the sequence of the polynucleotide encoding the FcR8 is shown in SEQ ID NO: 44.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR8 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences.
  • isoleucine of Phe29Ile is present at the 45th position
  • glutamic acid of Val117Glu is located at the 133rd position
  • serine of Phe171Ser is present at the 187th position.
  • FcR9 Phe29Ile, Gln48Arg, Val117Glu and Phe171Ser were selected from the amino acid substitutions involved in improving thermal stability, which were revealed in Example 6, and FcR9 integrated in FcR5a (Example 4 (c)) was selected.
  • FcR9 was produced by introducing a mutation that caused Gln48Arg to the polynucleotide encoding FcR8.
  • (B-4) A method similar to (a-4), using the PCR product m9p obtained in (b-3) as a template and an oligonucleotide consisting of the sequences of SEQ ID NO: 21 and SEQ ID NO: 22 as a PCR primer PCR was performed. This produced a polynucleotide encoding FcR9.
  • (B-5) After purifying the polynucleotide obtained in (b-4), digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • the amino acid sequence of FcR9 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 47, and the sequence of the polynucleotide encoding the FcR9 is shown in SEQ ID NO: 48.
  • SEQ ID NO: 47 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • FcR9 There is an amino acid sequence of FcR9 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine (Gly).
  • 211 to 216th histidine (His) is a tag sequence.
  • isoleucine of Phe29Ile is present at the 45th position
  • arginine of Gln48Arg is present at the 64th position
  • glutamic acid of Val117Glu is present at the 133rd position
  • serine of Phe171Ser is present at the 187th position.
  • FcR10 Phe29Ile, Gln48Arg, Tyr51Ser, Val117Glu and Phe171Ser were selected from the amino acid substitutions involved in the improvement of thermal stability, which were revealed in Example 6, and these substitutions were accumulated in FcR5a (Example 4 (c)).
  • FcR10 was prepared. Specifically, FcR10 was produced by introducing a mutation that caused Gln48Arg and Tyr51Ser into a polynucleotide encoding FcR8.
  • the purified PCR product was designated as m10F.
  • C-2 except that pET-FcR8 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 21 and SEQ ID NO: 50 (5′-GCTAGACCGCCCCGGCATTTAAGGGTCAC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m10R.
  • C-3) After mixing the two kinds of PCR products (m10F, m10R) obtained in (c-1) and (c-2), PCR was performed in the same manner as in (a-3), and m10F And m10R were linked. The obtained PCR product was designated as m10p.
  • C-4 The same method as (a-4), using the PCR product m10p obtained in (c-3) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR10.
  • C-5 After purifying the polynucleotide obtained in (c-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • the amino acid sequence of FcR10 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 51, and the sequence of the polynucleotide encoding the FcR10 is shown in SEQ ID NO: 52.
  • SEQ ID NO: 51 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • FcR10 There is an amino acid sequence of FcR10 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine (Gly).
  • 211 to 216th histidine (His) is a tag sequence.
  • isoleucine of Phe29Ile is located at position 45
  • arginine of Gln48Arg is located at position 64
  • serine of Tyr51Ser is located at position 67
  • glutamic acid of Val117Glu is located at position 133
  • serine of Phe171Ser is located at position 187.
  • FcR11 Phe29Ile, Gln48Arg, Tyr51Ser, Gln90Arg, Val117Glu, and Phe171Ser were selected from the amino acid substitutions involved in improving the thermal stability, which were revealed in Example 6, and these substitutions were FcR5a (Example 4 (c)).
  • FcR11 accumulated in the above was prepared. Specifically, FcR11 was produced by introducing a mutation that caused Gln90Arg to the polynucleotide encoding FcR10.
  • PCR was performed in the same manner as (a-1).
  • the purified PCR product was designated as m11R.
  • D-3 After mixing the two types of PCR products (m11F, m11R) obtained in (d-1) and (d-2), PCR was performed in the same manner as in (a-3). And m11R were linked. The obtained PCR product was designated as m11p.
  • (D-4) A method similar to (a-4), using the PCR product m11p obtained in (d-3) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR11.
  • (D-5) After purifying the polynucleotide obtained in (d-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • the amino acid sequence of FcR11 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 55, and the sequence of the polynucleotide encoding the FcR11 is shown in SEQ ID NO: 56.
  • SEQ ID NO: 55 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • isoleucine of Phe29Ile is at position 45
  • arginine of Gln48Arg is position 64
  • serine of Tyr51Ser is position 67
  • arginine of Gln90Arg is position 106
  • glutamic acid of Val117Glu is position 133
  • serine of Phe171Ser is position 187.
  • Example 8 Evaluation of acid stability of Fc binding protein (1) Wild type Fc binding protein prepared in Example 1, Fc binding protein selected in Example 6 (FcR7a), and Fc prepared in Example 7 Transformants expressing binding proteins (FcR8, FcR9, FcR10, FcR11) are inoculated into 3 mL of 2YT liquid medium each containing 50 ⁇ g / mL kanamycin and cultured aerobically at 37 ° C. overnight. Thus, preculture was performed.
  • Example 3 shows the antibody binding activity of the protein after acid treatment with glycine hydrochloride buffer (pH 3.0) and the antibody binding activity of the protein without acid treatment. It was measured by the described ELISA method. Thereafter, the residual activity was calculated by dividing the antibody binding activity when acid treatment was performed by the antibody binding activity when acid treatment was not performed.
  • Example 9 Preparation of FcR5a (FcR5aCys) Added with Cysteine Tag (1) PCR was performed using pET-FcR5a prepared in Example 4 (c) as a template. As a primer in the PCR, an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 57 (5′-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3 ′) was used. In PCR, after preparing a reaction solution having the composition shown in Table 2, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C.
  • the polynucleotide obtained in (1) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. Escherichia coli BL21 (DE3) strain was transformed with the ligation product.
  • pETmalE Japanese Patent Laid-Open No. 2011-206046
  • the obtained transformant was cultured in an LB medium containing 50 ⁇ g / mL kanamycin, and then the expression vector pET-FcR5aCys was extracted using QIAprep Spin Miniprep kit (Qiagen).
  • the nucleotide sequence of pET-FcR5aCys was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of the polypeptide expressed by the expression vector pET-FcR5aCys is shown in SEQ ID NO: 58, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 59, respectively.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR5a corresponding to the 17th to 192nd region of SEQ ID NO: 1
  • the 209th glycine (Gly) to the 216th Up to glycine (Gly) is the cysteine tag sequence.
  • Example 10 Preparation of FcR9 (FcR9Cys) Added with Cysteine Tag (1) Using the pET-FcR9 prepared in Example 7 (b) as a template, an oligonucleotide comprising the sequences described in SEQ ID NO: 21 and SEQ ID NO: 57 was subjected to PCR. PCR was performed in the same manner as in Example 9 (1) except that primers were used. (2) E. coli BL21 (DE3) strain was transformed by the same method as in Example 9 (2). (3) After the obtained transformant was cultured in the same manner as in Example 9 (3), the expression vector pET-FcR9Cys was extracted using QIAprep Spin Miniprep kit (Qiagen). (4) The nucleotide sequence of pET-FcR9Cys was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of the polypeptide expressed by the expression vector pET-FcR9Cys is shown in SEQ ID NO: 60, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 61, respectively.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR9 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th glycine (Gly) to the 216th Up to glycine (Gly) is the cysteine tag sequence.
  • Example 11 Preparation of FcR5aCys (1) 400 mL of 2YT liquid medium containing 50 ⁇ g / mL kanamycin (peptone 16 g / L, yeast extract) containing the transformant expressing FcR5aCys prepared in Example 9 in a 2 L baffle flask 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
  • 2YT liquid medium containing 50 ⁇ g / mL kanamycin (peptone 16 g / L, yeast extract) containing the transformant expressing FcR5aCys prepared in Example 9 in a 2 L baffle flask 10 g / L, sodium chloride 5 g / L
  • the liquid culture medium 1.8L containing / L was inoculated with 180 mL of the culture solution of (1), and main culture was performed using a 3 L fermenter (product of Biot). The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation.
  • the pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali.
  • the dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. .
  • fed-batch medium glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L
  • DO dissolved oxygen
  • the collected cells are suspended in 20 mM Tris-HCl buffer (pH 7.0) so as to be 5 mL / 1 g (cells), and an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation) ) was used to disrupt the cells at an output of about 150 W for about 10 minutes at 4 ° C.
  • the cell disruption solution was centrifuged twice at 8000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
  • the supernatant obtained in (5) is a VL32 ⁇ 250 column (Merck Millipore) packed with 140 mL of TOYOPEARL CM-650M (manufactured by Tosoh) equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.0). Manufactured at a flow rate of 5 mL / min. After washing with the buffer used for equilibration, elution was performed with 20 mM Tris-HCl buffer (pH 7.0) containing 0.5 M sodium chloride.
  • Example 12 Preparation of FcR5a Immobilized Gel and Antibody Separation
  • FcR5aCys prepared in Example 11 after activating the hydroxyl group on the surface of 2 mL of hydrophilic vinyl polymer for separation agent (Tosoh Pearl: Toyopearl) with iodoacetyl group was reacted to obtain an FcR5a-immobilized gel.
  • Example 13 Measurement of ADCC (Antibody Dependent Cytotoxic Activity) Activity of Antibody Separated on FcR5a Immobilized Gel
  • Monoclonal antibodies were separated under the elution conditions described in Example 12, and the elution pattern shown in FIG. Fraction A (FrA) and fraction B (FrB) were fractionated.
  • Fraction A (FrA) and fraction B (FrB) were fractionated.
  • Fraction B (FrB) Fraction A
  • FrB fraction B
  • PBS Phosphate Buffered Saline
  • the ADCC activity of the antibodies contained in FrA and FrB was measured by the following method.
  • (4-1) Using ADCC Assay Buffer prepared by mixing 1.4 mL of Low IgG Serum and 33.6 mL of RPMI1640 medium, the antibody contained in FrA and FrB and the monoclonal antibody before separation were 3 ⁇ g / mL. 8 dilution series were prepared with 1/3 dilution.
  • (4-2) Raji cells were prepared to about 5 ⁇ 10 5 cells / mL with ADCC Assay Buffer and added to a 96-well plate (3917: Corning) at 25 ⁇ L / well.
  • Luciferase Assay Reagent manufactured by Promega
  • a GloMax Multi Detection System manufactured by Promega
  • FIG. 3 shows the results of comparing the luminescence intensity of FrA and FrB fractionated under the elution conditions described in Example 12 and the monoclonal antibody before separation.
  • the result of FIG. 3 shows a value obtained by subtracting the emission intensity of the blank from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
  • FrA has almost the same luminescence intensity as that of the monoclonal antibody before separation, it can be said that the ADCC activity is almost the same.
  • FrB was improved by about 3.2 times compared with the monoclonal antibody before separation and 2.5 times compared with FrA. That is, it can be seen that FrB has higher ADCC activity than the monoclonal antibody and FrA before separation.
  • Example 14 Sugar chain analysis of antibody separated by FcR5a-immobilized gel (1) FrA and FrB fractionated in Example 13 (1) and the monoclonal antibody before separation were denatured by heat treatment at 100 ° C. for 10 minutes, Glycoamidase A / pepsin and pronase were sequentially treated, and a sugar chain fraction was obtained through purification by gel filtration. (2) After concentrating and drying the sugar chain obtained in (1) with an evaporator, 2-aminopyridine and then dimethylamine borane are successively acted on in an acetic acid solvent to form a fluorescent labeled sugar chain. Purified.
  • the neutral sugar chain fraction and the monosialylated sugar were obtained by using the fluorescently labeled sugar chain obtained in (2). Separated into chain fractions.
  • the neutral sugar chain fraction and monosialylated sugar chain fraction obtained in (3) were isolated into individual sugar chains using an ODS column. After obtaining the molecular weight information of the sugar chain isolated by MALDI-TOF-MS analysis, the sugar chain structure was assigned in comparison with the retention time of the ODS column chromatograph.
  • the assigned sugar chain structures (N1 to N6, M1, M2 and D1) are shown in FIG. 4, the composition ratio of neutral sugar chains is shown in Table 10, and the composition ratio of monosialylated and disialylated sugar chains is shown in Table 11.
  • Antibodies with sugar chain structures N4 + N4 'and N6 increased with FrB before separation and compared to FrA.
  • antibodies with N1, N2 + N3 ', N3 and N5 were reduced in FrB before separation and compared to FrA. That is, it can be seen that antibodies having N4 + N4 ′ and N6 sugar chains strongly bind to FcR5a, and antibodies having N1, N2 + N3 ′, N3 and N5 have weak binding to FcR5a.
  • antibodies with M1, M2 and D1 increased in FrB before separation and compared to FrA. That is, it can be seen that antibodies having M1, M2 and D1 sugar chains strongly bind to FcR5a.
  • Example 13 When the above results and the results of Example 13 are combined, it can be seen that an antibody having a sugar chain structure increased by FrB before separation and FrA has higher ADCC activity. That is, it can be seen that the FcR5a-immobilized gel can identify the difference in the sugar chain structure of the antibody and can separate the antibody with high ADCC activity based on the identification.
  • Example 15 Preparation of FcR9-immobilized gel and antibody separation (1) Using the transformant expressing FcR9Cys prepared in Example 10, Example 11 Incubation was carried out in the same manner as (1) to (4). (2) Purification was performed in the same manner as in Example 11 to obtain about 10 mg of high-purity FcR9Cys. (3) After obtaining an FcR9Cys-immobilized gel in the same manner as in Example 12 (1), 0.5 mL of the gel was packed into a stainless steel column of ⁇ 4.0 mm ⁇ 40 mm.
  • a column packed with FcR9-immobilized gel was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation) and equilibrated with 20 mM acetate buffer (pH 4.5).
  • a monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) diluted to 4.0 mg / mL with PBS (Phosphate Buffered Saline) (pH 7.4) was applied at 0.15 mL at a flow rate of 0.3 mL / min.
  • Example 16 ADCC activity measurement of antibody separated on FcR9-immobilized gel
  • Monoclonal antibodies were separated under the elution conditions of Example 15, and fractions A (FrA) and B (FrB) in the elution pattern shown in FIG. ) And fraction C (FrC).
  • the concentrations of the antibody contained in FrA, FrB and FrC, and the monoclonal antibody before separation were measured by absorbance at 280 nm, and ADCC activity was measured by the same method as in Example 13 (4).
  • the results are shown in FIG.
  • the result of FIG. 6 shows a value obtained by subtracting the blank emission intensity from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
  • FrA and FrB have slightly lower ADCC activity than the monoclonal antibody before separation.
  • FrC improved ADCC activity by about 1.6 times compared with the monoclonal antibody before separation. That is, it can be seen that FrC that is slowly eluted has higher ADCC activity than FrA and FrB that are eluted quickly and the monoclonal antibody before separation.
  • the gel immobilized with the Fc-binding protein of the present invention can distinguish the difference in the sugar chain structure of the antibody. Therefore, an antibody that strongly binds to FcR9 contained in FrC has high ADCC activity. It is suggested that the antibody has a sugar chain structure.
  • Example 17 Mutation Introduction to FcR9 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR9 prepared in Example 7 (b) by error-prone PCR.
  • (1) Error-prone PCR was performed using the expression vector pET-FcR9 prepared in Example 7 (b) as a template.
  • pET-FcR9 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared except that oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 were used as primers.
  • a heat treatment at 95 ° C.
  • reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
  • Example 18 Screening of Alkaline Stabilized Fc Binding Protein (1)
  • the random mutation library prepared in Example 17 was cultured by the method described in Examples 3 (1) to (2) to obtain Fc binding protein. Expressed. (2) After culturing, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 10-fold with pure water, mixed with an equal volume of 60 mM sodium hydroxide solution, and 1 at 30 ° C. . Alkali treatment by standing for 5 hours. Thereafter, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
  • the selected transformant was cultured in a 2YT liquid medium containing 50 ⁇ g / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
  • the nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
  • Table 12 shows a summary of amino acid substitution positions for FcR9 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4).
  • Met18Ile this notation is SEQ ID NO: 1 represents that methionine at 18th position (34th in SEQ ID NO: 37) is substituted with isoleucine, the same applies to the following
  • Glu21Lys Glu21Gly, Leu23Met, Gln33Pro, Lys46Glu, Phe61Tyr
  • Glu64Gly Ser65Arg, Ser68Pro, Asp77V , Val81Met, Asp82Ala, Gln101Leu, Glu103Val, His105Arg, Glu120Val, Ser178Arg and Asn180Ly Fc binding proteins or amino acid substitutions are at least
  • Example 19 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 18 involved in improving the alkali stability of Fc binding protein in FcR9. Accumulation of substituted amino acids was mainly performed using PCR, and two types of improved Fc-binding proteins shown in (a) and (b) below were prepared.
  • B FcR13 obtained by further performing amino acid substitution of Glu21Gly, Leu23Met, Ser68Pro and Ser178Arg to FcR9
  • a method for producing each improved Fc-binding protein will be described in detail.
  • FcR12 Glu21Gly, Leu23Met and Ser178Arg were selected from the amino acid substitutions involved in improving alkali stability, which were revealed in Example 18, and FcR12 in which these substitutions were accumulated in FcR9 (Example 7 (b)) was prepared. did. Specifically, FcR12 is obtained by introducing a mutation that causes Glu21Gly and Leu23Met to the polynucleotide containing the Ser178Arg mutation obtained in Example 18. Was made. (A-1) PCR was carried out using the polynucleotide obtained in Example 18 encoding Fc-binding protein containing Ser178Arg mutation in FcR9 as a template.
  • oligonucleotides having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 62 were used.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, and the second step at 55 ° C. for 5 seconds.
  • 30 cycles of the third step of 1 minute at 72 ° C. were performed for 30 cycles, and finally heat treatment was performed at 72 ° C. for 5 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m12p.
  • A-2) m12p obtained in (a-1) was digested with restriction enzymes NcoI and HindIII, and ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. This was used to transform E. coli BL21 (DE3) strain.
  • A-3) The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
  • FcR12 which is a polypeptide in which amino acid substitution has been performed at three positions on FcR9 (12 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR12 containing was obtained.
  • A-4 The nucleotide sequence of pET-FcR12 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR12 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 63
  • the sequence of the polynucleotide encoding the FcR12 is shown in SEQ ID NO: 64.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR12 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences.
  • SEQ ID NO: 63 Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, Phe75Lu.
  • FcR13 Glu21Gly, Leu23Met, Ser68Pro and Ser178Arg were selected from the amino acid substitutions involved in improving the alkali stability, which were revealed in Example 18, and these substitutions were accumulated in FcR9 (Example 7 (b)). Was made. Specifically, FcR13 was produced by introducing a mutation that caused Ser68Pro to the polynucleotide encoding FcR12.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m13F. (B-2) except that pET-FcR12 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 66 (5′-GTAGCTGCTCGCCCTGCTGGGAATCAGGCT-3 ′) was used as a PCR primer, PCR was performed in the same manner as (b-1).
  • the purified PCR product was designated as m13R.
  • (B-4) PCR was performed using the PCR product m13p obtained in (b-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding FcR13 in which an amino acid substitution at one position was introduced into FcR12 was prepared.
  • FcR13 which is a polypeptide in which amino acids are substituted at 4 positions (13 positions with respect to a wild-type Fc-binding protein) with respect to FcR9 by extracting a plasmid from the collected microbial cells (transformants) Plasmid pET-FcR13 containing was obtained.
  • B-7 The nucleotide sequence of pET-FcR13 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR13 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 67, and the sequence of the polynucleotide encoding the FcR13 is shown in SEQ ID NO: 68.
  • SEQ ID NO: 67 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR13 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the region from the 17th to the 192nd region of SEQ ID NO: 1), the 209th to the 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
  • Example 20 Evaluation of Alkali Stability of Fc Binding Protein
  • Fc binding protein (FcR5a) prepared in Example 4 (c) Fc binding protein (FcR9) prepared in Example 7 (b), and The transformant expressing the Fc binding protein (FcR12, FcR13) prepared in Example 19 was cultured by the method described in Example 8 (1) to (4), and FcR5a, FcR9, FcR12 and FcR13 were prepared.
  • the antibody binding activity of FcR5a, FcR9, FcR12 and FcR13 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR9 purified and quantified, and the concentration was measured.
  • Example 21 Mutation Introduction to FcR13 and Library Preparation Mutation was randomly introduced into the polynucleotide part encoding FcR13 prepared in Example 19 (b) by error-prone PCR.
  • Error-prone PCR was performed using the expression vector pET-FcR13 prepared in Example 19 (b) as a template.
  • a reaction solution similar to the composition shown in Table 3 was prepared except that pET-FcR13 was used as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 were used as primers.
  • reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
  • Example 22 Screening of alkali-stabilized Fc-binding protein (1)
  • the random mutation library prepared in Example 21 was cultured by the method described in Examples 3 (1) to (2) to obtain an Fc-binding protein. Expressed. (2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation was treated with alkali by the method shown below. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1M Tris buffer (pH 7.0).
  • ii Dilute 20 times with pure water, After mixing with 60 mM sodium hydroxide solution, left at 30 ° C.
  • the selected transformant was cultured in a 2YT liquid medium containing 50 ⁇ g / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
  • the nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
  • Table 14 summarizes the amino acid substitution positions for FcR13 and the residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4) (alkaline treatment is the conditions of (i)). And Table 15 (alkali treatment is the condition (ii)).
  • FcR14 Fc binding protein in which amino acid substitution of Gly147Val occurred was named FcR14, and an expression vector containing a polynucleotide encoding FcR14 was designated as pET-FcR14. Named.
  • the amino acid sequence of FcR14 is shown in SEQ ID NO: 69, and the sequence of the polynucleotide encoding FcR14 is shown in SEQ ID NO: 70.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR14 corresponding to the 17th to 192nd region of SEQ ID NO: 1
  • 209th to 210th glycine (Gly) Is a linker sequence
  • histidines (His) from 211 to 216 are tag sequences.
  • Glu21Gly glycine is position 37
  • Leu23Met methionine is position 39
  • Val27Glu glutamate is position 43
  • Phe29Ile isoleucine
  • Tyr35Asn asparagine is position 51
  • Gln48Arg arginine is position 64
  • Ser68Pro. Proline 84th Phe75Leu leucine 91st, Asn92Ser serine 108th
  • Val117Glu glutamic acid 133rd Glu121Gly glycine 137th
  • Gly147Val valine 163rd Phe171Ser serine 187th
  • Ser178Arg Exists at the 194th position.
  • Example 23 Production of Improved Fc Binding Protein Tyr51His and Glu54Asp were selected from among the amino acid substitutions involved in improving the alkali stability of Fc binding protein revealed in Example 22, and these substitutions were changed to FcR14. By accumulating, an improved Fc binding protein (FcR16) was produced. Hereinafter, the production method will be described in detail.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m16F.
  • the purified PCR product was designated as m16R.
  • reaction solution having the composition shown in Table 6.
  • the reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed by heat treatment at 72 ° C. for 5 minutes to obtain a PCR product m16p in which m16F and m16R were ligated.
  • PCR was performed using the PCR product m16p obtained in (3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction with the third step of 1 minute as one cycle were performed, and finally PCR was performed by heat treatment at 72 ° C. for 5 minutes.
  • FcR16 which is a polypeptide in which amino acid substitution has been performed at two positions on FcR14 (16 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected cells (transformants) Plasmid pET-FcR16 containing was obtained. (7) The nucleotide sequence of pET-FcR16 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR16 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 73, and the sequence of the polynucleotide encoding the FcR16 is shown in SEQ ID NO: 74.
  • SEQ ID NO: 73 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR13 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the region from the 17th to the 192nd region of SEQ ID NO: 1), the 209th to the 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
  • Example 24 Alkali Stability Evaluation of Fc Binding Protein
  • Fc binding protein (FcR13) prepared in Example 19 (b), Fc binding protein (FcR14) obtained in Example 22, and Example 23 FcR13, FcR14, and FcR16 were prepared by culturing the transformant expressing Fc-binding protein (FcR16) prepared in step 1 by culturing by the method described in (1) to (4) of Example 8 and extracting the protein. did.
  • the antibody binding activity of FcR13, FcR14 and FcR16 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR9 purified and quantified, and the concentration was measured.
  • Example 25 Mutation Introduction to FcR16 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR16 prepared in Example 23 by error-prone PCR.
  • (1) Error-prone PCR was performed using the expression vector pET-FcR16 prepared in Example 23 as a template.
  • pET-FcR16 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared, except that oligonucleotides having the sequences shown in SEQ ID NOs: 23 and 24 were used as primers.
  • Heat treatment at 95 ° C. for 2 minutes, 35 cycles of reaction with 95 ° C. for 30 seconds for the first step, 50 ° C.
  • the PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
  • reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
  • Example 26 Screening of alkali-stabilized Fc-binding protein (1)
  • the random mutation library prepared in Example 25 was cultured by the method described in Examples 3 (1) to (2) to obtain an Fc-binding protein. Expressed. (2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, mixed with an equal amount of 80 mM sodium hydroxide solution, and then at 30 ° C. The alkali treatment was carried out by leaving still for 2 hours. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
  • the selected transformant was cultured in a 2YT liquid medium containing 50 ⁇ g / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
  • the nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
  • Table 17 shows a summary of amino acid substitution positions for FcR13 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4).
  • the amino acid residue from the 33rd glycine to the 208th glutamine corresponding to the 17th to 192nd of SEQ ID NO: 1
  • Ala78Ser this notation indicates that the 78th alanine in SEQ ID NO: 1 (94th in SEQ ID NO: 37) is substituted with serine, the same applies hereinafter
  • Asp82Glu Gln101Leu, Gln101Arg, Thr140Ile
  • An Fc-binding protein in which at least one amino acid substitution of any one of Gln143His, Tyr158His, Lys161Arg, Lys165Glu, Thr185Ala, Asn187Asp, Asn187Tyr has occurred is more stable than Fc
  • Example 27 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 26 involved in improving the alkali stability of Fc binding protein in FcR16. Accumulation of substituted amino acids was mainly performed using PCR, and three types of improved Fc-binding proteins shown in (a) to (c) below were prepared.
  • A FcR19 in which Thr140Ile, Tyr158His and Lys165Glu were further substituted for FcR16
  • B FcR21 in which Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu were further substituted for FcR16
  • C FcR24 in which Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, Lys165Glu, Thr185Ala, and Asn187Asp were further substituted for FcR16
  • a method for producing each improved Fc-binding protein will be described in detail.
  • FcR19 Thr140Ile, Tyr158His and Lys165Glu were selected from the amino acid substitutions involved in improving alkali stability, which were revealed in Example 26, and FcR19 in which these substitutions were accumulated in FcR16 (Example 23) was prepared. Specifically, FcR19 was produced by carrying out mutation introduction
  • Example 26 (A-1) SEQ ID NO: 24 and SEQ ID NO: 75 (5′-ATTCCCAAAGCGACCGCTGGAGGACACGCGGC-3 ′), using as a template the polynucleotide obtained in Example 26 that encodes an Fc-binding protein containing Thr140Ile and Tyr158His mutations in FcR16
  • the reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, and then the first step at 98 ° C. for 10 seconds.
  • the reaction was performed by 30 cycles of the second step at 55 ° C. for 5 seconds and the third step at 72 ° C. for 1 minute for 30 cycles, and finally by heat treatment at 72 ° C. for 5 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m19F.
  • A-2 SEQ ID NO: 23 and SEQ ID NO: 76 (5′-ATAGCTGCCGCTGTCTCCCCACGGTCGCTTT-3 ′) using, as a template, a polynucleotide encoding an Fc-binding protein containing Thr140Ile and Tyr158His mutations in FcR16 obtained in Example 26 PCR was performed in the same manner as (a-1) except that the oligonucleotide having the sequence described in (1) was used as a PCR primer.
  • the purified PCR product was designated as m19R.
  • A-3 Two types of PCR products (m19F and m19R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was carried out to obtain a PCR product m19p in which m19F and m19R were linked.
  • (A-4) PCR was performed using the PCR product m19p obtained in (a-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding FcR19 having three amino acid substitutions introduced into FcR16 was prepared.
  • FcR19 which is a polypeptide in which amino acid substitution has been performed on FcR16 at three positions (19 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR19 containing was obtained.
  • A-7) The nucleotide sequence of pET-FcR19 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR19 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 77, and the sequence of the polynucleotide encoding the FcR19 is shown in SEQ ID NO: 78.
  • the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides
  • the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m21-2F.
  • (B-2) SEQ ID NO: 23 and SEQ ID NO: 80 (5′-TTGCAGGGTAGACTACTTTATGCAGGGCGGT-) using as a template a polynucleotide encoding an Fc-binding protein obtained in Example 26 and containing Asp82Glu, Gln101Leu and Asn187Asp mutations in FcR16 PCR was performed in the same manner as (b-1) except that the oligonucleotide having the sequence described in 3 ′) was used as a PCR primer.
  • the purified PCR product was designated as m21-2R.
  • reaction solution having the composition shown in Table 6.
  • the reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed to obtain a PCR product m21-2p in which m21-2F and m21-2R were ligated.
  • (B-4) PCR was performed using the PCR product m21-2p obtained in (b-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m21-1F.
  • (B-7) Two types of PCR products (m21-1F, m21-1R) obtained in (b-5) and (b-6) were mixed to prepare a reaction solution having the composition shown in Table 6.
  • the reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed to obtain a PCR product m21-1p in which m21-1F and m21-1R were ligated.
  • (B-8) PCR was performed using the PCR product m21-1p obtained in (b-7) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • the reaction solution having the composition shown in Table 5 was prepared except that the oligonucleotide having the sequence described above was used as a PCR primer, and then the reaction solution was heat-treated at 98 ° C. for 5 minutes, and the first step at 98 ° C. for 10 seconds, 55
  • the reaction was carried out by performing 30 cycles of the second step of 5 seconds at ° C and the third step of 1 minute at 72 ° C for 30 cycles, and finally heat treating at 72 ° C for 5 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m21F (Asn187Asp mutation was deleted by this operation).
  • B-10 A polynucleotide encoding FcR21-1 containing the mutations of Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, and Asn187Asp obtained in (b-8) in FcR16 is used as a template.
  • SEQ ID NO: 62 and SEQ ID NO: 76 PCR was carried out in the same manner as in (b-9) except that oligonucleotides having the sequences described were used as PCR primers.
  • the purified PCR product was designated as m21R.
  • reaction solution having the composition shown in Table 6.
  • the reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed to obtain a PCR product m21p in which m21F and m21R were linked.
  • (B-12) PCR was performed using the PCR product m21p obtained in (b-11) as a template and oligonucleotides having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 22 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • the amino acid sequence of FcR21 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 83, and the sequence of a polynucleotide encoding the FcR21 is shown in SEQ ID NO: 84.
  • SEQ ID NO: 83 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • FcR24 Among the amino acid substitutions involved in improving the alkali stability revealed in Example 26, Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, Lys165Glu, Thr185Ala and Asn187Asp were selected, and these substitutions were selected as FcR16 (Example 23). FcR24 accumulated in (1) was prepared. Specifically, FcR24 was prepared by introducing a mutation that causes Ala78Ser, Thr185Ala and Asn187Asp to the polynucleotide encoding FcR21.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen).
  • the purified PCR product was designated as m24-2F. (C-2) except that pET-FcR21 prepared in (b) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 23 and SEQ ID NO: 86 (5′-GCTATCTCCCACCGTCGCCGAATCAATAAG-3 ′) was used as a PCR primer.
  • PCR was performed in the same manner as in (c-1).
  • the purified PCR product was designated m24-2R.
  • (C-4) PCR was performed using the PCR product m24-2p obtained in (c-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • (C-5) The polynucleotide obtained by (c-4) and encoding FcR24-2 containing mutations of Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu in FcR16 as a template, SEQ ID NO: 24 and SEQ ID NO: 87 (5′-AAAAATGTGAGCAGCGAGGCCCGTGGATTT-3 ′) except that the PCR primer was used as a PCR primer, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes.
  • the amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m24F.
  • (C-6) The polynucleotide obtained by (c-4) and encoding FcR24-2 containing mutations of Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu in FcR16 was used as a template, and SEQ ID NO: 62 and SEQ ID NO: PCR was carried out in the same manner as in (c-5), except that an oligonucleotide having the sequence described in No. 88 (5′-GGTAATGGTAATATCCCACGCCCCGCGTGCT-3 ′) was used as a PCR primer. The purified PCR product was designated as m24R.
  • (C-8) PCR was performed using the PCR product m24p obtained in (c-7) as a template and oligonucleotides having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a plasmid pET-FcR24 containing a polynucleotide encoding FcR24, which is a polypeptide obtained by substituting eight amino acids for FcR16 was obtained.
  • C-11 The nucleotide sequence of pET-FcR24 was analyzed in the same manner as in Example 1 (5).
  • the amino acid sequence of FcR24 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 89, and the sequence of the polynucleotide encoding the FcR24 is shown in SEQ ID NO: 90.
  • SEQ ID NO: 89 the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences.
  • the amino acid sequence of FcR24 (corresponding to the 17th to 192nd region of SEQ ID NO: 1) from the 33rd glycine (Gly) to the 208th glutamine (Gln), and the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
  • Glu21Gly glycine is position 37
  • Leu23Met methionine is position 39
  • Val27Glu glutamate is position 43
  • Phe29Ile isoleucine is position 45
  • Tyr35Asn asparagine is position 51
  • Gln48Arg arginine is position 64
  • Tyr51His is also known in SEQ ID NO: 89.
  • Example 28 Evaluation of Alkali Stability of Fc Binding Protein (1) Expression of Fc binding protein (FcR16) prepared in Example 23 and Fc binding proteins (FcR19, FcR21 and FcR24) obtained in Example 27 FcR16, FcR19, FcR21 and FcR24 were prepared by culturing the transformant by the method described in Example 8 (1) to (4) and extracting the protein. (2) The antibody binding activity of FcR16, FcR19, FcR21 and FcR24 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR13 purified and quantified, and the concentration was measured.
  • Example 29 Mutation Introduction to FcR24 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR24 prepared in Example 27 (c) by error-prone PCR.
  • (1) Error prone PCR was performed using the expression vector pET-FcR24 prepared in Example 27 (c) as a template.
  • pET-FcR24 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared except that oligonucleotides having the sequences shown in SEQ ID NOs: 23 and 24 were used as primers.
  • Heat treatment at 95 ° C. for 2 minutes, 35 cycles of reaction with 95 ° C. for 30 seconds for the first step, 50 ° C.
  • the heat treatment was performed at 72 ° C. for 7 minutes.
  • the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
  • the PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
  • the reaction solution was introduced into E. coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 ⁇ g / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
  • Example 30 Screening of alkali-stabilized Fc binding protein (1) Random mutation library prepared in Example 29 was cultured by the method described in Example 3 (1) to (2) to thereby obtain Fc binding protein. Expressed. (2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, mixed with an equal amount of 300 mM sodium hydroxide solution, and then at 30 ° C. The alkali treatment was carried out by leaving still for 2 hours. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
  • the selected transformant was cultured in a 2YT liquid medium containing 50 ⁇ g / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
  • the nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
  • Table 19 shows a summary of amino acid substitution positions for FcR24 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4).
  • the amino acid residues from the 33rd glycine to the 208th glutamine corresponding to the 17th to 192nd of SEQ ID NO: 1
  • Lys40Gln this notation represents that 40th lysine in SEQ ID NO: 1 (56th in SEQ ID NO: 37) is substituted with glutamine, the same applies hereinafter
  • Lys46Asn Ala50Thr, Asn56Tyr, His62Leu, Ser65Gly, Tyr74His, Asp77Val, Gln90Leu, Lys119Thr, Lys119Glu, Asp122Glu, His137Gln, Thr (Ile) 140Met (This notation is the 140th position of SEQ ID NO:
  • Example 31 Production of Thr140 or Tyr158 Amino Acid Substituted Of the amino acid substitutions that contribute to the improvement of alkali stability of the Fc binding protein revealed in Example 26, the 140th position of SEQ ID NO: 1 (156th position of SEQ ID NO: 37) In particular, threonine (Thr140) was replaced with isoleucine (Ile) and 158th (174th in SEQ ID NO: 37) tyrosine (Tyr158) was replaced with histidine, whereby alkali stability was particularly improved.
  • Thr140 threonine
  • Ile isoleucine
  • Tyr158 tyrosine
  • Thr140 and Tyr158 were prepared in Example 27 (c) Fc-binding protein was prepared by substituting 174th in number 89 with another amino acid.
  • PCR was performed by performing 35 cycles of the second step of 30 seconds at 72 ° C and the third step of 90 seconds at 72 ° C for one cycle, and finally heat treating at 72 ° C for 7 minutes.
  • the obtained PCR product was designated as T140p1.
  • A-2 Using, as a primer, an oligonucleotide having the sequence described in SEQ ID NO: 23 and SEQ ID NO: 92 (5′-CCGTTTTGCAGGTAMNNCACTTTATGCAGGG-3 ′), which was prepared in Example 27 (c), using pET-FcR24 as a template After preparing a reaction solution having the same composition as shown in Table 3, the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C.
  • T140p2 Two types of PCR products (T140p1, T140p2) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed, and a PCR product T140p in which T140p1 and T140p2 were ligated was obtained.
  • A-4 PCR was performed using the PCR product T140p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding an Fc binding protein in which the 140th amino acid of the Fc binding protein (FcR24) was substituted with an arbitrary amino acid was obtained.
  • the obtained polynucleotide was designated as T140p3.
  • A-5 After purifying the polynucleotide obtained in (a-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • A-6 The obtained transformant was cultured in LB medium supplemented with 50 ⁇ g / mL kanamycin.
  • a plasmid was extracted from the collected cells (transformants), and the nucleotide sequence of the polynucleotide region was analyzed by the method described in Example 1 (5). As a result, Thr140 (SEQ ID NO: 89) of the Fc-binding protein FcR24 was analyzed. Then, a transformant expressing an Fc-binding protein in which 156th isoleucine) was substituted with Ala, Arg, Gly, Leu, Lys, Phe, Thr, Ser or Val was obtained.
  • PCR was performed by performing 35 cycles of the second step of 30 seconds at 72 ° C and the third step of 90 seconds at 72 ° C for one cycle, and finally heat treating at 72 ° C for 7 minutes.
  • the obtained PCR product was designated as Y158p1.
  • B-2 An oligonucleotide having the sequence described in SEQ ID NO: 23 and SEQ ID NO: 94 (5′-GTCGCTTTGGGAATNNNGAAGTCGGAGTTG-3 ′) prepared in Example 27 (c) as a template and used as a primer
  • the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C. for 30 seconds, second step at 50 ° C.
  • PCR was carried out by performing 35 cycles of a reaction in which the third step of 90 seconds at 90 ° C. was carried out, and finally heat treating at 72 ° C. for 7 minutes.
  • the obtained PCR product was designated as Y158p2.
  • B-3 Two types of PCR products (Y158p1, Y158p2) obtained in (b-1) and (b-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute.
  • PCR was performed to obtain a PCR product Y140p in which Y140p1 and Y140p2 were ligated.
  • B-4 PCR was performed using the PCR product Y140p obtained in (b-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C.
  • the reaction with the third step of 1 minute as one cycle was performed 30 cycles.
  • a polynucleotide encoding an Fc binding protein in which the 158th amino acid of the Fc binding protein (FcR24) was substituted with an arbitrary amino acid was obtained.
  • the obtained polynucleotide was designated as Y158p3.
  • B-5 After purifying the polynucleotide obtained in (b-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Unexamined Patent Publication No. 2011-206046) was used to transform Escherichia coli BL21 (DE3) strain.
  • B-6 The obtained transformant was cultured in an LB medium supplemented with 50 ⁇ g / mL kanamycin.
  • a plasmid was extracted from the collected cells (transformants), and the nucleotide sequence of the polynucleotide region was analyzed by the method described in Example 1 (5). As a result, Tyr158 (SEQ ID NO: 89) of the Fc-binding protein FcR24 was analyzed. Fc-binding protein in which 174th histidine is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val A transformant expressing was obtained.
  • Example 32 Evaluation of Thr140 or Tyr158 Amino Acid Substitution (1)
  • the transformant expressing the Fc-binding protein prepared in Example 31 was cultured by the method described in Example 8 (1) to (4).
  • the protein was extracted.
  • the antibody binding activity of the Fc binding protein in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4).
  • a calibration curve was prepared using FcR24 purified and quantified, and the concentration was measured.
  • Example 33 Preparation of Fc-binding protein-immobilized gel (1) Transformation obtained by transforming Escherichia coli with a plasmid containing a polynucleotide encoding a human Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 60 The body was cultured, and the Fc receptor protein was purified from the obtained cells to prepare a ligand to be immobilized on an insoluble carrier.
  • the human Fc binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 60 had the following amino acid substitutions (a) to (d) in the human Fc binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 58. It is a protein.
  • Buffer A is allowed to flow for 2 minutes after the addition of the monoclonal antibody solution, and from 2% to 40 minutes, from Buffer A 100% -10 mM glycine hydrochloride buffer (pH 3.0) (Buffer B) 0%
  • Buffer A 100% -10 mM glycine hydrochloride buffer (pH 3.0)
  • Buffer B 0%
  • the monoclonal antibody added to the column was separated and eluted with a gradient of Buffer A 0%-Buffer B 100%.
  • the eluted monoclonal antibody was detected with a UV detector (absorption at 280 nm).
  • Example 35 Separation of monoclonal antibody according to the present invention (part 2)
  • the same experiment as in Comparative Example 1 was performed except that 20 mM sodium acetate buffer (pH 5.0) supplemented with 100 mM or 200 mM potassium chloride was used as buffer A.
  • the results are shown in FIG.
  • the Rs value was calculated in the same manner as in Comparative Example 1 from the peak with the earlier elution time to Peak 1, Peak 2, and Peak 3, the results shown in Table 23 were obtained. From this result, even when potassium chloride was used instead of sodium chloride, the Rs value between peak 1 and peak 2 and / or the Rs value between peak 2 and peak 3 was improved as in the result of Example 34. I understand that.
  • Example 36 Separation of monoclonal antibody according to the present invention (part 3)
  • the same experiment as Comparative Example 1 was performed except that 20 mM sodium acetate buffer (pH 5.0) to which 100 mM sodium sulfate or ammonium sulfate was added as buffer A was used.
  • the results are shown in FIG.
  • the Rs value was calculated in the same manner as in Comparative Example 1 from the peak with the earlier elution time to Peak 1, Peak 2, and Peak 3, the results shown in Table 24 were obtained. From this result, even when sulfate ion is used instead of chloride ion, the Rs value between peak 1 and peak 2 or the Rs value between peak 2 and peak 3 is improved as in the result of Example 34. I understand.
  • Example 37 Production of Fc binding protein with amino acid substitution at one position Among the amino acid substitutions involved in improving the stability of Fc binding protein revealed in Example 3, the 27th valine (Val) of SEQ ID NO: 1, Regarding the 35th tyrosine (Tyr) and the 121st glutamic acid (Glu), Fc binding proteins substituted with other amino acids were prepared by the following methods, respectively.
  • PCR was performed in the same manner as in Example 4 (a-1).
  • the purified PCR product was 27 pR.
  • A-3 After mixing the two PCR products (27pF, 27pR) obtained in (A-1) and (A-2), PCR was performed in the same manner as in Example 4 (a-3). 27pF and 27pR were ligated. The obtained PCR product was designated as 27p.
  • Example 4 (a-4) and the PCR product 27p obtained in (A-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed in the same manner.
  • a polynucleotide encoding an Fc-binding protein in which the 27th valine of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
  • A-5) After purifying the polynucleotide obtained in (A-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No.
  • PCR was performed in the same manner as in Example 4 (a-1).
  • the purified PCR product was 35 pR.
  • B-3 After mixing the two types of PCR products (35pF, 35pR) obtained in (B-1) and (B-2), PCR was performed in the same manner as in Example 4 (a-3). And 35 pF and 35 pR were ligated. The obtained PCR product was 35p.
  • Tyr35Cys (Y35C), Tyr35Asp (Y35D), Tyr35Phe (Y35F), Tyr35Gly (Y35G), Tyr35Lys (Y35K), Tyr35Leu (Y35L), Tyr35Asn (Y35N), Tyr35Tr35Yr, YrN ), Tyr35Thr (Y35T), Tyr35Val (Y35V), or Tyr35Trp (Y35W), a polynucleotide encoding an Fc-binding protein in which an amino acid substitution occurred was obtained.
  • Example 4 (C-2) except that pET-eFcR prepared in Example 1 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 100 (5′-AATCGGATCMNNCTCTTTGAACACCCCACCG-3 ′) was used as a PCR primer, PCR was performed in the same manner as in Example 4 (a-1).
  • the purified PCR product was 121 pR.
  • C-3) After mixing the two kinds of PCR products (121pF, 121pR) obtained by (C-1) and (C-2), PCR was performed in the same manner as in Example 4 (a-3). In practice, 121pF and 121pR were ligated.
  • the obtained PCR product was designated as 121p.
  • C-4 The same as in Example 4 (a-4), using the PCR product 121p obtained in (C-3) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed.
  • a polynucleotide encoding an Fc binding protein in which the 121st glutamic acid of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
  • Example 38 Evaluation of Antibody Binding Activity of 1 Amino Acid-Substituted Fc Binding Protein (1) Expressing the wild-type Fc binding protein prepared in Example 1 and the Fc binding protein with one amino acid substitution prepared in Example 37 The transformant was cultured in the same manner as in Example 3 (1) and (2), and wild type Fc binding protein and Fc binding protein substituted with 1 amino acid were expressed. (2) The binding activity of the expressed Fc binding protein with one amino acid substitution was examined by the ELISA method described in Example 3 (3) and (4).
  • the 35th tyrosine of SEQ ID NO: 1 is converted to aspartic acid (Y35D), phenylalanine (Y35F), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S).
  • Y35D aspartic acid
  • Y35F phenylalanine
  • Y35G glycine
  • Y35K lysine
  • leucine Y35L
  • asparagine Y35N
  • proline Y35P
  • serine Y35S
  • Y35D, Y35G, Y35K, Y35L, Y35N, Y35P, Y35S, Y35T, and Y35W have significantly improved antibody binding activity compared to the wild-type Fc-binding protein.
  • the 35th tyrosine of SEQ ID NO: 1 was substituted with cysteine (Y35C) or arginine (Y35R), the antibody binding activity was almost equivalent to that of the wild type Fc binding protein.
  • E121G By replacing the 121st glutamic acid of SEQ ID NO: 1 with lysine (E121K), arginine (E121R), glycine (E121G), and histidine (E121H), the antibody binding activity was improved compared to the wild-type Fc-binding protein. . Among them, E121G showed significantly improved antibody binding activity as compared with wild-type Fc binding protein. On the other hand, when the glutamic acid at position 121 of SEQ ID NO: 1 was substituted with valine (E121V), the antibody-binding activity was almost equivalent to that of wild-type Fc binding protein, and when substituted with proline (E121P), wild-type Fc binding Antibody binding activity was reduced compared to the sex protein.
  • E121K lysine
  • E121R arginine
  • E121G glycine
  • E121H histidine
  • Example 39 Preparation of Fc-binding protein expression vector
  • the expression vector pTrc99a encodes a signal peptide in which the sixth proline (P) of the PelB signal peptide described in SEQ ID NO: 101 (MKYLLPTAAAGLLLLAAQPAMA) is substituted with serine (S)
  • SEQ ID NO: 101 MKYLLPTAAAGLLLLAAQPAMA
  • SEQ ID NO: 102 5′-CATGAAATACCTGCTGTCGCACCGCTGCTGCTGTGTCTGCTGCTCCCTCGCTGCCCAGCCGGCGGATGGCGCGCGCGCTCGCTCGC The temperature was lowered every 1 ° C. in 1 minute, and a double-stranded oligonucleotide was prepared by holding when the temperature reached 15 ° C.
  • PCR was performed using pET-eFcR prepared in Example 1 as a template.
  • an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 57 was used as a primer in the PCR.
  • the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C.
  • the amino acid sequence of the polypeptide expressed by the expression vector pTrc-eFcRCys is shown in SEQ ID NO: 106, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 107, respectively.
  • SEQ ID NO: 107 from the first methionine (Met) to the 22nd alanine (Ala) is a PelB signal peptide in which the 6th proline is substituted with serine, and the 199th from the 24th glycine (Gly).
  • Up to glutamine is the amino acid sequence of the Fc binding protein (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the cysteine tag sequence is from the 200th glycine (Gly) to the 207th glycine (Gly). It is.
  • Example 41 Preparation of FcRCys (1) 400 mL of 2YT liquid medium (peptone 16 g / L, yeast extract) containing 100 ⁇ g / mL carbenicillin in a 2 L baffle flask containing the transformant expressing FcRCys produced in Example 40 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
  • 2YT liquid medium peptone 16 g / L, yeast extract
  • the liquid culture medium 1.8L containing / L was inoculated with 180 mL of the culture solution of (1), and main culture was performed using a 3 L fermenter (manufactured by Biot). The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation.
  • the pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali.
  • the dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. .
  • fed-batch medium glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L
  • DO dissolved oxygen
  • the collected cells are suspended in 20 mM Tris-HCl buffer (pH 7.0) so as to be 5 mL / 1 g (cells), and an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation) ) was used to disrupt the cells at an output of about 150 W for about 10 minutes at 4 ° C.
  • the cell disruption solution was centrifuged twice at 8000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
  • Example 42 Preparation of Fc Binding Protein (FcR) Immobilized Gel and Antibody Separation
  • FcR Fc Binding Protein
  • Toyopearl Toyopearl
  • An FcR-immobilized gel was obtained by reacting 4 mg of FcRCys prepared in Example 41.
  • An FcR column was prepared by filling 0.5 mL of the FcR-immobilized gel prepared in (1) into a ⁇ 4.6 mm ⁇ 75 mm stainless steel column.
  • the FcR column prepared in (2) was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation), and equilibrated with 20 mM acetate buffer (pH 4.5).
  • a monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) diluted to 4.0 mg / mL with PBS (Phosphate Buffered Saline) (pH 7.4) was applied at 0.15 mL at a flow rate of 0.3 mL / min.
  • Example 43 Antibody Dependent Cytotoxicity (ADCC) Activity Measurement of Antibody Separated on FcR-Immobilized Gel
  • Monoclonal antibody was separated under the elution conditions described in Example 42, and fractions in the elution pattern described in FIG. The region of A (FrA) and fraction B (FrB) was fractionated.
  • PBS (10 mM disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride) while concentrating the fractionated FrA and FrB with an ultrafiltration membrane (Merck Millipore)
  • the buffer solution was exchanged to pH 7.4.
  • Raji cells were prepared to about 5 ⁇ 10 5 cells / mL with ADCC Assay Buffer and added to a 96-well plate (3917: Corning) at 25 ⁇ L / well. (4-3) Only 25 ⁇ L / well of FrA and FrB prepared in (2), the monoclonal antibody before separation, and blank ADCC Assay Buffer were added to the well to which Raji cells were added. (4-4) Effector cells (Promega) were prepared to about 3.0 ⁇ 10 5 cells / mL using ADCC Assay Buffer, and added to wells containing Raji cells and antibodies at 25 ⁇ L / well. Then, it was left still for 6 hours in a CO 2 incubator (5% CO 2 , 37 ° C.).
  • Luciferase Assay Reagent manufactured by Promega was added at 75 ⁇ L / well. After reacting at room temperature for 30 minutes, luminescence was measured with GloMax Multi Detection System (Promega).
  • FIG. 12 shows the results of comparing the luminescence intensities of FrA and FrB fractionated under the elution conditions described in Example 42 and the monoclonal antibody before separation.
  • the result of FIG. 12 shows a value obtained by subtracting the blank emission intensity from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
  • FrB has higher ADCC activity than the monoclonal antibody and FcA before separation.
  • the FcR-immobilized gel is based on the strength of ADCC activity. Can be separated.

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Abstract

Provided are: an Fc-binding protein having improved stability, particularly to heat and acid; a method for producing the protein; an antibody adsorbent using the protein; and a method for separating the antibodies, using the adsorbent. Specifically provided are: an Fc-binding protein having improved stability to heat and acid, achieved by substituting an amino-acid residue in a specific position in the extracellular region of human FcγRIIIa with another specific amino acid; a method for producing the protein; an antibody adsorbent using the protein; and a method for separating the antibodies, using the adsorbent.

Description

改良Ｆｃ結合性タンパク質、当該タンパク質の製造方法、当該タンパク質を用いた抗体吸着剤および当該吸着剤を用いた抗体の分離方法Improved Fc binding protein, method for producing the protein, antibody adsorbent using the protein, and antibody separation method using the adsorbent
　本発明は、免疫グロブリンに親和性のあるＦｃ結合性タンパク質に関する。より詳しくは、本発明は、熱や酸に対する安定性が野生型よりも高いＦｃ結合性タンパク質、当該タンパク質の製造方法、当該タンパク質を不溶性担体に固定化して得られる抗体吸着剤、および当該吸着剤を用いた抗体の分離方法に関する。 The present invention relates to an Fc binding protein having affinity for immunoglobulin. More specifically, the present invention relates to an Fc-binding protein having higher stability against heat and acid than wild type, a method for producing the protein, an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier, and the adsorbent. The present invention relates to a method for separating an antibody using.
　Ｆｃレセプターは、免疫グロブリン分子のＦｃ領域に結合する一群の分子である。個々の分子は、免疫グロブリンスーパーファミリーに属する認識ドメインによって、単一の、または同じグループの免疫グロブリンイソタイプをＦｃレセプター上の認識ドメインによって認識している。これによって、免疫応答においてどのアクセサリー細胞が動因されるかが決まってくる。Ｆｃレセプターは、さらにいくつかのサブタイプに分類することができ、ＩｇＧ（免疫グロブリンＧ）に対するレセプターであるＦｃγレセプター、ＩｇＥのＦｃ領域に結合するＦｃεレセプター、ＩｇＡのＦｃ領域に結合するＦｃαレセプター等がある。また各レセプターは更に細かく分類されており、Ｆｃγレセプターは、ＦｃγＲＩ、ＦｃγＲＩＩａ、ＦｃγＲＩＩｂ及びＦｃγＲＩＩＩａ、ＦｃγＲＩＩＩｂの存在が報告されている（非特許文献１）。 Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Individual molecules recognize a single or the same group of immunoglobulin isotypes by a recognition domain on the Fc receptor by a recognition domain belonging to the immunoglobulin superfamily. This determines which accessory cells are driven in the immune response. Fc receptors can be further classified into several subtypes, such as Fcγ receptors that are receptors for IgG (immunoglobulin G), Fcε receptors that bind to the Fc region of IgE, Fcα receptors that bind to the Fc region of IgA, etc. There is. Each receptor is further classified, and Fcγ receptors have been reported to include FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb (Non-patent Document 1).
　Ｆｃγレセプターの中でも、ＦｃγＲＩＩＩａはナチュラルキラー細胞（ＮＫ細胞）やマクロファージなどの細胞表面に存在しており、ヒト免疫機構の中でも重要なＡＤＣＣ（抗体依存性細胞傷害）活性に関与している重要なレセプターである。このＦｃγＲＩＩＩａとヒトＩｇＧとの親和性は結合の強さを示す結合定数（ＫＡ）が１０７Ｍ－１以下であることが報告されている（非特許文献２）。ヒトＦｃγＲＩＩＩａのアミノ酸配列（配列番号１）はＵｎｉＰｒｏｔ（Ａｃｃｅｓｓｉｏｎ　ｎｕｍｂｅｒ：Ｐ０８６３７）などの公的データベースに公表されている。また、ヒトＦｃγＲＩＩＩａの構造上の機能ドメイン、細胞膜を貫通するためのシグナルペプチド配列、細胞膜貫通領域の位置についても同様に公表されている。図１にヒトＦｃγＲＩＩＩａの構造略図を示す。なお、図１中の番号はアミノ酸番号を示しており、その番号は配列番号１に記載のアミノ酸番号に対応する。すなわち、配列番号１中の１番目のメチオニン（Ｍｅｔ）から１６番目のアラニン（Ａｌａ）までがシグナル配列（Ｓ）、１７番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までが細胞外領域（ＥＣ）、２０９番目のバリン（Ｖａｌ）から２２９番目のバリン（Ｖａｌ）までが細胞膜貫通領域（ＴＭ）および２３０番目のリジン（Ｌｙｓ）から２５４番目のリジン（Ｌｙｓ）までが細胞内領域（Ｃ）とされている。なおＦｃγＲＩＩＩａはＩｇＧ１からＩｇＧ４まであるヒトＩｇＧサブクラスのうち、特にＩｇＧ１とＩｇＧ３に対し強く結合する一方、ＩｇＧ２とＩｇＧ４に対する結合は弱いことが知られている。 Among Fcγ receptors, FcγRIIIa is present on the surface of natural killer cells (NK cells) and macrophages and is an important receptor involved in ADCC (antibody-dependent cellular cytotoxicity) activity, which is important in the human immune system. It is. The FcγRIIIa and affinity for human IgG has been reported that binding constant indicates the strength of the bond (KA) is 10 7 M -1 or less (Non-Patent Document 2). The amino acid sequence of human FcγRIIIa (SEQ ID NO: 1) is published in public databases such as UniProt (Accession number: P08637). Similarly, the functional domain in the structure of human FcγRIIIa, the signal peptide sequence for penetrating the cell membrane, and the position of the cell membrane penetrating region are also published. FIG. 1 shows a schematic diagram of the structure of human FcγRIIIa. In addition, the number in FIG. 1 has shown the amino acid number, and the number respond | corresponds to the amino acid number as described in sequence number 1. That is, the signal sequence (S) is from the first methionine (Met) to the 16th alanine (Ala) in SEQ ID NO: 1, and the extracellular region is from the 17th glycine (Gly) to the 208th glutamine (Gln). (EC), from the 209th valine (Val) to the 229th valine (Val) is the transmembrane region (TM) and from the 230th lysine (Lys) to the 254th lysine (Lys) is the intracellular region (C ). It is known that FcγRIIIa binds strongly to IgG1 and IgG3 among human IgG subclasses ranging from IgG1 to IgG4, but weakly binds to IgG2 and IgG4.
　また近年、ガンや免疫疾患等の治療に抗体を含む医薬品（抗体医薬）が用いられている。抗体医薬に用いる抗体は、遺伝子工学的手法により得られた、当該抗体を発現可能な細胞（たとえば、チャイニーズハムスター卵巣（ＣＨＯ）細胞等）を培養後、カラムクロマトグラフィー等を用いて高純度に精製し製造するが、近年の研究で前記抗体が、酸化、還元、異性化、糖鎖付加等の修飾を受けることで多様な分子の集合体となっていることが判明しており、薬効や安全性への影響が懸念されている。 In recent years, drugs containing antibodies (antibody drugs) have been used for the treatment of cancer and immune diseases. The antibody used for the antibody drug is purified to high purity using column chromatography after culturing cells capable of expressing the antibody (for example, Chinese hamster ovary (CHO) cells) obtained by genetic engineering techniques. However, in recent studies, it has been found that the antibody is a collection of various molecules by undergoing modifications such as oxidation, reduction, isomerization, and glycosylation. There are concerns about the effects on sex.
　抗体医薬に用いる抗体の分子構造を分析する方法として、従来より、ペプチドマッピング、二次元電気泳動による分析や糖鎖の切り出しを含むＬＣ－ＭＳ分析（非特許文献３）が実施されている。しかしながらいずれの方法も非常に煩雑な操作を伴う。より簡便な抗体の分子構造の分析方法としては、クロマトグラフィーによる分析があげられる。具体的には、ゲルろ過クロマトグラフィーを用いて、抗体を分子量に基づき分離することで凝集体や分解物を分離・定量することが可能である。またイオン交換クロマトグラフィーにより、抗体分子が有する電荷の違いを分離することができる。しかしながら前述したクロマトグラフィーによる分析では、抗体分子の微小な構造変化を識別できないため、得られる分析結果は限定的であった。 As a method for analyzing the molecular structure of an antibody used for an antibody drug, conventionally, LC-MS analysis (Non-Patent Document 3) including peptide mapping, analysis by two-dimensional electrophoresis, and glycan excision has been performed. However, both methods involve very complicated operations. As a simpler method for analyzing the molecular structure of an antibody, chromatographic analysis can be mentioned. Specifically, it is possible to separate and quantify aggregates and degradation products by separating antibodies based on molecular weight using gel filtration chromatography. In addition, the difference in charge of antibody molecules can be separated by ion exchange chromatography. However, the above-described chromatographic analysis has limited results because the minute structural changes of antibody molecules cannot be identified.
　一方、クロマトグラフィーの中でもアフィニティークロマトグラフィーによる分析は、不溶性担体に固定化されたアフィニティーリガンドと抗体との親和性に基づく分析が可能である。そのため抗体分子の微小な構造変化を識別することができる（特許文献１および非特許文献４）。しかしながら特許文献１および非特許文献４に記載の方法を用いて、工業的スケールで抗体分子を分離するのは事実上困難であり、改善が望まれていた。 On the other hand, analysis by affinity chromatography among chromatographies can be performed based on the affinity between an affinity ligand immobilized on an insoluble carrier and an antibody. Therefore, minute structural changes of the antibody molecule can be identified (Patent Document 1 and Non-Patent Document 4). However, it is practically difficult to separate antibody molecules on an industrial scale using the methods described in Patent Document 1 and Non-Patent Document 4, and improvements have been desired.
　さらに、抗体医薬で用いるヒトＩｇＧのうち、Ｆｃ領域の２９７番目のアスパラギン残基に付加するＮ型糖鎖の違いにより抗体依存性細胞傷害（ＡＤＣＣ）活性が変化することが知られており、特に糖鎖の一種であるフコースを除去した抗体でＡＤＣＣ活性が向上することが報告されている（非特許文献５）。 Furthermore, it is known that antibody-dependent cytotoxicity (ADCC) activity is changed by the difference in the N-type sugar chain added to the 297th asparagine residue in the Fc region of human IgG used in antibody pharmaceuticals. It has been reported that ADCC activity is improved by an antibody from which fucose, a kind of sugar chain, has been removed (Non-patent Document 5).
　抗体医薬においては、抗体が有するＡＤＣＣ活性の強さに重要な意味がある。しかしながら、抗体医薬は通常、動物細胞を宿主とした遺伝子組換え技術を用いて製造しており、宿主内での糖鎖付加を制御できないことから、一定のＡＤＣＣ活性を有した抗体を発現させるのは困難である。また発現した抗体の中から、ＡＤＣＣ活性の強さに基づき抗体を分離するには多くの時間と労力が必要である。 In antibody medicine, the strength of ADCC activity possessed by an antibody is important. However, antibody drugs are usually produced using genetic recombination techniques using animal cells as hosts, and because glycosylation in the host cannot be controlled, antibodies with a certain ADCC activity are expressed. It is difficult. In addition, it takes a lot of time and effort to separate antibodies from the expressed antibodies based on the strength of ADCC activity.
ＷＯ２０１３／１２０９２９号WO2013 / 120929
　本発明の課題は、特に熱や酸に対する安定性が向上したＦｃ結合性タンパク質、当該タンパク質の製造方法、および当該タンパク質を用いた抗体吸着剤を提供することにある。 An object of the present invention is to provide an Fc-binding protein having improved stability to heat and acid, a method for producing the protein, and an antibody adsorbent using the protein.
　また本発明の別の課題は、アフィニティーリガンドを固定化した担体を用いた抗体の分離方法において、前記抗体を、その分子構造の違いに基づき、簡便かつ高効率に分離できる方法を提供することにある。 Another object of the present invention is to provide a method for separating antibodies in a simple and highly efficient manner based on the difference in molecular structure in a method for separating antibodies using a carrier on which an affinity ligand is immobilized. is there.
　また本発明のさらに別の課題は、抗体依存性細胞傷害活性の強さに基づき抗体を分離する方法を提供することにある。 Still another object of the present invention is to provide a method for separating antibodies based on the strength of antibody-dependent cytotoxic activity.
　本発明者らは上記の課題を解決すべく鋭意検討した結果、ヒトＦｃγＲＩＩＩａにおける安定性向上に関与したアミノ酸残基を特定し、当該アミノ酸残基を他のアミノ酸残基に置換した変異体が、熱や酸に対して優れた安定性を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have identified an amino acid residue involved in stability improvement in human FcγRIIIa, and a mutant in which the amino acid residue is substituted with another amino acid residue, It has been found that it has excellent stability against heat and acid, and the present invention has been completed.
　また本発明者らは上記に示す別の課題を解決すべく鋭意検討した結果、アフィニティーリガンドを固定化した不溶性担体を充填したカラムの平衡化液に一定濃度の塩化物イオンまたは硫酸イオンを添加することで、抗体の分離能が向上することを見出し、本発明を完成させるに至った。 In addition, as a result of intensive studies to solve the other problems described above, the present inventors add a certain concentration of chloride ion or sulfate ion to the equilibration solution of the column packed with the insoluble carrier on which the affinity ligand is immobilized. As a result, it was found that the resolution of the antibody was improved, and the present invention was completed.
　また本発明者らは上記に示すさらに別の課題を解決すべく鋭意検討した結果、Ｆｃ結合性タンパク質を不溶性担体に固定化して得られる吸着剤を用いることで、抗体依存性細胞傷害（ＡＤＣＣ）活性の強さに基づいて抗体を分離できることを見出し、本発明を完成するに至った。 Further, as a result of intensive studies to solve the above-described further problems, the present inventors have used antibody-dependent cell cytotoxicity (ADCC) by using an adsorbent obtained by immobilizing an Fc-binding protein on an insoluble carrier. The inventors have found that antibodies can be separated based on the strength of activity, and have completed the present invention.
　すなわち、本願は以下の（Ａ）から（Ｔ）に記載の態様を包含する：
　（Ａ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において以下の（１）から（８４）のうち少なくともいずれか１つのアミノ酸置換が生じている、Ｆｃ結合性タンパク質。
（１）配列番号３７の４５番目のフェニルアラニンがイソロイシンまたはロイシンに置換
（２）配列番号３７の５５番目のグルタミン酸がグリシンに置換
（３）配列番号３７の６４番目のグルタミンがアルギニンに置換
（４）配列番号３７の６７番目のチロシンがセリンに置換
（５）配列番号３７の７７番目のフェニルアラニンがチロシンに置換
（６）配列番号３７の９３番目のアスパラギン酸がグリシンに置換
（７）配列番号３７の９８番目のアスパラギン酸がグルタミン酸に置換
（８）配列番号３７の１０６番目のグルタミンがアルギニンに置換
（９）配列番号３７の１２８番目のグルタミンがロイシンに置換
（１０）配列番号３７の１３３番目のバリンがグルタミン酸に置換
（１１）配列番号３７の１３５番目のリジンがアスパラギンまたはグルタミン酸に置換
（１２）配列番号３７の１５６番目のスレオニンがイソロイシンに置換
（１３）配列番号３７の１５８番目のロイシンがグルタミンに置換
（１４）配列番号３７の１８７番目のフェニルアラニンがセリンに置換
（１５）配列番号３７の１９１番目のロイシンがアルギニンに置換
（１６）配列番号３７の１９６番目のアスパラギンがセリンに置換
（１７）配列番号３７の２０４番目のイソロイシンがバリンに置換
（１８）配列番号３７の３４番目のメチオニンがイソロイシン、リジンまたはスレオニンに置換
（１９）配列番号３７の３７番目のグルタミン酸がグリシンまたはリジンに置換
（２０）配列番号３７の３９番目のロイシンがメチオニンまたはアルギニンに置換
（２１）配列番号３７の４９番目のグルタミンがプロリンに置換
（２２）配列番号３７の６２番目のリジンがイソロイシンまたはグルタミン酸に置換
（２３）配列番号３７の６４番目のグルタミンがトリプトファンに置換
（２４）配列番号３７の６７番目のチロシンがヒスチジンまたはアスパラギンに置換
（２５）配列番号３７の７０番目のグルタミン酸がグリシンまたはアスパラギン酸に置換
（２６）配列番号３７の７２番目のアスパラギンがセリンまたはイソロイシンに置換
（２７）配列番号３７の７７番目のフェニルアラニンがロイシンに置換
（２８）配列番号３７の８０番目のグルタミン酸がグリシンに置換
（２９）配列番号３７の８１番目のセリンがアルギニンに置換
（３０）配列番号３７の８３番目のイソロイシンがロイシンに置換
（３１）配列番号３７の８４番目のセリンがプロリンに置換
（３２）配列番号３７の８５番目のセリンがアスパラギンに置換
（３３）配列番号３７の８７番目のアラニンがスレオニンに置換
（３４）配列番号３７の９０番目のチロシンがフェニルアラニンに置換
（３５）配列番号３７の９１番目のフェニルアラニンがアルギニンに置換
（３６）配列番号３７の９３番目のアスパラギン酸がバリンまたはグルタミン酸に置換
（３７）配列番号３７の９４番目のアラニンがグルタミン酸に置換
（３８）配列番号３７の９７番目のバリンがメチオニンとグルタミン酸に置換
（３９）配列番号３７の９８番目のアスパラギン酸がアラニンに置換
（４０）配列番号３７の１０２番目のグルタミン酸がアスパラギン酸に置換
（４１）配列番号３７の１０６番目のグルタミンがロイシンに置換
（４２）配列番号３７の１０９番目のロイシンがグルタミンに置換
（４３）配列番号３７の１１７番目のグルタミンがロイシンに置換
（４４）配列番号３７の１１９番目のグルタミン酸がバリンに置換
（４５）配列番号３７の１２１番目のヒスチジンがアルギニンに置換
（４６）配列番号３７の１３０番目のプロリンがロイシンに置換
（４７）配列番号３７の１３５番目のリジンがチロシンに置換
（４８）配列番号３７の１３６番目のグルタミン酸がバリンに置換
（４９）配列番号３７の１４１番目のヒスチジンがグルタミンに置換
（５０）配列番号３７の１４６番目のセリンがスレオニンに置換
（５１）配列番号３７の１５４番目のリジンがアルギニンに置換
（５２）配列番号３７の１５９番目のグルタミンがヒスチジンに置換
（５３）配列番号３７の１６３番目のグリシンがバリンに置換
（５４）配列番号３７の１６５番目のリジンがメチオニンに置換
（５５）配列番号３７の１６７番目のフェニルアラニンがチロシンに置換
（５６）配列番号３７の１６９番目のヒスチジンがチロシンに置換
（５７）配列番号３７の１７４番目のチロシンがフェニルアラニンに置換
（５８）配列番号３７の１７７番目のリジンがアルギニンに置換
（５９）配列番号３７の１８５番目のセリンがグリシンに置換
（６０）配列番号３７の１９４番目のセリンがアルギニンに置換
（６１）配列番号３７の１９６番目のアスパラギンがリジンに置換
（６２）配列番号３７の２０１番目のスレオニンがアラニンに置換
（６３）配列番号３７の２０３番目のアスパラギンがイソロイシンまたはリジンに置換
（６４）配列番号３７の２０７番目のスレオニンがアラニンに置換
（６５）配列番号３７の９４番目のアラニンがセリンに置換
（６６）配列番号３７の９８番目のアスパラギン酸がグルタミン酸に置換
（６７）配列番号３７の１１７番目のグルタミンがアルギニンに置換
（６８）配列番号３７の１７４番目のチロシンがヒスチジンに置換
（６９）配列番号３７の１８１番目のリジンがグルタミン酸に置換
（７０）配列番号３７の２０３番目のアスパラギンがアスパラギン酸またはチロシンに置換
（７１）配列番号３７の５６番目のリジンがグルタミンに置換
（７２）配列番号３７の６２番目のリジンがアスパラギンに置換
（７３）配列番号３７の６６番目のアラニンがスレオニンに置換
（７４）配列番号３７の７２番目のアスパラギンがチロシンに置換
（７５）配列番号３７の７８番目のヒスチジンがロイシンに置換
（７６）配列番号３７の８１番目のセリンがグリシンに置換
（７７）配列番号３７の９０番目のチロシンがヒスチジンに置換
（７８）配列番号３７の１３８番目のアスパラギン酸がグルタミン酸に置換
（７９）配列番号３７の１５３番目のヒスチジンがグルタミンに置換
（８０）配列番号３７の１５６番目のスレオニンがアラニン、アルギニン、ロイシン、リジン、フェニルアラニン、セリン、バリンまたはメチオニンに置換
（８１）配列番号３７の１５７番目のチロシンがフェニルアラニンに置換
（８２）配列番号３７の１７４番目のチロシンがロイシン、システイン、イソロイシン、リジン、トリプトファンまたはバリンに置換
（８３）配列番号３７の２０６番目のイソロイシンがバリンに置換
（８４）配列番号３７の２０７番目のスレオニンがイソロイシンに置換
　（Ｂ）配列番号３９、配列番号４３、配列番号４７、配列番号５１、配列番号５５、配列番号６３、配列番号６７、配列番号６９、配列番号７３、配列番号７７、配列番号８３、配列番号８９のいずれかに記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含む、（Ａ）に記載のＦｃ結合性タンパク質。 That is, this application includes the aspects described in the following (A) to (T):
(A) The amino acid sequence of SEQ ID NO: 37 comprises the amino acid residues from the 33rd to the 208th, and the amino acid residues from the 33rd to the 208th of the following (1) to (84) An Fc binding protein wherein at least one amino acid substitution has occurred.
(1) 45th phenylalanine of SEQ ID NO: 37 is replaced with isoleucine or leucine (2) 55th glutamic acid of SEQ ID NO: 37 is replaced with glycine (3) 64th glutamine of SEQ ID NO: 37 is replaced with arginine (4) The 67th tyrosine of SEQ ID NO: 37 is replaced with serine (5) The 77th phenylalanine of SEQ ID NO: 37 is replaced with tyrosine (6) The 93rd aspartic acid of SEQ ID NO: 37 is replaced with glycine (7) of SEQ ID NO: 37 98th aspartic acid replaced with glutamic acid (8) 106th glutamine of SEQ ID NO: 37 replaced with arginine (9) 128th glutamine of SEQ ID NO: 37 replaced with leucine (10) 133rd valine of SEQ ID NO: 37 Is replaced with glutamic acid (11) The 135th lysine of SEQ ID NO: 37 is asparagine Or substituted with glutamic acid (12) 156th threonine of SEQ ID NO: 37 replaced with isoleucine (13) 158th leucine of SEQ ID NO: 37 replaced with glutamine (14) 187th phenylalanine of SEQ ID NO: 37 replaced with serine (15) 191st leucine of SEQ ID NO: 37 is replaced with arginine (16) 196th asparagine of SEQ ID NO: 37 is replaced with serine (17) 204th isoleucine of SEQ ID NO: 37 is replaced with valine (18) SEQ ID NO: 37th methionine of 37 was replaced with isoleucine, lysine or threonine (19) 37th glutamic acid of SEQ ID NO: 37 was replaced with glycine or lysine (20) 39th leucine of SEQ ID NO: 37 was replaced with methionine or arginine (21 49th glutamine of SEQ ID NO: 37 Replacement with proline (22) Replacement of 62nd lysine of SEQ ID NO: 37 with isoleucine or glutamic acid (23) Replacement of 64th glutamine of SEQ ID NO: 37 with tryptophan (24) Replacement of 67th tyrosine of SEQ ID NO: 37 with histidine or asparagine (25) The 70th glutamic acid of SEQ ID NO: 37 is replaced with glycine or aspartic acid (26) The 72nd asparagine of SEQ ID NO: 37 is replaced with serine or isoleucine (27) The 77th phenylalanine of SEQ ID NO: 37 is leucine (28) The 80th glutamic acid of SEQ ID NO: 37 is replaced with glycine (29) The 81st serine of SEQ ID NO: 37 is replaced with arginine (30) The 83rd isoleucine of SEQ ID NO: 37 is replaced with leucine (31) 84th serine of SEQ ID NO: 37 Replacement with proline (32) Replacement of 85th serine of SEQ ID NO: 37 with asparagine (33) Replacement of 87th alanine of SEQ ID NO: 37 with threonine (34) Replacement of 90th tyrosine of SEQ ID NO: 37 with phenylalanine (35 ) The 91st phenylalanine of SEQ ID NO: 37 is replaced with arginine (36) The 93rd aspartic acid of SEQ ID NO: 37 is replaced with valine or glutamic acid (37) The 94th alanine of SEQ ID NO: 37 is replaced with glutamic acid (38) The 97th valine of No. 37 is substituted with methionine and glutamic acid (39) The 98th aspartic acid of SEQ ID NO: 37 is substituted with alanine (40) The 102nd glutamic acid of SEQ ID NO: 37 is substituted with aspartic acid (41) 37 of 106th glutamine replaced by leucine (42) The 109th leucine in column No. 37 is replaced with glutamine (43) The 117th glutamine in SEQ ID NO: 37 is replaced with leucine (44) The 119th glutamic acid in SEQ ID NO: 37 is replaced with valine (45) 121 of SEQ ID NO: 37 The histidine is replaced with arginine (46) The 130th proline of SEQ ID NO: 37 is replaced with leucine (47) The 135th lysine of SEQ ID NO: 37 is replaced with tyrosine (48) The 136th glutamic acid of SEQ ID NO: 37 is valine (49) The 141st histidine of SEQ ID NO: 37 is replaced with glutamine (50) The 146th serine of SEQ ID NO: 37 is replaced with threonine (51) The 154th lysine of SEQ ID NO: 37 is replaced with arginine (52) The 159th glutamine of SEQ ID NO: 37 was replaced with histidine (53) SEQ ID NO: 3 The 163rd glycine of SEQ ID NO: 37 is replaced with methineine (55) the 167th phenylalanine of SEQ ID NO: 37 is replaced with tyrosine (56) the 169th histidine of SEQ ID NO: 37 (57) 174th tyrosine of SEQ ID NO: 37 replaced with phenylalanine (58) 177th lysine of SEQ ID NO: 37 replaced with arginine (59) 185th serine of SEQ ID NO: 37 replaced with glycine ( 60) 194th serine of SEQ ID NO: 37 is replaced with arginine (61) 196th asparagine of SEQ ID NO: 37 is replaced with lysine (62) 201st threonine of SEQ ID NO: 37 is replaced with alanine (63) SEQ ID NO: 37 The asparagine at position 203 is substituted with isoleucine or lysine (64) 207 threonine of column number 37 is replaced with alanine (65) 94th alanine of SEQ ID NO: 37 is replaced with serine (66) 98th aspartic acid of SEQ ID NO: 37 is replaced with glutamic acid (67) 117th glutamine is replaced with arginine (68) 174th tyrosine of SEQ ID NO: 37 is replaced with histidine (69) 181st lysine of SEQ ID NO: 37 is replaced with glutamic acid (70) 203rd asparagine of SEQ ID NO: 37 is Substitution with aspartic acid or tyrosine (71) Replacement of 56th lysine of SEQ ID NO: 37 with glutamine (72) Replacement of 62nd lysine of SEQ ID NO: 37 with asparagine (73) Replacement of 66th alanine of SEQ ID NO: 37 with threonine Substitution (74) Asparagine at position 72 in SEQ ID NO: 37 replaced with tyrosine 75) 78th histidine of SEQ ID NO: 37 is replaced with leucine (76) 81st serine of SEQ ID NO: 37 is replaced with glycine (77) 90th tyrosine of SEQ ID NO: 37 is replaced with histidine (78) SEQ ID NO: 37 138th aspartic acid is replaced with glutamic acid (79) 153rd histidine of SEQ ID NO: 37 is replaced with glutamine (80) 156th threonine of SEQ ID NO: 37 is alanine, arginine, leucine, lysine, phenylalanine, serine, valine Alternatively, methionine is substituted (81) 157th tyrosine of SEQ ID NO: 37 is replaced with phenylalanine (82) 174th tyrosine of SEQ ID NO: 37 is replaced with leucine, cysteine, isoleucine, lysine, tryptophan or valine (83) SEQ ID NO: 37 The 206th isoro Syn replaced with valine (84) 207 threonine of SEQ ID NO: 37 replaced with isoleucine (B) SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 67 Fc according to (A), comprising amino acid residues from position 33 to position 208 of the amino acid sequence according to any one of SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 83, SEQ ID NO: 89 Binding protein.
　（Ｃ）配列番号３９、配列番号４３、配列番号４７、配列番号５１、配列番号５５、配列番号６３、配列番号６７、配列番号６９、配列番号７３、配列番号７７、配列番号８３、配列番号８９のいずれかに記載のアミノ酸配列からなる、（Ｂ）に記載のＦｃ結合性タンパク質。 (C) SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 83, SEQ ID NO: 89 The Fc binding protein according to (B), comprising the amino acid sequence according to any one of the above.
　（Ｄ）（Ａ）に記載のＦｃ結合性タンパク質において、さらに以下の（８５）から（８８）のうち少なくともいずれか１つのアミノ酸置換が生じている、Ｆｃ結合性タンパク質。
（８５）配列番号３７の８２番目のロイシンがヒスチジンまたはアルギニンに置換
（８６）配列番号３７の１６３番目のグリシンがアスパラギン酸に置換
（８７）配列番号３７の１７４番目のチロシンがヒスチジンに置換
（８８）配列番号３７の１９２番目のバリンがフェニルアラニンに置換
　（Ｅ）（Ａ）から（Ｄ）のいずれかに記載のＦｃ結合性タンパク質を不溶性担体に固定化して得られる吸着剤。 (D) The Fc binding protein according to (A), further comprising at least one amino acid substitution of (85) to (88) below:
(85) 82nd leucine of SEQ ID NO: 37 was replaced with histidine or arginine (86) 163rd glycine of SEQ ID NO: 37 was replaced with aspartic acid (87) 174th tyrosine of SEQ ID NO: 37 was replaced with histidine (88 ) Substitution of 192th valine of SEQ ID NO: 37 with phenylalanine (E) An adsorbent obtained by immobilizing the Fc-binding protein according to any one of (A) to (D) on an insoluble carrier.
　（Ｆ）（Ｅ）に記載の吸着剤を充填したカラムに平衡化液を添加してカラムを平衡化する工程と、前記平衡化したカラムに抗体を含む溶液を添加して前記抗体を前記担体に吸着させる工程と、前記担体に吸着した抗体を溶出液を用いて溶出させる工程とを含む、抗体の分離方法。 (F) a step of adding an equilibration solution to the column packed with the adsorbent according to (E) to equilibrate the column; and adding a solution containing an antibody to the equilibrated column to allow the antibody to move to the carrier A method for separating antibodies, comprising a step of adsorbing to a carrier and a step of eluting the antibody adsorbed on the carrier using an eluent.
　（Ｇ）平衡化液が３０ｍＭ以上の塩化物イオンまたは硫酸イオンを含む、（Ｆ）に記載の分離方法。 (G) The separation method according to (F), wherein the equilibration solution contains 30 mM or more of chloride ions or sulfate ions.
　（Ｈ）（Ｅ）に記載の吸着剤を用いた、抗体依存性細胞傷害活性の強さに基づき抗体を分離する方法。 (H) A method for separating antibodies based on the strength of antibody-dependent cytotoxic activity using the adsorbent described in (E).
　（Ｉ）（Ｆ）から（Ｈ）のいずれかに記載の分離方法で得られる抗体。 (I) An antibody obtained by the separation method according to any one of (F) to (H).
　（Ｊ）（Ｅ）に記載の吸着剤を用いて抗体を分離することで、抗体が有する糖鎖構造の違いを識別する方法。 (J) A method for discriminating differences in sugar chain structures of antibodies by separating the antibodies using the adsorbent described in (E).
　（Ｋ）（Ｅ）に記載の吸着剤を用いた糖鎖の分離方法。 (K) A method for separating sugar chains using the adsorbent according to (E).
　（Ｌ）（Ｋ）に記載の分離方法で得られる糖鎖。 (L) A sugar chain obtained by the separation method described in (K).
　（Ｍ）（Ａ）から（Ｄ）のいずれかに記載のＦｃ結合性タンパク質をコードするポリヌクレオチド。 (M) A polynucleotide encoding the Fc-binding protein according to any one of (A) to (D).
　（Ｎ）（Ｋ）に記載のポリヌクレオチドを含む発現ベクター。 (N) An expression vector comprising the polynucleotide according to (K).
　（Ｏ）（Ｌ）に記載の発現ベクターで宿主を形質転換して得られる形質転換体。 (O) A transformant obtained by transforming a host with the expression vector described in (L).
　（Ｐ）宿主が大腸菌である、（Ｏ）に記載の形質転換体。 (P) The transformant according to (O), wherein the host is E. coli.
　（Ｑ）（Ｏ）または（Ｐ）に記載の形質転換体を培養することによりＦｃ結合性タンパク質を発現させ、その培養物から発現されたＦｃ結合性タンパク質を回収する、Ｆｃ結合性タンパク質の製造方法。 (Q) Production of Fc-binding protein, wherein Fc-binding protein is expressed by culturing the transformant described in (O) or (P), and Fc-binding protein expressed from the culture is recovered. Method.
　（Ｒ）Ｆｃ結合性タンパク質を固定化した不溶性担体を充填したカラムに平衡化液を添加してカラムを平衡化する工程と、前記平衡化したカラムに抗体を含む溶液を添加して前記抗体を前記担体に吸着させる工程と、前記担体に吸着した抗体を溶出液を用いて溶出させる工程とを含む、抗体の分離方法であって、前記平衡化液が３０ｍＭ以上の塩化物イオンまたは硫酸イオンを含む、前記分離方法。 (R) a step of equilibrating the column by adding an equilibration solution to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized; and adding a solution containing an antibody to the equilibrated column, An antibody separation method comprising a step of adsorbing on the carrier and a step of eluting the antibody adsorbed on the carrier using an eluent, wherein the equilibration solution contains chloride ions or sulfate ions of 30 mM or more. Including the separation method.
　（Ｓ）Ｆｃ結合性タンパク質を不溶性担体に固定化して得られる吸着剤を用いた、抗体依存性細胞傷害活性の強さに基づき抗体を分離する方法。 (S) A method of separating antibodies based on the strength of antibody-dependent cytotoxic activity using an adsorbent obtained by immobilizing an Fc-binding protein on an insoluble carrier.
　（Ｔ）Ｆｃ結合性タンパク質がヒトＦｃγＲＩＩＩａである、（Ｒ）または（Ｓ）に記載の方法。 (T) The method according to (R) or (S), wherein the Fc binding protein is human FcγRIIIa.
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
　本発明のＦｃ結合性タンパク質は、抗体のＦｃ領域に結合性を持つタンパク質であり、配列番号１に記載のアミノ酸配列からなるヒトＦｃγＲＩＩＩａの細胞外領域（図１のＥＣの領域）のうち、少なくとも１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を含むタンパク質であって、当該１７番目から１９２番目までのアミノ酸残基において特定位置におけるアミノ酸置換が生じたタンパク質である。したがって、本発明のＦｃ結合性タンパク質は、細胞外領域のＮ末端側にあるシグナルペプチド領域（図１のＳ）の全てまたは一部を含んでもよいし、細胞外領域のＣ末端側にある細胞膜貫通領域（図１のＴＭ）および細胞外領域（図１のＣ）の全てまたは一部を含んでもよい。前記特定位置におけるアミノ酸置換は、具体的には、配列番号１に記載のアミノ酸配列において、Ｖａｌ２７Ｇｌｕ（この表記は、配列番号１の２７番目（配列番号３７では４３番目）のバリンがグルタミン酸に置換されていることを表す、以下同様）、Ｔｙｒ３５Ａｓｎ、Ｐｈｅ７５Ｌｅｕ、Ａｓｎ９２Ｓｅｒ、Ｇｌｕ１２１Ｇｌｙ、Ｐｈｅ２９Ｉｌｅ、Ｐｈｅ２９Ｌｅｕ、Ｇｌｕ３９Ｇｌｙ、Ｇｌｎ４８Ａｒｇ、Ｔｙｒ５１Ｓｅｒ、Ｐｈｅ６１Ｔｙｒ、Ａｓｐ７７Ｇｌｙ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ９０Ａｒｇ、Ｇｌｎ１１２Ｌｅｕ、Ｖａｌ１１７Ｇｌｕ、Ｌｙｓ１１９Ａｓｎ、Ｌｙｓ１１９Ｇｌｕ、Ｔｈｒ１４０Ｉｌｅ、Ｌｅｕ１４２Ｇｌｎ、Ｐｈｅ１７１Ｓｅｒ、Ｌｅｕ１７５Ａｒｇ、Ａｓｎ１８０Ｓｅｒ、Ｉｌｅ１８８Ｖａｌ、Ｍｅｔ１８Ｉｌｅ、Ｍｅｔ１８Ｌｙｓ、Ｍｅｔ１８Ｔｈｒ、Ｇｌｕ２１Ｇｌｙ、Ｇｌｕ２１Ｌｙｓ、Ｌｅｕ２３Ｍｅｔ、Ｌｅｕ２３Ａｒｇ、Ｇｌｎ３３Ｐｒｏ、Ｌｙｓ４６Ｉｌｅ、Ｌｙｓ４６Ｇｌｕ、Ｇｌｎ４８Ｔｒｐ、Ｔｙｒ５１Ｈｉｓ、Ｔｙｒ５１Ａｓｎ、Ｇｌｕ５４Ｇｌｙ、Ｇｌｕ５４Ａｓｐ、Ａｓｎ５６Ｓｅｒ、Ａｓｎ５６Ｉｌｅ、Ｐｈｅ６１Ｌｅｕ、Ｇｌｕ６４Ｇｌｙ、Ｓｅｒ６５Ａｒｇ、Ｉｌｅ６７Ｌｅｕ、Ｓｅｒ６８Ｐｒｏ、Ｓｅｒ６９Ａｓｎ、Ａｌａ７１Ｔｈｒ、Ｔｙｒ７４Ｐｈｅ、Ｐｈｅ７５Ａｒｇ、Ａｓｐ７７Ｖａｌ、Ａｓｐ７７Ｇｌｕ、Ａｌａ７８Ｇｌｕ、Ｖａｌ８１Ｍｅｔ、Ｖａｌ８１Ｇｌｕ、Ａｓｐ８２Ａｌａ、Ｇｌｕ８６Ａｓｐ、Ｇｌｎ９０Ｌｅｕ、Ｌｅｕ９３Ｇｌｎ、Ｇｌｎ１０１Ｌｅｕ、Ｇｌｕ１０３Ｖａｌ、Ｈｉｓ１０５Ａｒｇ、Ｐｒｏ１１４Ｌｅｕ、Ｌｙｓ１１９Ｔｙｒ、Ｇｌｕ１２０Ｖａｌ、Ｈｉｓ１２５Ｇｌｎ、Ｓｅｒ１３０Ｔｈｒ、Ｌｙｓ１３８Ａｒｇ、Ｇｌｎ１４３Ｈｉｓ、Ｇｌｙ１４７Ｖａｌ、Ｌｙｓ１４９Ｍｅｔ、Ｐｈｅ１５１Ｔｙｒ、Ｈｉｓ１５３Ｔｙｒ、Ｔｙｒ１５８Ｐｈｅ、Ｌｙｓ１６１Ａｒｇ、Ｓｅｒ１６９Ｇｌｙ、Ｓｅｒ１７８Ａｒｇ、Ａｓｎ１８０Ｌｙｓ、Ｔｈｒ１８５Ａｌａ、Ａｓｎ１８７Ｉｌｅ、Ａｓｎ１８７Ｌｙｓ、Ｔｈｒ１９１Ａｌａ、Ａｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ａｒｇ、Ｔｙｒ１５８Ｈｉｓ、Ｌｙｓ１６５Ｇｌｕ、Ａｓｎ１８７Ａｓｐ、Ａｓｎ１８７Ｔｙｒ、Ｌｙｓ４０Ｇｌｎ、Ｌｙｓ４６Ａｓｎ、Ａｌａ５０Ｔｈｒ、Ａｓｎ５６Ｔｙｒ、Ｈｉｓ６２Ｌｅｕ、Ｓｅｒ６５Ｇｌｙ、Ｔｙｒ７４Ｈｉｓ、Ａｓｐ１２２Ｇｌｕ、Ｈｉｓ１３７Ｇｌｎ、Ｔｈｒ１４０Ａｌａ、Ｔｈｒ１４０Ａｒｇ、Ｔｈｒ１４０Ｌｅｕ、Ｔｈｒ１４０Ｌｙｓ、Ｔｈｒ１４０Ｐｈｅ、Ｔｈｒ１４０Ｓｅｒ、Ｔｈｒ１４０Ｖａｌ、Ｔｈｒ１４０Ｍｅｔ、Ｔｙｒ１４１Ｐｈｅ、Ｔｙｒ１５８Ｌｅｕ、Ｔｙｒ１５８Ｃｙｓ、Ｔｙｒ１５８Ｉｌｅ、Ｔｙｒ１５８Ｌｙｓ、Ｔｙｒ１５８Ｔｒｐ、Ｔｙｒ１５８Ｖａｌ、Ｉｌｅ１９０Ｖａｌ、Ｔｈｒ１９１Ｉｌｅのうち、少なくともいずれかの１つの置換である。なお、野生型ＦｃγＲＩＩＩａには、Ｌｅｕ６６Ｈｉｓ、Ｌｅｕ６６Ａｒｇ、Ｇｌｙ１４７Ａｓｐ、Ｔｙｒ１５８Ｈｉｓ、Ｖａｌ１７６Ｐｈｅのうち、いずれか１つ以上のアミノ酸置換が生じた変異体が知られているが、前記特定位置におけるアミノ酸置換以外にこれらのアミノ酸置換を含んでいてもよい。 The Fc-binding protein of the present invention is a protein having a binding property to the Fc region of an antibody, and at least of the extracellular region (EC region of FIG. 1) of human FcγRIIIa comprising the amino acid sequence set forth in SEQ ID NO: 1. A protein comprising amino acid residues from the 17th glycine to the 192nd glutamine, wherein the amino acid substitution at a specific position occurs in the 17th to 192th amino acid residues. Therefore, the Fc binding protein of the present invention may include all or part of the signal peptide region (S in FIG. 1) on the N-terminal side of the extracellular region, or the cell membrane on the C-terminal side of the extracellular region. All or part of the penetrating region (TM in FIG. 1) and the extracellular region (C in FIG. 1) may be included. Specifically, the amino acid substitution at the specific position is, in the amino acid sequence shown in SEQ ID NO: 1, Val27Glu (in this notation, the 27th valine of SEQ ID NO: 1 (43th in SEQ ID NO: 37) is substituted with glutamic acid. indicating that is, the same applies hereinafter), Tyr35Asn, Phe75Leu, Asn92Ser, Glu121Gly, Phe29Ile, Phe29Leu, Glu39Gly, Gln48Arg, Tyr51Ser, Phe61Tyr, Asp77Gly, Asp82Glu, Gln90Arg, Gln112Leu, Val117Glu, Lys119Asn, Lys119Glu, Thr140Ile, Leu142Gln, Phe171Ser, Leu175Arg, Asn180Ser, Ile188Val, M t18Ile, Met18Lys, Met18Thr, Glu21Gly, Glu21Lys, Leu23Met, Leu23Arg, Gln33Pro, Lys46Ile, Lys46Glu, Gln48Trp, Tyr51His, Tyr51Asn, Glu54Gly, Glu54Asp, Asn56Ser, Asn56Ile, Phe61Leu, Glu64Gly, Ser65Arg, Ile67Leu, Ser68Pro, Ser69Asn, Ala71Thr, Tyr74Phe, Phe75Arg, Asp77Val, Asp77Glu, Ala78Glu, Val81Met, Val81Glu, Asp82Ala, Glu86Asp, Gln90Leu, Leu93Gln, Gln101Leu, Glu103Val His105Arg, Pro114Leu, Lys119Tyr, Glu120Val, His125Gln, Ser130Thr, Lys138Arg, Gln143His, Gly147Val, Lys149Met, Phe151Tyr, His153Tyr, Tyr158Phe, Lys161Arg, Ser169Gly, Ser178Arg, Asn180Lys, Thr185Ala, Asn187Ile, Asn187Lys, Thr191Ala, Ala78Ser, Asp82Glu, Gln101Arg, Tyr158His, Lys165Glu, Asn187Asp, Asn187Tyr, Lys40Gln, Lys46Asn, Ala50Thr, Asn56Tyr, His62Leu, Ser65Gly, T yr74His, Asp122Glu, His137Gln, Thr140Ala, Thr140Arg, Thr140Leu, Thr140Lys, Thr140Phe, Thr140Ser, Thr140Val, Thr140Met, Tyr141Phe, Tyr158Leu, Tyr158Cys, Tyr158Ile, Tyr158Lys, Tyr158Trp, Tyr158Val, Ile190Val, among Thr191Ile, at least one in one substitution is there. In addition, among wild-type FcγRIIIa, mutants in which any one or more amino acid substitutions are known among Leu66His, Leu66Arg, Gly147Asp, Tyr158His, and Val176Phe are known. An amino acid substitution may be included.
　アミノ酸置換を行なうことで本発明のＦｃ結合性タンパク質を作製する際、特定位置のアミノ酸残基については、抗体結合活性を有する限り前述したアミノ酸以外のアミノ酸に置換してもよい。その一例として、両アミノ酸の物理的性質と化学的性質またはそのどちらかが類似したアミノ酸間で置換する保守的置換があげられる。保守的置換は、Ｆｃ結合性タンパク質に限らず一般に、置換が生じているものと置換が生じていないものとの間でタンパク質の機能が維持されることが当業者において知られている。保守的置換の一例としては、グリシンとアラニン間、アスパラギン酸とグルタミン酸間、セリンとプロリン間、またはグルタミン酸とアラニン間に生じる置換があげられる（タンパク質の構造と機能，メディカル・サイエンス・インターナショナル社，９，２００５）。 When the Fc-binding protein of the present invention is produced by amino acid substitution, the amino acid residue at a specific position may be substituted with an amino acid other than those described above as long as it has antibody binding activity. One example is a conservative substitution that substitutes between amino acids whose physical and / or chemical properties of both amino acids are similar. Conservative substitutions are not limited to Fc-binding proteins, and are generally known to those skilled in the art to maintain protein function between those with substitutions and those without substitutions. Examples of conservative substitutions include substitutions that occur between glycine and alanine, between aspartic acid and glutamic acid, between serine and proline, or between glutamic acid and alanine (protein structure and function, Medical Science International, 9 2005).
　本発明のＦｃ結合性タンパク質において、置換するアミノ酸の数に特に制限はない。一例として、以下の（ａ）から（ｌ）に示すＦｃ結合性タンパク質があげられる。これらのＦｃ結合性タンパク質は熱、酸またはアルカリに対する安定性が向上する点で好ましい。
（ａ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、４５番目のフェニルアラニンがイソロイシンに、１３３番目のバリンがグルタミン酸にそれぞれ置換されているＦｃ結合性タンパク質（配列番号３９に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｂ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、４５番目のフェニルアラニンがイソロイシンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号４３に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｃ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号４７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｄ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがセリンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号５１に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｅ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがセリンに、１０６番目のグルタミンがアルギニンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号５５に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｆ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号６３に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｇ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、８４番目のセリンがプロリンに、１３３番目のバリンがグルタミン酸に、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号６７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｈ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、８４番目のセリンがプロリンに、１３３番目のバリンがグルタミン酸に、１６３番目のグリシンがバリンに、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号６９に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｉ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがヒスチジン、７０番目のグルタミン酸がアスパラギン酸に、８４番目のセリンがプロリンに、１３３番目のバリンがグルタミン酸に、１６３番目のグリシンがバリンに、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号７３に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｊ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがヒスチジン、７０番目のグルタミン酸がアスパラギン酸に、８４番目のセリンがプロリンに、１３３番目のバリンがグルタミン酸に、１５６番目のスレオニンがイソロイシンに、１６３番目のグリシンがバリンに、１７４番目のチロシンがヒスチジンに、１８１番目のリジンがグルタミン酸に、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号７７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｋ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがヒスチジン、７０番目のグルタミン酸がアスパラギン酸に、８４番目のセリンがプロリンに、９８番目のアスパラギン酸がグルタミン酸に、１１７番目のグルタミンがロイシンに、１３３番目のバリンがグルタミン酸に、１５６番目のスレオニンがイソロイシンに、１６３番目のグリシンがバリンに、１７４番目のチロシンがヒスチジンに、１８１番目のリジンがグルタミン酸に、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンにそれぞれ置換されているＦｃ結合性タンパク質（配列番号８３に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。
（ｌ）配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、３７番目のグルタミン酸がグリシンに、３９番目のロイシンがメチオニンに、４５番目のフェニルアラニンがイソロイシンに、６４番目のグルタミンがアルギニンに、６７番目のチロシンがヒスチジン、７０番目のグルタミン酸がアスパラギン酸に、８４番目のセリンがプロリンに、９４番目のアラニンがセリンに、９８番目のアスパラギン酸がグルタミン酸に、１１７番目のグルタミンがロイシンに、１３３番目のバリンがグルタミン酸に、１５６番目のスレオニンがイソロイシンに、１６３番目のグリシンがバリンに、１７４番目のチロシンがヒスチジンに、１８１番目のリジンがグルタミン酸に、１８７番目のフェニルアラニンがセリンに、１９４番目のセリンがアルギニンに、２０１番目のスレオニンがアラニンに、２０３番目のアスパラギンがアスパラギン酸にそれぞれ置換されているＦｃ結合性タンパク質（配列番号８９に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸配列を含むＦｃ結合性タンパク質）。 In the Fc binding protein of the present invention, the number of amino acids to be substituted is not particularly limited. As an example, Fc-binding proteins shown in the following (a) to (l) can be mentioned. These Fc-binding proteins are preferable in terms of improving stability against heat, acid or alkali.
(A) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid residues from the 33rd to the 208th amino acid residues are included, and the 45th phenylalanine is the isoleucine in the 33rd to 208th amino acid residues. Fc binding protein (Fc binding protein containing the amino acid sequence from the 33rd position to the 208th position in the amino acid sequence shown in SEQ ID NO: 39) wherein
(B) including the 33rd to 208th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 37, and the 45th phenylalanine is the isoleucine in the 33rd to 208th amino acid residues. Fc-binding protein in which valine is substituted with glutamic acid and 187th phenylalanine is substituted with serine (Fc-binding protein containing the amino acid sequence from the 33rd to the 208th of the amino acid sequence described in SEQ ID NO: 43).
(C) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid residue from the 33rd to the 208th amino acid residue, and in the amino acid residue from the 33rd to the 208th, the 45th phenylalanine is the isoleucine, the 64th Fc-binding protein in which glutamine is substituted with arginine, 133th valine is substituted with glutamic acid, and 187th phenylalanine is substituted with serine (amino acid sequence from 33rd to 208th of the amino acid sequence described in SEQ ID NO: 47) Fc binding protein).
(D) In the amino acid sequence of SEQ ID NO: 37, the amino acid residues from the 33rd to the 208th amino acid residues are included, and in the amino acid residues from the 33rd to the 208th, the 45th phenylalanine is the isoleucine, the 64th Of Fc-binding protein in which glutamine of arginine, 67th tyrosine is substituted with serine, 133rd valine is substituted with glutamic acid, and 187th phenylalanine is substituted with serine (33 of the amino acid sequence shown in SEQ ID NO: 51) Fc-binding protein comprising the amino acid sequence from position No. to position 208).
(E) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid residue from the 33rd to the 208th amino acid residue, and in the amino acid residue from the 33rd to the 208th, the 45th phenylalanine is the isoleucine, the 64th Fc-binding protein wherein SEQ ID NO: 55 is substituted with glutamine of arginine, 67th tyrosine with serine, 106th glutamine with arginine, 133rd valine with glutamic acid, and 187th phenylalanine with serine. Fc-binding protein comprising the amino acid sequence of the 33rd to 208th amino acids in the amino acid sequence described in 1).
(F) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid residue from the 33rd to the 208th amino acid residue, and in the amino acid residue from the 33rd to the 208th, the 37th glutamic acid is the glycine, the 39th Leucine is replaced with methionine, 45th phenylalanine with isoleucine, 64th glutamine with arginine, 133th valine with glutamic acid, 187th phenylalanine with serine, and 194th serine with arginine. Fc binding protein (Fc binding protein containing the amino acid sequence from the 33rd to 208th among the amino acid sequences of sequence number 63).
(G) including the 33rd to 208th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 37, and in the 33rd to 208th amino acid residues, the 37th glutamic acid is glycine, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 84th serine is proline, 133th valine is glutamic acid, 187th phenylalanine is serine, 194th serine Are Fc-binding proteins each substituted with arginine (Fc-binding protein comprising the amino acid sequence from the 33rd to the 208th of the amino acid sequence shown in SEQ ID NO: 67).
(H) In the amino acid sequence of SEQ ID NO: 37, the amino acid residue from the 33rd to the 208th amino acid residue, and the 37th glutamic acid in the amino acid residue from the 33rd to the 208th to the glycine, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 84th serine is proline, 133th valine is glutamic acid, 163rd glycine is valine, 187th phenylalanine Is an Fc-binding protein in which serine is substituted for serine and 194th serine is substituted for arginine (an Fc-binding protein comprising the amino acid sequence from the 33rd to the 208th of the amino acid sequence shown in SEQ ID NO: 69).
(I) The amino acid sequence of SEQ ID NO: 37 comprises the amino acid residues from the 33rd to the 208th, and the 37th glutamic acid is the glycine in the 33rd to 208th amino acid residues, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 133th valine Is a glutamic acid, 163rd glycine is replaced by valine, 187th phenylalanine is replaced by serine, and 194th serine is replaced by arginine (from the 33rd amino acid sequence of SEQ ID NO: 73) Amino acid sequence up to 208th Fc-binding protein that contains).
(J) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid sequence includes the 33rd to 208th amino acid residues, and in the 33rd to 208th amino acid residues, the 37th glutamic acid is glycine, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 133th valine Is glutamic acid, 156th threonine is isoleucine, 163rd glycine is valine, 174th tyrosine is histidine, 181st lysine is glutamic acid, 187th phenylalanine is serine, 194th serine is arginine In each Has been that Fc binding proteins (Fc-binding protein comprising an amino acid sequence from the 33 th of the amino acid sequence set forth to 208th in SEQ ID NO: 77).
(K) includes the 33rd to 208th amino acid residues in the amino acid sequence set forth in SEQ ID NO: 37, and the 37th glutamic acid is the glycine in the 33rd to 208th amino acid residues, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 98th asparagine Acid is glutamic acid, 117th glutamine is leucine, 133th valine is glutamic acid, 156th threonine is isoleucine, 163rd glycine is valine, 174th tyrosine is histidine, 181st lysine is In glutamic acid, 18 Fc binding protein in which the phenylalanine at position No. 1 is substituted with serine and the serine at position 194 is substituted with arginine (Fc binding protein comprising the amino acid sequence from the 33rd position to the 208th position in the amino acid sequence of SEQ ID NO: 83) .
(L) In the amino acid sequence shown in SEQ ID NO: 37, the amino acid sequence includes the 33rd to 208th amino acid residues. In the 33rd to 208th amino acid residues, the 37th glutamic acid is the glycine, the 39th Leucine is methionine, 45th phenylalanine is isoleucine, 64th glutamine is arginine, 67th tyrosine is histidine, 70th glutamic acid is aspartic acid, 84th serine is proline, 94th alanine Is serine, 98th aspartic acid is glutamic acid, 117th glutamine is leucine, 133th valine is glutamic acid, 156th threonine is isoleucine, 163rd glycine is valine, and 174th tyrosine is 181st in histidine Fc-binding protein in which lysine is substituted with glutamic acid, 187th phenylalanine is substituted with serine, 194th serine with arginine, 201st threonine with alanine, and 203rd asparagine with aspartic acid (SEQ ID NO: 89) Fc-binding protein comprising the amino acid sequence of the 33rd to 208th amino acids in the amino acid sequence described in 1).
　本発明のＦｃ結合性タンパク質は、そのＮ末端側またはＣ末端側に、夾雑物質存在下の溶液から分離する際に有用なオリゴペプチドをさらに付加してもよい。前記オリゴペプチドとしては、ポリヒスチジン、ポリリジン、ポリアルギニン、ポリグルタミン酸、ポリアスパラギン酸等があげられる。また本発明のＦｃ結合性タンパク質をクロマトグラフィー用の支持体等の固相に固定化する際に有用な、システインを含むオリゴペプチドを、本発明のＦｃ結合性タンパク質のＮ末端側またはＣ末端側にさらに付加してもよい。Ｆｃ結合性タンパク質のＮ末端側またはＣ末端側に付加するオリゴペプチドの長さは、本発明のＦｃ結合性タンパク質のＩｇＧ結合性や安定性を損なわない限り特に制限はない。前記オリゴペプチドを本発明のＦｃ結合性タンパク質に付加させる際は、前記オリゴペプチドをコードするポリヌクレオチドを作製後、当業者に周知の方法を用いて遺伝子工学的にＦｃ結合性タンパク質のＮ末端側またはＣ末端側に付加させてもよいし、化学的に合成した前記オリゴペプチドを本発明のＦｃ結合性タンパク質のＮ末端側またはＣ末端側に化学的に結合させて付加させてもよい。さらに本発明のＦｃ結合性タンパク質のＮ末端側には、宿主での効率的な発現を促すためのシグナルペプチドを付加してもよい。宿主が大腸菌の場合における前記シグナルペプチドの例としては、ＰｅｌＢ（配列番号１０１）、ＤｓｂＡ、ＭａｌＥ（ＵｎｉＰｒｏｔ　Ｎｏ．Ｐ０ＡＥＸ９に記載のアミノ酸配列のうち１番目から２６番目までの領域）、ＴｏｒＴなどといったペリプラズムにタンパク質を分泌させるシグナルペプチドを例示することができる（特開２０１１－０９７８９８号公報）。 The Fc-binding protein of the present invention may further be added with an oligopeptide useful for separation from a solution in the presence of a contaminant substance on the N-terminal side or C-terminal side. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid and the like. Further, an oligopeptide containing cysteine useful for immobilizing the Fc-binding protein of the present invention on a solid phase such as a support for chromatography is used as an N-terminal side or C-terminal side of the Fc-binding protein of the present invention. It may be further added to. The length of the oligopeptide added to the N-terminal side or C-terminal side of the Fc binding protein is not particularly limited as long as the IgG binding property and stability of the Fc binding protein of the present invention are not impaired. When the oligopeptide is added to the Fc-binding protein of the present invention, a polynucleotide encoding the oligopeptide is prepared and then genetically engineered using a method well known to those skilled in the art to the N-terminal side of the Fc-binding protein. Alternatively, it may be added to the C-terminal side, or the chemically synthesized oligopeptide may be added by chemically binding to the N-terminal side or C-terminal side of the Fc-binding protein of the present invention. Furthermore, a signal peptide for promoting efficient expression in the host may be added to the N-terminal side of the Fc binding protein of the present invention. Examples of the signal peptide when the host is Escherichia coli include periplasm such as PelB (SEQ ID NO: 101), DsbA, MalE (the first to 26th region of the amino acid sequence described in UniProt No. P0AEX9), TorT, and the like. An example is a signal peptide that secretes a protein (Japanese Patent Laid-Open No. 2011-097898).
　本発明のＦｃ結合性タンパク質には糖鎖を有していても有していなくても構わない。糖鎖を有するＦｃ結合性タンパク質を得るためには、動物細胞、酵母や昆虫細胞等を宿主として用いればよい。さらに人工的に合成した糖鎖を修飾してもよい。また、糖鎖を有していないＦｃ結合性タンパク質を得るためには、大腸菌等糖鎖付加が起こらない宿主として用いればよい。さらに糖鎖を有したＦｃ結合性タンパク質から糖鎖を除去する操作を行なうことで、糖鎖を有していないＦｃ結合性タンパク質を得ることもできる。 The Fc binding protein of the present invention may or may not have a sugar chain. In order to obtain an Fc-binding protein having a sugar chain, animal cells, yeasts, insect cells and the like may be used as hosts. Furthermore, an artificially synthesized sugar chain may be modified. Further, in order to obtain an Fc-binding protein having no sugar chain, it may be used as a host in which sugar chain addition does not occur, such as Escherichia coli. Further, by performing an operation of removing a sugar chain from an Fc binding protein having a sugar chain, an Fc binding protein having no sugar chain can be obtained.
　本発明のポリヌクレオチドの作製方法の一例として、
（Ｉ）本発明のＦｃ結合性タンパク質のアミノ酸配列からヌクレオチド配列に変換し、当該ヌクレオチド配列を含むポリヌクレオチドを人工的に合成する方法や、
（ＩＩ）Ｆｃ結合性タンパク質の全体または部分配列を含むポリヌクレオチドを直接人工的に、またはＦｃ結合性タンパク質のｃＤＮＡ等からＰＣＲ法といったＤＮＡ増幅法を用いて調製し、調製した当該ポリヌクレオチドを適当な方法で連結する方法、が例示できる。
前記（Ｉ）の方法において、アミノ酸配列からヌクレオチド配列に変換する際、形質転換させる宿主におけるコドンの使用頻度を考慮して変換するのが好ましい。一例として、宿主が大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）の場合は、アルギニン（Ａｒｇ）ではＡＧＡ／ＡＧＧ／ＣＧＧ／ＣＧＡが、イソロイシン（Ｉｌｅ）ではＡＴＡが、ロイシン（Ｌｅｕ）ではＣＴＡが、グリシン（Ｇｌｙ）ではＧＧＡが、プロリン（Ｐｒｏ）ではＣＣＣが、それぞれ使用頻度が少ないため（いわゆるレアコドンであるため）、それらのコドンを避けるように変換すればよい。コドンの使用頻度の解析は公的データベース（例えば、かずさＤＮＡ研究所のホームページにあるＣｏｄｏｎ　Ｕｓａｇｅ　Ｄａｔａｂａｓｅなど）を利用することによっても可能である。 As an example of a method for producing the polynucleotide of the present invention,
(I) a method of artificially synthesizing a polynucleotide containing the nucleotide sequence by converting the amino acid sequence of the Fc-binding protein of the present invention into a nucleotide sequence,
(II) A polynucleotide containing the whole or a partial sequence of an Fc binding protein is prepared artificially or from a cDNA of the Fc binding protein using a DNA amplification method such as PCR, and the prepared polynucleotide is appropriately used. The method of connecting by various methods can be illustrated.
In the method (I), when converting from an amino acid sequence to a nucleotide sequence, the conversion is preferably performed in consideration of the frequency of codon usage in the host to be transformed. For example, when the host is Escherichia coli, AGA / AGG / CGG / CGA is used for arginine (Arg), ATA is used for isoleucine (Ile), CTA is used for leucine (Leu), and GGA is used for glycine (Gly). However, since CCC is less frequently used in proline (Pro) (because it is a so-called rare codon), it may be converted so as to avoid those codons. Analysis of codon usage frequency can also be performed by using a public database (for example, Codon Usage Database on the website of Kazusa DNA Research Institute).
　本発明のポリヌクレオチドへ変異を導入する場合、エラープローンＰＣＲ法を用いることができる。エラープローンＰＣＲ法における反応条件は、Ｆｃ結合性タンパク質をコードするポリヌクレオチドに所望の変異を導入できる条件であれば特に限定はなく、例えば、基質である４種類のデオキシヌクレオチド（ｄＡＴＰ／ｄＴＴＰ／ｄＣＴＰ／ｄＧＴＰ）の濃度を不均一にし、ＭｎＣｌ２を０．０１から１０ｍＭ（好ましくは０．１から１ｍＭ）の濃度でＰＣＲ反応液に添加してＰＣＲを行なうことで、ポリヌクレオチドに変異を導入することができる。またエラープローンＰＣＲ法以外の変異導入方法としては、Ｆｃ結合性タンパク質の全体または部分配列を含むポリヌクレオチドに、変異原となる薬剤を接触・作用させたり、紫外線を照射したりして、ポリヌクレオチドに変異を導入して作製する方法があげられる。当該方法において変異原として使用する薬剤としては、ヒドロキシルアミン、Ｎ－メチル－Ｎ’－ニトロ－Ｎ－ニトロソグアニジン、亜硝酸、亜硫酸、ヒドラジン等、当業者が通常用いる変異原性薬剤を用いればよい。 When introducing a mutation into the polynucleotide of the present invention, an error-prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as a desired mutation can be introduced into a polynucleotide encoding an Fc binding protein. For example, four types of deoxynucleotides (dATP / dTTP / dCTP) that are substrates are used. / DGTP) concentration is heterogeneous, and MnCl 2 is added to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to introduce a mutation into the polynucleotide. be able to. Further, as a method for introducing a mutation other than the error-prone PCR method, the polynucleotide containing the entire or partial sequence of the Fc-binding protein is contacted / acted with a drug as a mutagen, or irradiated with ultraviolet rays, to obtain a polynucleotide. And a method of producing a mutation by introducing a mutation. As a drug used as a mutagen in this method, a mutagenic drug commonly used by those skilled in the art such as hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine, nitrous acid, sulfite, hydrazine may be used. .
　本発明のＦｃ結合性タンパク質を発現させる宿主には特に制限はなく、一例として、動物細胞（ＣＨＯ細胞、ＨＥＫ細胞、Ｈｅｌａ細胞、ＣＯＳ細胞等）、酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅ、Ｐｉｃｈｉａ　ｐａｓｔｏｒｉｓ、Ｈａｎｓｅｎｕｌａ　ｐｏｌｙｍｏｒｐｈａ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｊａｐｏｎｉｃｕｓ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｏｃｔｏｓｐｏｒｕｓ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ等）、昆虫細胞（Ｓｆ９、Ｓｆ２１等）、大腸菌（ＪＭ１０９株、ＢＬ２１（ＤＥ３）株、Ｗ３１１０株等）や枯草菌があげられる。なお動物細胞や大腸菌を宿主として用いると生産性の面で好ましく、大腸菌を宿主として用いるとさらに好ましい。 The host that expresses the Fc-binding protein of the present invention is not particularly limited, and examples thereof include animal cells (CHO cells, HEK cells, Hela cells, COS cells, etc.), yeasts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schomisacios). and japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, insect cells (Sf9, Sf21 etc.), E. coli (JM109 strain, BL21 (DE3) strain, W3110 strain etc.) and Bacillus subtilis. Use of animal cells or Escherichia coli as a host is preferable in terms of productivity, and more preferable when Escherichia coli is used as a host.
　本発明のポリヌクレオチドを用いて宿主を形質転換する場合、本発明のポリヌクレオチドそのものを用いてもよいが、発現ベクター（例えば、原核細胞や真核細胞の形質転換に通常用いるバクテリオファージ、コスミドやプラスミド等）の適切な位置に本発明のポリヌクレオチドを挿入したものを用いると、より好ましい。なお当該発現ベクターは、形質転換する宿主内で安定に存在し複製できるものであれば特に制限はなく、大腸菌を宿主とする場合は、ｐＥＴプラスミドベクター、ｐＵＣプラスミドベクター、ｐＴｒｃプラスミドベクター、ｐＣＤＦプラスミドベクター、ｐＢＢＲプラスミドベクターを例示することができる。また前記適切な位置とは、発現ベクターの複製機能、所望の抗生物質マーカー、伝達性に関わる領域を破壊しない位置を意味する。前記発現ベクターに本発明のポリヌクレオチドを挿入する際は、発現に必要なプロモータといった機能性ポリヌクレオチドに連結される状態で挿入すると好ましい。当該プロモータの例として、宿主が大腸菌の場合は、ｔｒｐプロモータ、ｔａｃプロモータ、ｔｒｃプロモータ、ｌａｃプロモータ、Ｔ７プロモータ、ｒｅｃＡプロモータ、ｌｐｐプロモータ、さらにはλファージのλＰＬプロモータ、λＰＲプロモータがあげられ、宿主が動物細胞の場合は、ＳＶ４０プロモーター、ＣＭＶプロモーター、ＣＡＧプロモーターがあげられる。 When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but an expression vector (for example, bacteriophage, cosmid, or the like commonly used for transformation of prokaryotic cells or eukaryotic cells) may be used. It is more preferable to use a plasmid or the like in which the polynucleotide of the present invention is inserted at an appropriate position. The expression vector is not particularly limited as long as it is stably present in the host to be transformed and can be replicated. When Escherichia coli is used as a host, pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector A pBBR plasmid vector can be exemplified. The appropriate position means a position where the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility are not destroyed. When inserting the polynucleotide of the present invention into the expression vector, it is preferable to insert it in a state linked to a functional polynucleotide such as a promoter required for expression. Examples of the promoter include trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, λ phage λPL promoter, λPR promoter when the host is Escherichia coli. In the case of animal cells, examples include SV40 promoter, CMV promoter, and CAG promoter.
　前記方法により作製した、本発明のポリヌクレオチドを挿入した発現ベクター（以下、本発明の発現ベクターとする）を用いて宿主を形質転換するには、当業者が通常用いる方法で行なえばよい。例えば、宿主としてＥｓｃｈｅｒｉｃｈｉａ属に属する微生物（大腸菌ＪＭ１０９株、大腸菌ＢＬ２１（ＤＥ３）株、大腸菌Ｗ３１１０株等）を選択する場合には、公知の文献（例えば、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ，２５６，１９９２）に記載の方法等により形質転換すればよい。なお宿主が動物細胞である場合にはエレクトロポレーションやリポフェクションを用いればよい。前述した方法で形質転換して得られた形質転換体は、適切な方法でスクリーニングすることにより、本発明のＦｃ結合性タンパク質を発現可能な形質転換体（以下、本発明の形質転換体とする）を取得することができる。 In order to transform a host using the expression vector inserted with the polynucleotide of the present invention (hereinafter referred to as the expression vector of the present invention) produced by the above-described method, a person skilled in the art may carry out the method. For example, when a microorganism belonging to the genus Escherichia (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) is selected as a host, known literature (eg, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). And the like. When the host is an animal cell, electroporation or lipofection may be used. By transforming the transformant obtained by the above-described method with an appropriate method, a transformant capable of expressing the Fc-binding protein of the present invention (hereinafter referred to as the transformant of the present invention). ) Can be obtained.
　本発明の形質転換体から本発明の発現ベクターを調製するには、形質転換に用いた宿主に適した方法で、本発明の形質転換体から本発明の発現ベクターを抽出し調製すればよい。
例えば、本発明の形質転換体の宿主が大腸菌の場合、形質転換体を培養して得られる培養物からアルカリ抽出法またはＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）等の市販の抽出キットを用いて調製すればよい。 In order to prepare the expression vector of the present invention from the transformant of the present invention, the expression vector of the present invention may be extracted from the transformant of the present invention and prepared by a method suitable for the host used for transformation.
For example, when the host of the transformant of the present invention is Escherichia coli, it can be prepared from a culture obtained by culturing the transformant using an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (Qiagen). That's fine.
　本発明の形質転換体を培養し、得られた培養物から本発明のＦｃ結合性タンパク質を回収することで、本発明のＦｃ結合性タンパク質を製造することができる。なお本明細書において培養物とは、培養された本発明の形質転換体の細胞そのもののほか、培養に用いた培地も含まれる。本発明のタンパク質製造方法で用いる形質転換体は、対象宿主の培養に適した培地で培養すればよく、宿主が大腸菌の場合は、必要な栄養源を補ったＬＢ（Ｌｕｒｉａ－Ｂｅｒｔａｎｉ）培地が好ましい培地の一例としてあげられる。なお、本発明の発現ベクターの導入の有無により本発明の形質転換体を選択的に増殖させるために、培地に当該ベクターに含まれる薬剤耐性遺伝子に対応した薬剤を添加して培養すると好ましい。例えば、当該ベクターがカナマイシン耐性遺伝子を含んでいる場合は、培地にカナマイシンを添加すればよい。また培地には、炭素、窒素および無機塩供給源の他に、適当な栄養源を添加してもよく、所望により、グルタチオン、システイン、シスタミン、チオグリコレートおよびジチオスレイトールからなる群から選択される一種類以上の還元剤を含んでもよい。さらにグリシンといった前記形質転換体から培養液へのタンパク質分泌を促す試薬を添加してもよく、具体的には、宿主が大腸菌の場合、培地に対してグリシンを２％（ｗ／ｖ）以下で添加すると好ましい。培養温度は宿主が大腸菌の場合、一般に１０℃から４０℃、好ましくは２０℃から３７℃、より好ましくは２５℃前後であるが、発現させるタンパク質の特性により選択すればよい。培地のｐＨは宿主が大腸菌の場合、ｐＨ６．８からｐＨ７．４、好ましくはｐＨ７．０前後である。また本発明のベクターに誘導性のプロモータが含まれている場合は、本発明のＦｃ結合性タンパク質が良好に発現できるような条件下で誘導をかけると好ましい。誘導剤としてはＩＰＴＧ（ｉｓｏｐｒｏｐｙｌ－β－Ｄ－ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ）を例示することができる。宿主が大腸菌の場合、培養液の濁度（６００ｎｍにおける吸光度）を測定し、約０．５から１．０となったときに適当量のＩＰＴＧを添加後、引き続き培養することで、Ｆｃ結合性タンパク質の発現を誘導することができる。ＩＰＴＧの添加濃度は０．００５から１．０ｍＭの範囲から適宜選択すればよいが、０．０１から０．５ｍＭの範囲が好ましい。ＩＰＴＧ誘導に関する種々の条件は当該技術分野において周知の条件で行なえばよい。 The Fc-binding protein of the present invention can be produced by culturing the transformant of the present invention and recovering the Fc-binding protein of the present invention from the obtained culture. In the present specification, the culture includes not only the cultured cells of the transformant of the present invention itself but also a medium used for the culture. The transformant used in the protein production method of the present invention may be cultured in a medium suitable for culturing the target host. When the host is Escherichia coli, an LB (Luria-Bertani) medium supplemented with necessary nutrient sources is preferable. An example of the medium is given. In order to selectively proliferate the transformant of the present invention depending on the presence or absence of introduction of the expression vector of the present invention, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the medium and culture. For example, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to the carbon, nitrogen and inorganic salt sources, an appropriate nutrient source may be added to the medium, and if desired, the medium is selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol. One or more reducing agents may be included. Furthermore, a reagent that promotes protein secretion from the transformant to the culture solution such as glycine may be added. Specifically, when the host is Escherichia coli, glycine is contained in the medium at 2% (w / v) or less. Addition is preferred. When the host is Escherichia coli, the culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., more preferably around 25 ° C., but may be selected depending on the characteristics of the protein to be expressed. When the host is Escherichia coli, the pH of the medium is pH 6.8 to pH 7.4, preferably around pH 7.0. In addition, when an inducible promoter is included in the vector of the present invention, it is preferable that the induction be performed under conditions that allow the Fc-binding protein of the present invention to be expressed well. Examples of the inducer include IPTG (isopropyl-β-D-thiogalactopyranoside). When the host is Escherichia coli, the turbidity (absorbance at 600 nm) of the culture solution is measured, and when it reaches about 0.5 to 1.0, an appropriate amount of IPTG is added, followed by further culturing, thereby allowing Fc binding. Protein expression can be induced. The addition concentration of IPTG may be appropriately selected from the range of 0.005 to 1.0 mM, but is preferably in the range of 0.01 to 0.5 mM. Various conditions relating to the IPTG induction may be performed under conditions well known in the art.
　本発明の形質転換体を培養して得られた培養物から本発明のＦｃ結合性タンパク質を回収するには、本発明の形質転換体における本発明のＦｃ結合性タンパク質の発現形態に適した方法で、当該培養物から分離／精製して本発明のＦｃ結合性タンパク質を回収すればよい。例えば、培養上清に発現する場合は菌体を遠心分離操作によって分離し、得られる培養上清から本発明のＦｃ結合性タンパク質を精製すればよい。また、細胞内（ペリプラズムを含む）に発現する場合には、遠心分離操作により菌体を集めた後、酵素処理剤や界面活性剤等を添加したり、超音波やフレンチプレス等を用いて菌体を破砕して本発明のＦｃ結合性タンパク質を抽出した後、精製すればよい。本発明のＦｃ結合性タンパク質を精製するには、当該技術分野において公知の方法を用いればよく、一例として液体クロマトグラフィーを用いた分離／精製があげられる。液体クロマトグラフィーには、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、ゲルろ過クロマトグラフィー、アフィニティークロマトグラフィー等があり、これらのクロマトグラフィーを組み合わせて精製操作を行なうことにより、本発明のＦｃ結合性タンパク質を高純度に調製することができる。 In order to recover the Fc-binding protein of the present invention from the culture obtained by culturing the transformant of the present invention, a method suitable for the expression form of the Fc-binding protein of the present invention in the transformant of the present invention Thus, the Fc-binding protein of the present invention may be recovered by separating / purifying from the culture. For example, when expressed in the culture supernatant, the cells are separated by centrifugation, and the Fc-binding protein of the present invention may be purified from the obtained culture supernatant. In the case of expression in cells (including periplasm), the bacterial cells are collected by centrifugation, and then added with an enzyme treatment agent, a surfactant, etc., or by using ultrasonic waves, a French press, etc. What is necessary is just to refine | purify, after crushing a body and extracting Fc binding protein of this invention. In order to purify the Fc-binding protein of the present invention, a method known in the art may be used, and an example is separation / purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. By performing a purification operation by combining these chromatography, the Fc binding property of the present invention can be obtained. Proteins can be prepared with high purity.
　得られた本発明のＦｃ結合性タンパク質のＩｇＧに対する結合活性を測定する方法としては、例えばＩｇＧに対する結合活性をＥｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｏｒｂｅｎｔ　Ａｓｓａｙ（以下、ＥＬＩＳＡと表記）法や表面プラズモン共鳴法などを用いて測定すればよい。結合活性の測定に使用するＩｇＧは、ヒトＩｇＧが好ましく、ヒトＩｇＧ１やヒトＩｇＧ３が特に好ましい。 As a method for measuring the binding activity of the obtained Fc-binding protein of the present invention to IgG, for example, the binding activity to IgG is measured using the Enzyme-Linked ImmunoSorbent Assay (hereinafter referred to as ELISA) method or the surface plasmon resonance method. Just measure. The IgG used for the measurement of the binding activity is preferably human IgG, and human IgG1 and human IgG3 are particularly preferable.
　本発明のＦｃ結合性タンパク質を不溶性担体に結合させることで、本発明の吸着剤を製造することができる。前記不溶性担体には特に限定はなく、アガロース、アルギネート（アルギン酸塩）、カラゲナン、キチン、セルロース、デキストリン、デキストラン、デンプンといった多糖質を原料とした担体や、ポリビニルアルコール、ポリメタクレート、ポリ（２－ヒドロキシエチルメタクリレート）、ポリウレタン、ポリアクリル酸、ポリスチレン、ポリアクリルアミド、ポリメタクリルアミド、ビニルポリマーといった合成高分子を原料とした担体や、ジルコニア、ゼオライト、シリカ、皮膜シリカといったセラミックスを原料とした担体が例示できる。中でも、多糖質を原料とした担体や合成高分子を原料とした担体が不溶性担体として好ましい。前記好ましい担体の一例として、トヨパール（東ソー製）等の水酸基を導入したポリメタクリレートゲル、Ｓｅｐｈａｒｏｓｅ（ＧＥヘルスケア製）等のアガロースゲル、セルファイン（ＪＮＣ製）等のセルロースゲルがあげられる。不溶性担体の形状については特に限定はなく、粒状物または非粒状物、多孔性または非多孔性、いずれであってもよい。 The adsorbent of the present invention can be produced by binding the Fc binding protein of the present invention to an insoluble carrier. The insoluble carrier is not particularly limited, and carriers made from polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextran, starch, polyvinyl alcohol, polymethacrylate, poly (2- Examples include carriers made of synthetic polymers such as hydroxyethyl methacrylate), polyurethane, polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, and vinyl polymers, and carriers made of ceramics such as zirconia, zeolite, silica, and coated silica. it can. Of these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carrier include polymethacrylate gel introduced with a hydroxyl group such as Toyopearl (manufactured by Tosoh Corporation), agarose gel such as Sepharose (manufactured by GE Healthcare), and cellulose gel such as Cellufine (manufactured by JNC). The shape of the insoluble carrier is not particularly limited, and may be granular or non-particulate, porous or non-porous.
　Ｆｃ結合性タンパク質を不溶性担体に固定化するには、不溶性担体にＮ－ヒドロキシコハク酸イミド（ＮＨＳ）活性化エステル基、エポキシ基、カルボキシル基、マレイミド基、ハロアセチル基、トレシル基、ホルミル基、ハロアセトアミド等の活性基を付与し、当該活性基を介してヒトＦｃ結合性タンパク質と不溶性担体とを共有結合させることで固定化すればよい。活性基を付与した担体は市販の担体をそのまま用いてもよいし、適切な反応条件で担体表面に活性基を導入して調製してもよい。活性基を付与した市販の担体としてはＴＯＹＯＰＥＡＲＬ　ＡＦ－Ｅｐｏｘｙ－６５０Ｍ、ＴＯＹＯＰＥＡＲＬ　ＡＦ－Ｔｒｅｓｙｌ－６５０Ｍ（いずれも東ソー製）、ＨｉＴｒａｐ　ＮＨＳ－ａｃｔｉｖａｔｅｄ　ＨＰ　Ｃｏｌｕｍｎｓ、ＮＨＳ－ａｃｔｉｖａｔｅｄ　Ｓｅｐｈａｒｏｓｅ　４　Ｆａｓｔ　Ｆｌｏｗ、Ｅｐｏｘｙ－ａｃｔｉｖａｔｅｄ　Ｓｅｐｈａｒｏｓｅ　６Ｂ（いずれもＧＥヘルスケア製）、ＳｕｌｆｏＬｉｎｋ　Ｃｏｕｐｌｉｎｇ　Ｒｅｓｉｎ（サーモサイエンティフィック製）が例示できる。 In order to immobilize the Fc binding protein on an insoluble carrier, N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, halo What is necessary is just to fix | immobilize by providing active groups, such as acetamide, and covalently bonding a human Fc binding protein and an insoluble carrier through the said active group. The carrier provided with the active group may be a commercially available carrier as it is, or may be prepared by introducing an active group on the surface of the carrier under appropriate reaction conditions. Commercially available carriers to which an active group has been added include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (all manufactured by Tosoh), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4F-SepacteF 4eFeSeFeSeFeSeFeSeFeSeFeSeFeSeFeSeFeSeFeSe? (Both manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Scientific).
　一方、担体表面に活性基を導入する方法としては、担体表面に存在する水酸基やエポキシ基、カルボキシル基、アミノ基等に対して２個以上の活性部位を有する化合物の一方を反応させる方法が例示できる。当該化合物の一例のうち、担体表面の水酸基やアミノ基にエポキシ基を導入する化合物としては、エピクロロヒドリン、エタンジオールジグリシジルエーテル、ブタンジオールジグリシジルエーテル、ヘキサンジオールジグリシジルエーテルが例示できる。前記化合物により担体表面にエポキシ基を導入した後、担体表面にカルボキシル基を導入する化合物としては、２－メルカプト酢酸、３－メルカプトプロピオン酸、４－メルカプト酪酸、６－メルカプト酪酸、グリシン、３－アミノプロピオン酸、４－アミノ酪酸、６－アミノヘキサン酸を例示できる。 On the other hand, examples of the method for introducing an active group on the surface of the carrier include a method in which one of compounds having two or more active sites reacts with a hydroxyl group, an epoxy group, a carboxyl group, an amino group, etc. present on the surface of the carrier. it can. Among examples of the compound, examples of the compound that introduces an epoxy group into the hydroxyl group or amino group on the surface of the carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of the compound that introduces an epoxy group on the carrier surface with the compound and then introduces a carboxyl group on the carrier surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3- Examples thereof include aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.
　担体表面に存在する水酸基やエポキシ基、カルボキシル基、アミノ基にマレイミド基を導入する化合物としては、Ｎ－（ε－マレイミドカプロン酸）ヒドラジド、Ｎ－（ε－マレイミドプロピオン酸）ヒドラジド、４－［４－Ｎ－マレイミドフェニル］酢酸ヒドラジド、２－アミノマレイミド、３－アミノマレイミド、４－アミノマレイミド、６－アミノマレイミド、１－（４－アミノフェニル）マレイミド、１－（３－アミノフェニル）マレイミド、４－（マレイミド）フェニルイソシアナート、２－マレイミド酢酸、３－マレイミドプロピオン酸、４－マレイミド酪酸、６－マレイミドヘキサン酸、（Ｎ－［α―マレイミドアセトキシ］スクシンイミドエステル）、（ｍ－マレイミドベンゾイル）Ｎ－ヒドロキシスクシンイミドエステル、（スクシンイミジル－４－［マレイミドメチル］シクロヘキサンー１－カルボニル－［６－アミノヘキサン酸］）、（スクシンイミジル－４－［マレイミドメチル］シクロヘキサンー１－カルボン酸）、（ｐ－マレイミドベンゾイル）Ｎ－ヒドロキシスクシンイミドエステル、（ｍ－マレイミドベンゾイル）Ｎ－ヒドロキシスクシンイミドエステル、３－マレイミドプロピオン酸Ｎ－スクシンイミジルを例示できる。 Examples of the compound that introduces a maleimide group into the hydroxyl group, epoxy group, carboxyl group or amino group present on the surface of the carrier include N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidopropionic acid) hydrazide, 4- [ 4-N-maleimidophenyl] acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (maleimido) phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, (N- [α-maleimidoacetoxy] succinimide ester), (m-maleimidobenzoyl) N-hydroxysuccinimide ester (Succinimidyl-4- [maleimidomethyl] cyclohexane-1-carbonyl- [6-aminohexanoic acid]), (succinimidyl-4- [maleimidomethyl] cyclohexane-1-carboxylic acid), (p-maleimidobenzoyl) N-hydroxy Examples thereof include succinimide ester, (m-maleimidobenzoyl) N-hydroxysuccinimide ester, and 3-maleimidopropionate N-succinimidyl.
　担体表面に存在する水酸基やアミノ基にハロアセチル基を導入する化合物としては、クロロ酢酸、ブロモ酢酸、ヨード酢酸、クロロ酢酸クロリド、ブロモ酢酸クロリド、ブロモ酢酸ブロミド、クロロ酢酸無水物、ブロモ酢酸無水物、ヨード酢酸無水物、２－（ヨードアセトアミド）酢酸－Ｎ－ヒドロキシスクシンイミドエステル、３－（ブロモアセトアミド）プロピオン酸－Ｎ－ヒドロキシスクシンイミドエステル、４－（ヨードアセチル）アミノ安息香酸－Ｎ－ヒドロキシスクシンイミドエステルを例示できる。なお担体表面に存在する水酸基やアミノ基にω－アルケニルアルカングリシジルエーテルを反応させた後、ハロゲン化剤でω－アルケニル部位をハロゲン化し活性化する方法も例示できる。ω－アルケニルアルカングリシジルエーテルとしては、アリルグリシジルエーテル、３－ブテニルグリシジルエーテル、４－ペンテニルグリシジルエーテルを例示でき、ハロゲン化剤としてはＮ－クロロスクシンイミド、Ｎ－ブロモスクシンイミド、Ｎ－ヨードスクシンイミドを例示できる。 Compounds that introduce a haloacetyl group into the hydroxyl group or amino group present on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, Iodoacetic anhydride, 2- (iodoacetamido) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamido) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be illustrated. An example is a method in which ω-alkenyl alkanglycidyl ether is reacted with a hydroxyl group or amino group present on the surface of the carrier, and then the ω-alkenyl moiety is halogenated with a halogenating agent to activate. Examples of ω-alkenyl alkanglycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.
　担体表面に活性基を導入する方法の別の例として、担体表面に存在するカルボキシル基に対して縮合剤と添加剤を用いて活性化基を導入する方法がある。縮合剤としては１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）、ジシクロヘキシルカルボジアミド、カルボニルジイミダゾールを例示できる。また添加剤としてはＮ－ヒドロキシコハク酸イミド（ＮＨＳ）、４－ニトロフェノール、１－ヒドロキシベンズトリアゾールを例示できる。 As another example of the method for introducing an active group on the surface of the carrier, there is a method for introducing an activating group into the carboxyl group present on the surface of the carrier using a condensing agent and an additive. Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.
　また前述した以外に、担体表面に活性基を導入する化合物としては、トレシルクロリド（活性化基としてトレシル基を形成）、ビニルブロミド（活性化基としてビニル基を形成）を例示することができる。 In addition to those described above, examples of the compound that introduces an active group on the surface of the carrier include tresyl chloride (forms a tresyl group as an activating group) and vinyl bromide (forms a vinyl group as an activating group). .
　本発明のＦｃ結合性タンパク質を不溶性担体に固定化する際用いる緩衝液としては、酢酸緩衝液、リン酸緩衝液、ＭＥＳ緩衝液、ＨＥＰＥＳ緩衝液、トリス緩衝液、ホウ酸緩衝液を例示できる。固定化させるときの反応温度は、５℃から５０℃までの温度範囲の中から活性基の反応性や本発明のＦｃ結合性タンパク質の安定性を考慮の上、適宜設定すればよく、好ましくは１０℃から３５℃の範囲である。 Examples of the buffer used when the Fc-binding protein of the present invention is immobilized on an insoluble carrier include acetate buffer, phosphate buffer, MES buffer, HEPES buffer, Tris buffer, and borate buffer. The reaction temperature at the time of immobilization may be appropriately set in consideration of the reactivity of the active group and the stability of the Fc-binding protein of the present invention from the temperature range from 5 ° C. to 50 ° C., preferably It is in the range of 10 ° C to 35 ° C.
　本発明の分離方法は、Ｆｃ結合性タンパク質を固定化した不溶性担体を充填したカラムに平衡化液を添加してカラムを平衡化する工程と、前記平衡化したカラムに抗体を含む溶液を添加して前記抗体を前記担体に吸着させる工程と、前記担体に吸着した抗体を溶出液を用いて溶出させる工程とを含む、抗体の分離方法において、前記平衡化液が３０ｍＭ以上の塩化物イオンまたは硫酸イオンを含むことを特徴としている。本発明により、抗体成分の分離度をＲｓ値換算で１．１倍から１．８倍に向上させることができる。従って、今まで検出できなかった抗体分子構造の微小な差異も検出でき、分析の精度を向上させることができる。なお前記平衡化液に含まれる塩化物イオンまたは硫酸イオンの濃度は３０ｍＭ以上であればよいが、３０ｍＭ以上１５００ｍＭ以下であると好ましく、３０ｍＭ以上１０００ｍＭ以下であるとより好ましく、３０ｍＭ以上５００ｍＭ以下であるとさらに好ましく、５０ｍＭ以上５００ｍＭ以下であるとさらにより好ましい。 The separation method of the present invention comprises a step of equilibrating a column by adding an equilibration solution to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized, and a solution containing an antibody is added to the equilibrated column. In the antibody separation method, the step of adsorbing the antibody to the carrier and the step of eluting the antibody adsorbed to the carrier using an eluate, the equilibration solution is 30 mM or more of chloride ions or sulfuric acid. It is characterized by containing ions. According to the present invention, the degree of separation of antibody components can be improved from 1.1 times to 1.8 times in terms of Rs value. Therefore, minute differences in antibody molecular structures that could not be detected can be detected, and the accuracy of analysis can be improved. The concentration of chloride ion or sulfate ion contained in the equilibration solution may be 30 mM or more, preferably 30 mM or more and 1500 mM or less, more preferably 30 mM or more and 1000 mM or less, and 30 mM or more and 500 mM or less. And more preferably 50 mM or more and 500 mM or less.
　前記平衡化液を用いて前記吸着した抗体を溶出させるには、前記抗体とＦｃ結合性タンパク質との親和性を弱める溶出液を用いて溶出させればよい。一例として、平衡化液として３０ｍＭ以上の塩化物イオンまたは硫酸イオンを含むｐＨ５．０から６．９の弱酸性緩衝液を、溶出液としてｐＨ２．５から４．５の酸性緩衝液をそれぞれ用いたグラジエント溶出法があげられる。緩衝剤としては公知の緩衝剤の中から、作成する緩衝液のｐＨなどに基づき適宜選択すればよく、一例として、リン酸、酢酸、ギ酸、ＭＥＳ（２－Ｍｏｒｐｈｏｌｉｎｏｅｔｈａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ）、ＭＯＰＳ（３－Ｍｏｒｐｈｏｌｉｎｏｐｒｏｐａｎｅｓｕｌｆｏｎｉｃ　ａｃｉｄ）、クエン酸、コハク酸、グリシン、ピペラジンがあげられる。 In order to elute the adsorbed antibody using the equilibration solution, it may be eluted using an elution solution that weakens the affinity between the antibody and the Fc-binding protein. As an example, a weakly acidic buffer solution having a pH of 5.0 to 6.9 containing 30 mM or more of chloride ions or sulfate ions was used as an equilibration solution, and an acidic buffer solution having a pH of 2.5 to 4.5 was used as an eluent. A gradient elution method is mentioned. The buffer may be appropriately selected from known buffers based on the pH of the buffer to be prepared. Examples of the buffer include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinosulfonic acid), and MOPS (3-Morpholinosulfonic sulfonic acid). acid), citric acid, succinic acid, glycine, and piperazine.
　本発明の分離方法は、Ｆｃ結合性タンパク質と親和性を有する、糖鎖を付加した抗体のＦｃ領域を少なくとも含んだ抗体であれば分離することができる。一例として、抗体医薬に用いる抗体として一般的に用いられているキメラ抗体、ヒト化抗体、ヒト抗体やそれらのアミノ酸置換体があげられる。また二重特異性抗体（バイスペシフィック抗体）、糖鎖を付加した抗体のＦｃ領域と他のタンパク質との融合抗体、糖鎖を付加した抗体のＦｃ領域と薬物との複合体（ＡＤＣ）などの人工的に構造改変した抗体であっても、本発明の分離方法で分離することができる。 The separation method of the present invention can be separated if it is an antibody having at least the Fc region of an antibody to which a sugar chain is added and has an affinity for an Fc binding protein. Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substitutions thereof that are generally used as antibodies used in antibody pharmaceuticals. In addition, bispecific antibodies (bispecific antibodies), fusion antibodies of Fc regions of antibodies with added sugar chains and other proteins, complexes of Fc regions of drugs with added sugar chains and drugs (ADC), etc. Even an antibody whose structure has been artificially modified can be separated by the separation method of the present invention.
　また本発明の分離方法は、例えば、Ｆｃ結合性タンパク質を不溶性担体に固定化して得られる吸着剤を充填したカラムに、抗体を含む緩衝液をポンプ等の送液手段を用いて添加することで、抗体を前記吸着剤に特異的に吸着させた後、適切な溶出液をカラムに添加することで、前記吸着した抗体をＡＤＣＣ活性の強さに基づき抗体を分離することができる。なお、抗体を含む緩衝液をカラムに添加する前に、適切な緩衝液を用いてカラムを平衡化すると、抗体をより高純度に分離できるため好ましい。緩衝液としてはリン酸緩衝液等、無機塩を成分とした緩衝液を例示することができる。なお緩衝液のｐＨは、ｐＨ３から１０、好ましくはｐＨ５から８である。前記吸着剤に吸着した抗体を、ＡＤＣＣ活性の強さに基づき溶出させるには、抗体とリガンド（Ｆｃ結合性タンパク質）との相互作用を弱めればよく、具体的には、緩衝液によるｐＨ変化、カウンターペプチド、温度変化、塩濃度変化が例示できる。前記吸着剤に吸着した抗体を、ＡＤＣＣ活性の強さに基づき溶出させるための溶出液の具体例として、前記吸着剤に抗体を吸着させる際に用いた溶液よりも酸性側の緩衝液があげられる。緩衝液の種類としては酸性側に緩衝能を有するクエン酸緩衝液、グリシン塩酸緩衝液、酢酸緩衝液を例示できる。緩衝液のｐＨは、抗体が有する機能を損なわない範囲で設定すればよく、好ましくはｐＨ２．５から６．０、より好ましくはｐＨ３．０から５．０、さらに好ましくはｐＨ３．３から４．０である。 In addition, the separation method of the present invention includes, for example, adding a buffer solution containing an antibody to a column packed with an adsorbent obtained by immobilizing an Fc binding protein on an insoluble carrier using a liquid delivery means such as a pump. After the antibody is specifically adsorbed to the adsorbent, an appropriate eluate is added to the column, whereby the adsorbed antibody can be separated based on the strength of ADCC activity. Note that it is preferable to equilibrate the column with an appropriate buffer before adding the buffer containing the antibody to the column because the antibody can be separated with higher purity. Examples of the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution. The pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8. In order to elute the antibody adsorbed on the adsorbent based on the strength of ADCC activity, it is only necessary to weaken the interaction between the antibody and the ligand (Fc binding protein). , Counter peptide, temperature change, salt concentration change. A specific example of the eluate for eluting the antibody adsorbed on the adsorbent based on the strength of ADCC activity is a buffer solution on the acidic side of the solution used when the antibody is adsorbed on the adsorbent. . Examples of the buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side. The pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.
　本発明のＦｃ結合性タンパク質を不溶性担体に固定化して得られる本発明の吸着剤を用いて、糖鎖を有した抗体を分離するには、例えば、本発明の吸着剤を充填したカラムに糖鎖を有した抗体を含む緩衝液をポンプ等の送液手段を用いて添加することで、糖鎖を有した抗体を本発明の吸着剤に特異的に吸着させた後、適切な溶出液をカラムに添加することで、糖鎖を有した抗体を溶出すればよい。なお、糖鎖を有した抗体を含む緩衝液をカラムに添加する前に、適切な緩衝液を用いてカラムを平衡化すると、糖鎖を有した抗体をより高純度に分離できるため好ましい。緩衝液としてはリン酸緩衝液等、無機塩を成分とした緩衝液を例示することができる。なお緩衝液のｐＨは、ｐＨ３から１０、好ましくはｐＨ５から８である。本発明の吸着剤に吸着した、糖鎖を有した抗体を溶出させるには、糖鎖を有した抗体とリガンド（本発明のＦｃ結合性タンパク質）との相互作用を弱めればよく、具体的には、緩衝液によるｐＨ変化、カウンターペプチド、温度変化、塩濃度変化が例示できる。本発明の吸着剤に吸着した、糖鎖を有した抗体を溶出させるための溶出液の具体例として、本発明の吸着剤に糖鎖を有した抗体を吸着させる際に用いた溶液よりも酸性側の緩衝液があげられる。緩衝液の種類としては酸性側に緩衝能を有するクエン酸緩衝液、グリシン塩酸緩衝液、酢酸緩衝液を例示できる。緩衝液のｐＨは、抗体が有する機能を損なわない範囲で設定すればよく、好ましくはｐＨ２．５から６．０、より好ましくはｐＨ３．０から５．０、さらに好ましくはｐＨ３．３から４．０である。 In order to separate an antibody having a sugar chain using the adsorbent of the present invention obtained by immobilizing the Fc-binding protein of the present invention on an insoluble carrier, for example, a column packed with the adsorbent of the present invention is loaded with sugar. A buffer solution containing an antibody having a chain is added using a liquid delivery means such as a pump to specifically adsorb the antibody having a sugar chain to the adsorbent of the present invention, and then an appropriate eluate is added. By adding to the column, the antibody having a sugar chain may be eluted. Note that it is preferable to equilibrate the column with an appropriate buffer before adding a buffer containing an antibody having a sugar chain to the column, because the antibody having a sugar chain can be separated with higher purity. Examples of the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution. The pH of the buffer solution is pH 3 to 10, preferably pH 5 to 8. In order to elute the antibody having a sugar chain adsorbed on the adsorbent of the present invention, it is only necessary to weaken the interaction between the antibody having the sugar chain and the ligand (Fc binding protein of the present invention). Examples thereof include pH change due to buffer, counter peptide, temperature change, and salt concentration change. As a specific example of the eluate for eluting the antibody having a sugar chain adsorbed to the adsorbent of the present invention, it is more acidic than the solution used for adsorbing the antibody having a sugar chain to the adsorbent of the present invention. Side buffer. Examples of the buffer solution include a citrate buffer solution, a glycine hydrochloride buffer solution, and an acetate buffer solution having a buffer capacity on the acidic side. The pH of the buffer may be set within a range that does not impair the function of the antibody, preferably pH 2.5 to 6.0, more preferably pH 3.0 to 5.0, still more preferably pH 3.3 to 4. 0.
　なお糖鎖を有した抗体を含む溶液から、本発明の吸着剤を用いて前記抗体を分離する際、前記抗体が有する糖鎖構造の違いにより、抗体の溶出位置（溶出フラクション）が異なる。従って、本発明の吸着剤を用いて抗体を分離することで、抗体が有する糖鎖構造の違いを識別することができる。識別可能な糖鎖の構造に特に限定はなく、一例として、ＣＨＯ細胞といった動物由来の細胞や、ピキア酵母やサッカロミセス酵母といった酵母を宿主として抗体を発現させたときに付加される糖鎖や、ヒト抗体が有する糖鎖や、化学合成法で抗体に付加した糖鎖があげられる。また本発明の吸着剤は、抗体が有する糖鎖構造の違いに基づき分離できることから、糖鎖そのものの分離にも利用できる。 When the antibody is separated from the solution containing the antibody having a sugar chain using the adsorbent of the present invention, the elution position (elution fraction) of the antibody varies depending on the sugar chain structure of the antibody. Therefore, by separating the antibody using the adsorbent of the present invention, the difference in the sugar chain structure of the antibody can be identified. There are no particular limitations on the structure of the glycans that can be identified. For example, sugar chains added when antibodies are expressed using animal-derived cells such as CHO cells, yeasts such as Pichia yeast and Saccharomyces yeast, and humans. Examples include sugar chains possessed by antibodies and sugar chains added to antibodies by chemical synthesis methods. Moreover, since the adsorbent of the present invention can be separated based on the difference in the sugar chain structure of the antibody, it can also be used for separation of the sugar chain itself.
　なお本発明の吸着剤により抗体の分離、ＡＤＣＣ活性の強さに基づく抗体の分離および抗体の糖鎖構造の違いを識別できると前述したが、当該吸着剤に用いるＦｃ結合性タンパク質として、ＦｃγＲＩＩＩａ以外のＦｃレセプター（ＦｃγＲＩ、ＦｃγＲＩＩａ、ＦｃγＲＩＩｂ、ＦｃγＲＩＩＩａ、ＦｃγＲＩＩＩｂ、ＦｃＲｎ）を用いた場合でも同様に糖鎖構造の違いを識別できる。 As described above, the adsorbent of the present invention can identify antibody separation, antibody separation based on the strength of ADCC activity, and the difference in the sugar chain structure of the antibody. Even when the Fc receptors (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcRn) are used, the difference in the sugar chain structure can be similarly identified.
　本発明のＦｃ結合性タンパク質は、ヒトＦｃγＲＩＩＩａの細胞外領域中の特定位置におけるアミノ酸残基を他のアミノ酸残基に置換したタンパク質である。本発明のＦｃ結合性タンパク質は野生型のヒトＦｃγＲＩＩＩａと比較し熱および酸に対する安定性が向上している。そのため、本発明のＦｃ結合性タンパク質はイムノグロブリンを分離するための吸着剤のリガンドとして有用である。 The Fc-binding protein of the present invention is a protein in which an amino acid residue at a specific position in the extracellular region of human FcγRIIIa is substituted with another amino acid residue. The Fc-binding protein of the present invention has improved heat and acid stability compared to wild-type human FcγRIIIa. Therefore, the Fc binding protein of the present invention is useful as a ligand of an adsorbent for separating immunoglobulin.
　また本発明の分離方法は、薬効に基づく抗体または抗体のＦｃ領域を含む分子の分離をクロマトグラフィーを用いて簡便、かつ精度よく分離することができる。従って本発明により、抗体医薬の製造工程管理や品質管理をより精度よく行なえる。 In addition, the separation method of the present invention can easily and accurately separate an antibody based on a medicinal effect or a molecule containing an Fc region of an antibody using chromatography. Therefore, according to the present invention, manufacturing process management and quality control of antibody drugs can be performed with higher accuracy.
　また本発明の分離方法は、Ｆｃ結合性タンパク質（例えば糖鎖が付加されていないヒトＦｃγＲＩＩＩａ）を不溶性担体に固定化して得られる吸着剤を用いることで、抗体を抗体依存性細胞傷害活性（ＡＤＣＣ）の強さに基づいて分離することができる。 Further, the separation method of the present invention uses an adsorbent obtained by immobilizing an Fc-binding protein (for example, human FcγRIIIa to which a sugar chain is not added) on an insoluble carrier, so that the antibody is antibody-dependent cytotoxic activity (ADCC). ) Can be separated on the basis of strength.
ヒトＦｃγＲＩＩＩａの概略図である。図中の数字は配列番号１に記載のアミノ酸配列の番号を示している。図中のＳはシグナル配列、ＥＣは細胞外領域、ＴＭは細胞膜貫通領域、Ｃは細胞内領域を示している。1 is a schematic diagram of human FcγRIIIa. The numbers in the figure indicate the amino acid sequence numbers described in SEQ ID NO: 1. In the figure, S represents a signal sequence, EC represents an extracellular region, TM represents a transmembrane region, and C represents an intracellular region. ＦｃＲ５ａ固定化ゲルを用いた抗体の溶出パターンを示した図である。図中のＦｒＡ、ＦｒＢはそれぞれフラクションＡ、フラクションＢの位置を示している。It is the figure which showed the elution pattern of the antibody using FcR5a fixed gel. FrA and FrB in the figure indicate the positions of fraction A and fraction B, respectively. ＦｃＲ５ａ固定化ゲルで分離した抗体のＡＤＣＣ活性を測定した結果を示した図である。It is the figure which showed the result of having measured the ADCC activity of the antibody isolate | separated with the FcR5a fixed gel. 抗体に付加した糖鎖構造の一覧を示した図である。図中のＮ１からＮ６は表１０のＮ１からＮ６に対応し、Ｍ１、Ｍ２およびＤ１は表１１のＭ１、Ｍ２およびＤ１に対応している。It is the figure which showed the list | wrist of the sugar chain structure added to the antibody. N1 to N6 in the figure correspond to N1 to N6 in Table 10, and M1, M2 and D1 correspond to M1, M2 and D1 in Table 11, respectively. ＦｃＲ９固定化ゲルを用いた抗体の溶出パターンを示した図である。図中のＦｒＡ、ＦｒＢ、ＦｒＣはそれぞれフラクションＡ、フラクションＢ、フラクションＣの位置を示している。It is the figure which showed the elution pattern of the antibody using FcR9 fixed gel. FrA, FrB, and FrC in the figure indicate the positions of fraction A, fraction B, and fraction C, respectively. ＦｃＲ９固定化ゲルで分離した抗体のＡＤＣＣ活性を測定した結果を示した図である。It is the figure which showed the result of having measured the ADCC activity of the antibody isolate | separated with the FcR9 fixed gel. 塩化ナトリウムを添加した／または添加しない緩衝液（平衡化液）を用いてモノクローナル抗体を分離して得られたクロマトグラフである。It is the chromatograph obtained by isolate | separating a monoclonal antibody using the buffer solution (equilibration liquid) which added / does not add sodium chloride. 塩化カリウムを添加した緩衝液（平衡化液）を用いてモノクローナル抗体を分離して得られたクロマトグラフである。It is the chromatograph obtained by isolate | separating a monoclonal antibody using the buffer solution (equilibration liquid) which added potassium chloride. 硫酸ナトリウムおよび硫酸アンモニウムを添加した緩衝液（平衡化液）を用いてモノクローナル抗体を分離して得られたクロマトグラフである。It is the chromatograph obtained by isolate | separating a monoclonal antibody using the buffer solution (equilibration liquid) which added sodium sulfate and ammonium sulfate. １アミノ酸置換したＦｃ結合性タンパク質の抗体結合活性を評価した結果を示す図である。図中の野生型はアミノ酸置換の無いＦｃ結合性タンパク質を示している。It is a figure which shows the result of having evaluated the antibody binding activity of Fc binding protein substituted by 1 amino acid. The wild type in the figure indicates an Fc binding protein without amino acid substitution. ＦｃＲ固定化ゲルを用いた抗体の溶出パターンを示した図である。図中のＦｒＡ、ＦｒＢはそれぞれフラクションＡ、フラクションＢの位置を示している。It is the figure which showed the elution pattern of the antibody using FcR fixed gel. FrA and FrB in the figure indicate the positions of fraction A and fraction B, respectively. ＦｃＲ固定化ゲルで分離した抗体のＡＤＣＣ活性を測定した結果を示した図である。It is the figure which showed the result of having measured the ADCC activity of the antibody isolate | separated with the FcR fixed gel.
　以下、本発明をさらに詳細に説明するために実施例を示すが、本発明は実施例に限定されるものではない。 Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to the examples.
　実施例１　Ｆｃ結合性タンパク質発現ベクターの作製
（１）配列番号１に記載のヒトＦｃγＲＩＩＩａアミノ酸配列のうち、１７番目のグリシン（Ｇｌｙ）から１９２番目のグルタミン（Ｇｌｎ）までのアミノ酸配列を基に、ＤＮＡｗｏｒｋｓ法（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，３０，ｅ４３，２００２）を用いて、コドンをヒト型から大腸菌型に変換したヌクレオチド配列を設計した。設計したヌクレオチド配列を配列番号２に示す。
（２）配列番号２に記載の配列を含むポリヌクレオチドを作製するために、配列番号３から２０に記載の配列からなるオリゴヌクレオチドを合成し、前記オリゴヌクレオチドを用いて、下記に示す二段階ＰＣＲを行なった。
（２－１）一段階目のＰＣＲは、表１に示す組成の反応液を調製し、当該反応液を９８℃で５分熱処理後、９８℃で１０秒間の第１ステップ、６２℃で５秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を１０サイクル繰り返すことでポリヌクレオチドを合成し、これをＦｃＲｐ１とした。なお表１中のＤＮＡミックスとは、配列番号３から２０に記載の配列からなる１８種類のオリゴヌクレオチドをそれぞれ一定量サンプリングし混合した溶液を意味する。 Example 1 Preparation of Fc-binding protein expression vector (1) Based on the amino acid sequence from the 17th glycine (Gly) to the 192nd glutamine (Gln) of the human FcγRIIIa amino acid sequence described in SEQ ID NO: 1, Using the DNAworks method (Nucleic Acids Res., 30, e43, 2002), a nucleotide sequence in which a codon was converted from a human type to an E. coli type was designed. The designed nucleotide sequence is shown in SEQ ID NO: 2.
(2) In order to prepare a polynucleotide containing the sequence shown in SEQ ID NO: 2, an oligonucleotide consisting of the sequences shown in SEQ ID NO: 3 to 20 was synthesized, and the two-step PCR shown below using the oligonucleotide Was done.
(2-1) In the first stage PCR, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, and 5 ° C. at 62 ° C. A polynucleotide was synthesized by repeating a reaction in which the second step for 2 seconds and the third step for 90 seconds at 72 ° C. were repeated 10 cycles, and this was designated as FcRp1. The DNA mix in Table 1 means a solution obtained by sampling a predetermined amount of each of 18 types of oligonucleotides having the sequences described in SEQ ID NOs: 3 to 20 and mixing them.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（２－２）二段階目のＰＣＲは、（２－１）で合成したＦｃＲｐ１を鋳型とし、配列番号２１（５’－ＴＡＧＣＣＡＴＧＧＧＣＡＴＧＣＧＴＡＣＣＧＡＡＧＡＴＣＴＧＣＣＧＡＡＡＧＣ－３’）および配列番号２２（５’－ＣＣＣＡＡＧＣＴＴＡＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＡＴＧＧＣＣＣＣＣＴＴＧＧＧＴＡＡＴＧＧＴＡＡＴＡＴＴＣＡＣＧＧＴＣＴＣＧＣＴＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして実施した。具体的には、表２に示す組成の反応液を調製し、当該反応液を９８℃で５分熱処理後、９８℃で１０秒間の第１ステップ、６２℃で５秒間の第２ステップ、７２℃で１．５分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。 (2-2) The second-stage PCR uses FcRp1 synthesized in (2-1) as a template, SEQ ID NO: 21 (5′-TAGCCATGGGCATGCCGTACGAGAATCTGCCCGAAAGCTCTGTGGATGTCCCTGTGGGTAATCTGTG An oligonucleotide consisting of the sequence described in 1 was used as a PCR primer. Specifically, a reaction solution having the composition shown in Table 2 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, then the first step at 98 ° C. for 10 seconds, the second step at 62 ° C. for 5 seconds, 72 The reaction was carried out by repeating 30 cycles of the third step of 1.5 minutes at ° C. for 30 cycles.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
（３）（２）で得られたポリヌクレオチドを精製し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、当該ライゲーション産物を用いて大腸菌ＢＬ２１株（ＤＥ３）を形質転換した。
（４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンンを含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて、発現ベクターｐＥＴ－ｅＦｃＲを抽出した。
（５）（４）で作製した発現ベクターｐＥＴ－ｅＦｃＲのうち、ヒトＦｃγＲＩＩＩａをコードするポリヌクレオチドおよびその周辺の領域について、チェーンターミネータ法に基づくＢｉｇ　Ｄｙｅ　Ｔｅｒｍｉｎａｔｏｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　ＦＳ　ｒｅａｄ　Ｒｅａｃｔｉｏｎ　ｋｉｔ（ライフサイエンス製）を用いてサイクルシークエンス反応に供し、全自動ＤＮＡシークエンサーＡＢＩ　Ｐｒｉｓｍ　３７００　ＤＮＡ　ａｎａｌｙｚｅｒ（ライフサイエンス製）にてヌクレオチド配列を解析した。なお当該解析の際、配列番号２３（５’－ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧ－３’）または配列番号２４（５’－ＴＡＴＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧ－３’）に記載の配列からなるオリゴヌクレオチドをシークエンス用プライマーとして使用した。 (3) The polynucleotide obtained in (2) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. Escherichia coli BL21 strain (DE3) was transformed with the ligation product.
(4) The obtained transformant was cultured in an LB medium containing 50 μg / mL kanamycin, and then the expression vector pET-eFcR was extracted using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) Big Dye Terminator Cycle Sequencing FS read Reaction kit (manufactured by Life Science) based on the chain terminator method for the polynucleotide encoding human FcγRIIIa and its surrounding region in the expression vector pET-eFcR prepared in (4) The nucleotide sequence was analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (manufactured by Life Science). In the analysis, an oligonucleotide having the sequence described in SEQ ID NO: 23 (5′-TAATACGACTCACTATAGGGG ′) or SEQ ID NO: 24 (5′-TATGCTAGTTATTGCTCAG-3 ′) was used as a sequencing primer.
　発現ベクターｐＥＴ－ｅＦｃＲで発現されるポリペプチドのアミノ酸配列を配列番号２５に、当該ポリペプチドをコードするポリヌクレオチドの配列を配列番号２６に、それぞれ示す。なお配列番号２５において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがヒトＦｃγＲＩＩＩａの細胞外領域（配列番号１の１７番目から１９２番目までの領域）であり、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。 The amino acid sequence of the polypeptide expressed by the expression vector pET-eFcR is shown in SEQ ID NO: 25, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26, respectively. In SEQ ID NO: 25, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. , From the 33rd glycine (Gly) to the 208th glutamine (Gln) is the extracellular region of human FcγRIIIa (the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine ( Gly) is a linker sequence, and 211 to 216th histidine (His) is a tag sequence.
　実施例２　Ｆｃ結合性タンパク質への変異導入およびライブラリーの作製
　実施例１で作製したＦｃ結合性タンパク質発現ベクターｐＥＴ－ｅＦｃＲのうち、Ｆｃ結合性タンパク質をコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例１で作製したｐＥＴ－ｅＦｃＲを用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、表３に示す組成の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、６０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。前記エラープローンＰＣＲによりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入され、その平均変異導入率は１．２６％であった。 Example 2 Mutation Introduction to Fc Binding Protein and Library Preparation Of the Fc binding protein expression vector pET-eFcR prepared in Example 1, the polynucleotide portion encoding the Fc binding protein was subjected to error-prone PCR. Mutation was randomly introduced.
(1) Error prone PCR was performed using pET-eFcR prepared in Example 1 as a template. In error-prone PCR, after preparing a reaction solution having the composition shown in Table 3, the reaction solution is heat-treated at 95 ° C. for 2 minutes, the first step at 95 ° C. for 30 seconds, the second step at 60 ° C. for 30 seconds, 72 The reaction was carried out by performing 35 cycles of the third step of 90 seconds at 90 ° C., and finally heat-treating at 72 ° C. for 7 minutes. By the error-prone PCR, mutations were successfully introduced into the polynucleotide encoding the Fc binding protein, and the average mutation introduction rate was 1.26%.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養（３７℃で１８時間）後、プレート上に形成したコロニーをランダム変異体ライブラリーとした。
実施例３　熱安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例２で作製したランダム変異体ライブラリー（形質転換体）を、５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地（ペプトン１６ｇ／Ｌ、酵母エキス１０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）２００μＬに接種し、９６穴ディープウェルプレートを用いて、３０℃で一晩振とう培養した。
（２）培養後、５μＬの培養液を５００μＬの０．０５ｍＭのＩＰＴＧ（ｉｓｏｐｒｏｐｙｌ－β－Ｄ－ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ）、０．３％のグリシンおよび５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地に植え継ぎ、９６穴ディープウェルプレートを用いて、さらに２０℃で一晩振とう培養した。
（３）培養後、遠心操作によって得られた培養上清を１５０ｍＭの塩化ナトリウムを含む２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）で２倍に希釈した。希釈した溶液を４５℃で１０分間熱処理を行なった。
（４）（３）の熱処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（３）の熱処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、それぞれ下記に示すＥＬＩＳＡ法にて測定し、熱処理を行なった時のＦｃ結合性タンパク質の抗体結合活性を、熱処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４－１）ヒト抗体であるガンマグロブリン製剤（化学及血清療法研究所製）を、９６穴マイクロプレートのウェルに１μｇ／ｗｅｌｌで固定化し（４℃で１８時間）、固定化終了後、２％（ｗ／ｖ）のＳＫＩＭ　ＭＩＬＫ（ＢＤ製）および１５０ｍＭの塩化ナトリウムを含んだ２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）によりブロッキングした。
（４－２）洗浄緩衝液（０．０５％［ｗ／ｖ］のＴｗｅｅｎ　２０、１５０ｍＭのＮａＣｌを含む２０ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．４））で洗浄後、抗体結合活性を評価するＦｃ結合性タンパク質を含む溶液を添加し、Ｆｃ結合性タンパク質と固定化ガンマグロブリンとを反応させた（３０℃で１時間）。
（４－３）反応終了後、前記洗浄緩衝液で洗浄し、１００ｎｇ／ｍＬに希釈したＡｎｔｉ－６Ｈｉｓ抗体（Ｂｅｔｈｙｌ　Ｌａｂｏｒａｔｏｒｉｅｓ製）を１００μＬ／ｗｅｌｌで添加した。
（４－４）３０℃で１時間反応させ、前記洗浄緩衝液で洗浄した後、ＴＭＢ　Ｐｅｒｏｘｉｄａｓｅ　Ｓｕｂｓｔｒａｔｅ（ＫＰＬ製）を５０μＬ／ｗｅｌｌで添加した。１Ｍのリン酸を５０μＬ／ｗｅｌｌで添加することで発色を止め、マイクロプレートリーダー（テカン製）にて４５０ｎｍの吸光度を測定した。
（５）（４）の方法で約２７００株の形質転換体を評価し、その中から野生型（アミノ酸置換のない）Ｆｃ結合性タンパク質と比較して熱安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。前記選択した形質転換体を培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（６）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）の記載と同様の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 (2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into E. coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin (18 hours at 37 ° C.), and then placed on the plate. The formed colonies were used as a random mutant library.
Example 3 Screening of heat-stabilized Fc binding protein (1) The random mutant library (transformant) prepared in Example 2 was subjected to 2YT liquid medium containing 50 μg / mL kanamycin (peptone 16 g / L, yeast). (Extract 10 g / L, sodium chloride 5 g / L) was inoculated into 200 μL, and cultured with shaking at 30 ° C. overnight using a 96-well deep well plate.
(2) After culturing, 5 μL of the culture broth was subcultured in 2 μT of 2YT liquid medium containing 500 μL of 0.05 mM IPTG (isopropyl-β-D-thiogalactopylanoside), 0.3% glycine and 50 μg / mL kanamycin. Further, the culture was further performed overnight at 20 ° C. using a well-well plate.
(3) After culture, the culture supernatant obtained by centrifugation was diluted 2-fold with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. The diluted solution was heat-treated at 45 ° C. for 10 minutes.
(4) The ELISA method shown below for the antibody binding activity of the Fc binding protein when the heat treatment of (3) is performed and the antibody binding activity of the Fc binding protein when the heat treatment of (3) is not performed The residual activity was calculated by dividing the antibody binding activity of the Fc binding protein when subjected to heat treatment by the antibody binding activity of the Fc binding protein when not subjected to heat treatment.
(4-1) A gamma globulin preparation (manufactured by Chemistry and Serum Therapy Laboratories), which is a human antibody, was fixed to a well of a 96-well microplate at 1 μg / well (18 hours at 4 ° C.). Blocking was performed with 20 mM Tris-HCl buffer (pH 7.4) containing SKIM MILK (manufactured by BD) and 150 mM sodium chloride.
(4-2) Fc for evaluating antibody binding activity after washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Tween 20, 150 mM NaCl) A solution containing a binding protein was added to react the Fc binding protein with the immobilized gamma globulin (1 hour at 30 ° C.).
(4-3) After completion of the reaction, Anti-6His antibody (manufactured by Bethyl Laboratories) diluted with 100 ng / mL was washed with the washing buffer and added at 100 μL / well.
(4-4) The mixture was reacted at 30 ° C. for 1 hour, washed with the washing buffer, and then TMB Peroxidase Substrate (manufactured by KPL) was added at 50 μL / well. Color development was stopped by adding 1 M phosphoric acid at 50 μL / well, and absorbance at 450 nm was measured with a microplate reader (Tecan).
(5) About 2700 strains of transformants were evaluated by the method of (4), and an Fc binding protein having improved thermal stability compared to a wild type (without amino acid substitution) Fc binding protein was selected. Transformants that express were selected. The selected transformant was cultured, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(6) Analyzing the nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector in the same manner as described in Example 1 (5), and identifying the amino acid mutation site did.
　（５）で選択した形質転換体が発現するＦｃ結合性タンパク質の、野生型（アミノ酸置換のない）Ｆｃ結合性タンパク質に対するアミノ酸置換位置および熱処理後の残存活性（％）をまとめたものを表４に示す。配列番号１に記載のアミノ酸配列のうち、１７番目のグリシンから１９２番目のグルタミンまでのアミノ酸残基を含み、かつ当該１７番目から１９２番目までのアミノ酸残基において、Ｍｅｔ１８Ａｒｇ（この表記は、配列番号１の１８番目のメチオニンがアルギニンに置換されていることを表す、以下同様）、Ｖａｌ２７Ｇｌｕ、Ｐｈｅ２９Ｌｅｕ、Ｐｈｅ２９Ｓｅｒ、Ｌｅｕ３０Ｇｌｎ、Ｔｙｒ３５Ａｓｎ、Ｔｙｒ３５Ａｓｐ、Ｔｙｒ３５Ｓｅｒ、Ｔｙｒ３５Ｈｉｓ、Ｌｙｓ４６Ｉｌｅ、Ｌｙｓ４６Ｔｈｒ、Ｇｌｎ４８Ｈｉｓ、Ｇｌｎ４８Ｌｅｕ、Ａｌａ５０Ｈｉｓ、Ｔｙｒ５１Ａｓｐ、Ｔｙｒ５１Ｈｉｓ、Ｇｌｕ５４Ａｓｐ、Ｇｌｕ５４Ｇｌｙ、Ａｓｎ５６Ｔｈｒ、Ｇｌｎ５９Ａｒｇ、Ｐｈｅ６１Ｔｙｒ、Ｇｌｕ６４Ａｓｐ、Ｓｅｒ６５Ａｒｇ、Ａｌａ７１Ａｓｐ、Ｐｈｅ７５Ｌｅｕ、Ｐｈｅ７５Ｓｅｒ、Ｐｈｅ７５Ｔｙｒ、Ａｓｐ７７Ａｓｎ、Ａｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ａｓｐ８２Ｖａｌ、Ｇｌｎ９０Ａｒｇ、Ａｓｎ９２Ｓｅｒ、Ｌｅｕ９３Ａｒｇ、Ｌｅｕ９３Ｍｅｔ、Ｔｈｒ９５Ａｌａ、Ｔｈｒ９５Ｓｅｒ、Ｌｅｕ１１０Ｇｌｎ、Ａｒｇ１１５Ｇｌｎ、Ｔｒｐ１１６Ｌｅｕ、Ｐｈｅ１１８Ｔｙｒ、Ｌｙｓ１１９Ｇｌｕ、Ｇｌｕ１２０Ｖａｌ、Ｇｌｕ１２１Ａｓｐ、Ｇｌｕ１２１Ｇｌｙ、Ｐｈｅ１５１Ｓｅｒ、Ｐｈｅ１５１Ｔｙｒ、Ｓｅｒ１５５Ｔｈｒ、Ｔｈｒ１６３Ｓｅｒ、Ｓｅｒ１６７Ｇｌｙ、Ｓｅｒ１６９Ｇｌｙ、Ｐｈｅ１７１Ｔｙｒ、Ａｓｎ１８０Ｌｙｓ、Ａｓｎ１８０Ｓｅｒ、Ａｓｎ１８０Ｉｌｅ、Ｔｈｒ１８５Ｓｅｒ、Ｇｌｎ１９２Ｌｙｓのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、野生型のＦｃ結合性タンパク質と比較し熱安定性が向上しているといえる。 Table 4 summarizes the amino acid substitution position and the remaining activity (%) after heat treatment of the Fc binding protein expressed by the transformant selected in (5) with respect to the wild type (without amino acid substitution) Fc binding protein. Shown in Among the amino acid sequences described in SEQ ID NO: 1, the amino acid residues from the 17th glycine to the 192nd glutamine and the 17th to 192nd amino acid residues are Met18Arg (this notation is SEQ ID NO: 1 represents that 18th methionine of 1 is substituted with arginine, the same applies below), Val27Glu, Phe29Leu, Phe29Ser, Leu30Gln, Tyr35Asn, Tyr35Asp, Tyr35Sr, Tyr35His, Lys46Thr, Lys46Thr, Lys46Thr, , Glu54Asp, Glu54Gly, Asn56Thr, Gln59Arg, Phe61Tyr, Glu64Asp, Ser6 Arg, Ala71Asp, Phe75Leu, Phe75Ser, Phe75Tyr, Asp77Asn, Ala78Ser, Asp82Glu, Asp82Val, Gln90Arg, Asn92Ser, Leu93Arg, Leu93Met, Thr95Ala, Thr95Ser, Leu110Gln, Arg115Gln, Trp116Leu, Phe118Tyr, Lys119Glu, Glu120Val, Glu121Asp, Glu121Gly, Phe151Ser, Phe151Tyr, Any of Ser155Thr, Thr163Ser, Ser167Gly, Ser169Gly, Phe171Tyr, Asn180Lys, Asn180Ser, Asn180Ile, Thr185Ser, Gln192Lys Fc binding proteins Kano amino acid substitution is at least one occurs, it can be said that the thermal stability as compared to wild-type Fc binding protein is improved.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示す、アミノ酸置換されたＦｃ結合性タンパク質のうち、最も残存活性の高い、Ｖａｌ２７ＧｌｕおよびＴｙｒ３５Ａｓｎのアミノ酸置換が生じたＦｃ結合性タンパク質をＦｃＲ２と命名し、ＦｃＲ２をコードするポリヌクレオチドを含む発現ベクターをｐＥＴ－ＦｃＲ２と命名した。ＦｃＲ２のアミノ酸配列を配列番号２７に、ＦｃＲ２をコードするポリヌクレオチドの配列を配列番号２８に示す。なお配列番号２７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ２のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号２７において、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目の位置にそれぞれ存在する。 Among the Fc-binding proteins with amino acid substitutions shown in Table 4, the Fc-binding protein with the highest residual activity, in which the amino acid substitution of Val27Glu and Tyr35Asn occurred, was named FcR2 and contained a polynucleotide encoding FcR2. The vector was named pET-FcR2. The amino acid sequence of FcR2 is shown in SEQ ID NO: 27, and the sequence of the polynucleotide encoding FcR2 is shown in SEQ ID NO: 28. In SEQ ID NO: 27, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR2 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. In SEQ ID NO: 27, glutamic acid of Val27Glu is present at the 43rd position, and asparagine of Tyr35Asn is present at the 51st position.
　実施例４　アミノ酸置換Ｆｃ結合性タンパク質の作製
　実施例３で判明した、Ｆｃ結合性タンパク質の熱安定性向上に関与するアミノ酸置換を集積することで、さらなる安定性向上を図った。置換アミノ酸の集積は、主にＰＣＲを用いて行ない、以下の（ａ）から（ｃ）に示す３種類のＦｃ結合性タンパク質を作製した。
（ａ）ＦｃＲ２に対し、さらにＰｈｅ７５Ｌｅｕのアミノ酸置換を行なったＦｃＲ３
（ｂ）ＦｃＲ２に対し、さらにＰｈｅ７５ＬｅｕおよびＧｌｕ１２１Ｇｌｙのアミノ酸置換を行なったＦｃＲ４
（ｃ）ＦｃＲ４に対し、さらにＡｓｎ９２Ｓｅｒのアミノ酸置換を行なったＦｃＲ５ａ
以下、各Ｆｃ結合性タンパク質の作製方法を詳細に説明する。 Example 4 Production of Amino Acid-Substituted Fc Binding Protein The stability was further improved by accumulating amino acid substitutions that were found in Example 3 and involved in improving the thermal stability of the Fc binding protein. Accumulation of substituted amino acids was mainly performed using PCR, and three types of Fc-binding proteins shown in (a) to (c) below were prepared.
(A) FcR3 obtained by further performing amino acid substitution of Phe75Leu on FcR2
(B) FcR4 obtained by further performing amino acid substitution of Phe75Leu and Glu121Gly on FcR2.
(C) FcR5a in which Asn92Ser amino acid substitution was further performed on FcR4
Hereinafter, a method for producing each Fc-binding protein will be described in detail.
　（ａ）ＦｃＲ３
　実施例３で明らかになった、熱安定性向上に関与するアミノ酸置換の中から、Ｖａｌ２７Ｇｌｕ、Ｔｙｒ３５ＡｓｎおよびＰｈｅ７５Ｌｅｕを選択し、それらの置換を野生型のＦｃ結合性タンパク質に集積したＦｃＲ３を作製した。具体的には、ＦｃＲ２をコードするポリヌクレオチドに対してＰｈｅ７５Ｌｅｕを生じさせる変異導入を行なうことにより、ＦｃＲ３を作製した。
（ａ－１）実施例３で取得した、ｐＥＴ－ＦｃＲ２を鋳型としてＰＣＲを実施した。当該ＰＣＲにおけるプライマーは、配列番号２４および配列番号２９（５’－ＡＧＣＣＡＧＧＣＧＡＧＣＡＧＣＴＡＣＣＴＴＡＴＴＧＡＴＧＣＧ－３’）に記載の配列からなるオリゴヌクレオチドを用いた。ＰＣＲは、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で７分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ３Ｆとした。 (A) FcR3
Val27Glu, Tyr35Asn, and Phe75Leu were selected from among the amino acid substitutions involved in improving thermal stability, which were revealed in Example 3, and FcR3 in which these substitutions were accumulated in a wild-type Fc-binding protein was prepared. Specifically, FcR3 was produced by introducing a mutation causing Phe75Leu into a polynucleotide encoding FcR2.
(A-1) PCR was performed using pET-FcR2 obtained in Example 3 as a template. As primers for the PCR, oligonucleotides having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29 (5′-AGCCAGGCGAGCAGCTACCCTTTATTGATGCG-3 ′) were used. In PCR, after preparing a reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction in which the third step for 1 minute was set to 1 cycle were performed by heat treatment at 72 ° C. for 7 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m3F.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（ａ－２）実施例３で取得した、ｐＥＴ－ＦｃＲ２を鋳型とし、配列番号２３および配列番号３０（５’－ＣＣＡＣＣＧＴＣＧＣＣＧＣＡＴＣＡＡＴＡＡＧＧＴＡＧＣＴＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様に行なった。精製したＰＣＲ産物をｍ３Ｒとした。
（ａ－３）（ａ－１）および（ａ－２）で得られた２種類のＰＣＲ産物（ｍ３Ｆ、ｍ３Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ３Ｆとｍ３Ｒを連結したＰＣＲ産物ｍ３ｐを得た。 (A-2) Except that pET-FcR2 obtained in Example 3 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 30 (5′-CCACCGTCGCCCGCATCAATAAGGTTAGCTGC-3 ′) was used as a PCR primer. , (A-1). The purified PCR product was designated as m3R.
(A-3) Two types of PCR products (m3F and m3R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed, and a PCR product m3p in which m3F and m3R were linked was obtained.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（ａ－４）（ａ－３）で得られたＰＣＲ産物ｍ３ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ２に１箇所アミノ酸置換を導入したＦｃＲ３をコードするポリヌクレオチドを作製した。 (A-4) PCR was performed using the PCR product m3p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR3 in which an amino acid substitution at one site was introduced into FcR2 was prepared.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
（ａ－５）（ａ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、野生型Ｆｃ結合性タンパク質に対して３箇所アミノ酸置換したポリペプチドである、ＦｃＲ３をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ３を得た。
（ａ－７）ｐＥＴ－ＦｃＲ３のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 (A-5) After purifying the polynucleotide obtained in (a-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR3 containing a polynucleotide encoding FcR3, which is a polypeptide obtained by substituting three amino acids for the wild-type Fc-binding protein, is obtained. It was.
(A-7) The nucleotide sequence of pET-FcR3 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ３のアミノ酸配列を配列番号３１に、前記ＦｃＲ３をコードするポリヌクレオチドの配列を配列番号３２に示す。なお配列番号３１において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ３のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号３１において、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目の位置にそれぞれ存在する。 The amino acid sequence of FcR3 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 31, and the sequence of the polynucleotide encoding the FcR3 is shown in SEQ ID NO: 32. In SEQ ID NO: 31, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR3 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. In SEQ ID NO: 31, glutamic acid of Val27Glu is present at the 43rd position, asparagine of Tyr35Asn is present at the 51st position, and leucine of Phe75Leu is present at the 91st position.
　（ｂ）ＦｃＲ４
　実施例３で明らかになったＦｃ結合性タンパク質の安定性向上に関与するアミノ酸置換の中から、Ｖａｌ２７Ｇｌｕ、Ｔｙｒ３５Ａｓｎ、Ｐｈｅ７５ＬｅｕおよびＧｌｕ１２１Ｇｌｙを選択し、それらの置換を野生型のＦｃ結合性タンパク質に集積したＦｃＲ４を作製した。具体的には、ＦｃＲ２をコードするポリヌクレオチドに対してＰｈｅ７５ＬｅｕおよびＧｌｕ１２１Ｇｌｙを生じさせる変異導入を行なうことにより、ＦｃＲ４を作製した。
（ｂ－１）（ａ－２）と同様の方法でＰＣＲ産物ｍ３Ｒを得た。また実施例３で取得した、Ａｌａ７１Ａｓｐ、Ｐｈｅ７５ＬｅｕおよびＧｌｕ１２１Ｇｌｙのアミノ酸置換を含んだＦｃ結合性タンパク質（表４）を発現するプラスミドを鋳型とし、配列番号２４および配列番号２９に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－１）と同様の方法でＰＣＲを行なうことでＰＣＲ産物ｍ４Ｒを得た。
（ｂ－２）（ｂ－１）により得られた２種類のＰＣＲ産物（ｍ３Ｒ、ｍ４Ｒ）を混合後、（ａ－３）と同様の方法にてＰＣＲを行ない、ｍ３Ｒとｍ４Ｒを連結した。得られたＰＣＲ産物をｍ４ｐとした。
（ｂ－３）（ｂ－２）で得られたＰＣＲ産物ｍ４ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－４）と同様の方法でＰＣＲを行なった。これによりＦｃＲ４をコードするポリヌクレオチドを作製した。
（ｂ－４）（ｂ－３）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｂ－５）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、野生型Ｆｃ結合性タンパク質に対して４箇所アミノ酸置換したポリペプチドである、ＦｃＲ４をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ４を得た。
（ｂ－６）ｐＥＴ－ＦｃＲ４のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 (B) FcR4
Val27Glu, Tyr35Asn, Phe75Leu and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, and these substitutions were accumulated in the wild-type Fc binding protein. FcR4 was produced. Specifically, FcR4 was produced by introducing a mutation that causes Phe75Leu and Glu121Gly to the polynucleotide encoding FcR2.
(B-1) A PCR product m3R was obtained in the same manner as (a-2). Further, an oligonucleotide comprising the sequences described in SEQ ID NO: 24 and SEQ ID NO: 29, which was obtained in Example 3, using the plasmid expressing Fc-binding protein (Table 4) containing amino acid substitutions of Ala71Asp, Phe75Leu and Glu121Gly as a template. PCR product m4R was obtained by PCR in the same manner as in (a-1) using as a PCR primer.
(B-2) After mixing the two types of PCR products (m3R, m4R) obtained in (b-1), PCR was performed in the same manner as in (a-3) to link m3R and m4R. The obtained PCR product was designated as m4p.
(B-3) A method similar to (a-4), using the PCR product m4p obtained in (b-2) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR4.
(B-4) After purification of the polynucleotide obtained in (b-3), digestion with restriction enzymes NcoI and HindIII and expression vector pETmalE previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-5) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected cells (transformants), a plasmid pET-FcR4 containing a polynucleotide encoding FcR4, which is a polypeptide obtained by substituting the wild-type Fc binding protein with 4 amino acids, is obtained. It was.
(B-6) The nucleotide sequence of pET-FcR4 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ４のアミノ酸配列を配列番号３３に、前記ＦｃＲ４をコードするポリヌクレオチドの配列を配列番号３４に示す。なお、配列番号３３において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ４のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号３３において、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目の位置にそれぞれ存在する。 The amino acid sequence of FcR4 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 33, and the sequence of a polynucleotide encoding the FcR4 is shown in SEQ ID NO: 34. In SEQ ID NO: 33, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, the amino acid sequence of FcR4 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. Further, in SEQ ID NO: 33, glutamic acid of Val27Glu exists at position 43, asparagine of Tyr35Asn exists at position 51, leucine of Phe75Leu exists at position 91, and glycine of Glu121Gly exists at position 137.
　（ｃ）ＦｃＲ５ａ
　実施例３で明らかになったＦｃ結合性タンパク質の安定性向上に関与するアミノ酸置換の中から、Ｖａｌ２７Ｇｌｕ、Ｔｙｒ３５Ａｓｎ、Ｐｈｅ７５Ｌｅｕ、Ａｓｎ９２ＳｅｒおよびＧｌｕ１２１Ｇｌｙを選択し、それらの置換を野生型のＦｃ結合性タンパク質に集積したＦｃＲ５ａを作製した。具体的には、（ｂ）で作製したＦｃＲ４をコードするポリヌクレオチドに対してＡｓｎ９２Ｓｅｒを生じさせる変異導入を行なうことにより、ＦｃＲ５ａを作製した。
（ｃ－１）（ｂ）で作製した、ｐＥＴ－ＦｃＲ４を鋳型とし、配列番号２２および配列番号３５（５’－ＧＡＡＴＡＴＣＧＴＴＧＣＣＡＧＡＣＣＡＧＣＣＴＧＡＧＣＡＣＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ５ａＦとした。
（ｃ－２）（ｂ）で作製したｐＥＴ－ＦｃＲ４を鋳型とし、配列番号２１および配列番号３６（５’－ＧＡＴＣＧＣＴＣＡＧＧＧＴＧＣＴＣＡＧＧＣＴＧＧＴＣＴＧＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ５ａＲとした。
（ｃ－３）（ｃ－１）および（ｃ－２）で得られた２種類のＰＣＲ産物（ｍ５ａＦ、ｍ５ａＲ）を混合後、（ａ－３）と同様の方法にてＰＣＲを行ない、ｍ５ａＦとｍ５ａＲを連結した。得られたＰＣＲ産物をｍ５ａｐとした。
（ｃ－４）（ｃ－３）で得られたＰＣＲ産物ｍ５ａｐを鋳型とし、配列番号２１および配列番号２２に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－４）と同様の方法でＰＣＲを行なった。これによりＦｃＲ５ａをコードするポリヌクレオチドを作製した。
（ｃ－５）（ｃ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｃ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、野生型Ｆｃ結合性タンパク質に対して５箇所アミノ酸置換したポリペプチドである、ＦｃＲ５ａをコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ５ａを得た。
（ｃ－７）ｐＥＴ－ＦｃＲ５ａのヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 (C) FcR5a
Val27Glu, Tyr35Asn, Phe75Leu, Asn92Ser, and Glu121Gly were selected from the amino acid substitutions involved in improving the stability of the Fc binding protein revealed in Example 3, and these substitutions were changed to the wild type Fc binding protein. Accumulated FcR5a was produced. Specifically, FcR5a was prepared by introducing a mutation that caused Asn92Ser to the polynucleotide encoding FcR4 prepared in (b).
(C-1) except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 22 and SEQ ID NO: 35 (5′-GAATATCGTTGCCAGACCAGCCTGAGCACC-3 ′) was used as a PCR primer. PCR was performed in the same manner as in (a-1). The purified PCR product was designated as m5aF.
(C-2) except that pET-FcR4 prepared in (b) was used as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 36 (5′-GATCGCTCCAGGGTGCTCAGGCTGGTTCTGGC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m5aR.
(C-3) After mixing the two kinds of PCR products (m5aF, m5aR) obtained in (c-1) and (c-2), PCR was performed in the same manner as in (a-3), and m5aF And m5aR were ligated. The obtained PCR product was designated as m5ap.
(C-4) A method similar to (a-4), using the PCR product m5ap obtained in (c-3) as a template and an oligonucleotide comprising the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR5a.
(C-5) After purifying the polynucleotide obtained in (c-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(C-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmid pET-FcR5a containing a polynucleotide encoding FcR5a, which is a polypeptide obtained by substituting amino acid at five positions with respect to the wild-type Fc-binding protein, is obtained by extracting a plasmid from the collected bacterial cells (transformants). It was.
(C-7) The nucleotide sequence of pET-FcR5a was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ５ａのアミノ酸配列を配列番号３７に、前記ＦｃＲ５ａをコードするポリヌクレオチドの配列を配列番号３８に示す。なお、配列番号３７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ５ａのアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号３７において、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目の位置にそれぞれ存在する。 The amino acid sequence of FcR5a added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 37, and the sequence of the polynucleotide encoding the FcR5a is shown in SEQ ID NO: 38. In SEQ ID NO: 37, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, the amino acid sequence of FcR5a from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. Also, in SEQ ID NO: 37, glutamic acid of Val27Glu is located at position 43, asparagine of Tyr35Asn is located at position 51, leucine of Phe75Leu is located at position 91, serine of Asn92Ser is located at position 108, and glycine of Glu121Gly is located at position 137.
　実施例５　ＦｃＲ５ａへの変異導入およびライブラリーの作製
　実施例４（ｃ）で作製したＦｃＲ５ａをコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例４（ｃ）で作製した発現ベクターｐＥＴ－ＦｃＲ５ａを用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、鋳型にｐＥＴ－ＦｃＲ５ａを用いた以外は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、６０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。この反応によりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入された。
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養後、プレート上に形成したコロニーをランダム変異ライブラリーとした。 Example 5 Mutation Introduction to FcR5a and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR5a prepared in Example 4 (c) by error-prone PCR.
(1) Error-prone PCR was performed using the expression vector pET-FcR5a prepared in Example 4 (c) as a template. In error-prone PCR, after preparing a reaction solution having the same composition as shown in Table 3 except that pET-FcR5a was used as a template, the reaction solution was heat-treated at 95 ° C. for 2 minutes, and the first step at 95 ° C. for 30 seconds. The reaction was carried out by performing 35 cycles of a second step of 60 ° C. for 30 seconds and a third step of 72 ° C. for 90 seconds for one cycle, and finally heat treatment at 72 ° C. for 7 minutes. By this reaction, the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
(2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
　実施例６　熱安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例５で作製したランダム変異ライブラリーを実施例３（１）から（２）に記載の方法で培養することでＦｃ結合性タンパク質を発現させた。
（２）培養後、遠心操作によって得られた、Ｆｃ結合性タンパク質を含む培養上清を純水にて２０倍に希釈し、更に０．１Ｍの炭酸ナトリウム緩衝液（ｐＨ１０．０）で２０倍に希釈した。その後、希釈した溶液を４０℃で１５分間熱処理を行ない、１Ｍのトリス緩衝液（ｐＨ７．０）でｐＨを中性付近に戻した。
（３）（２）の熱処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（２）の熱処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法にて測定し、熱処理を行なったときのＦｃ結合性タンパク質の抗体結合活性を、熱処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４）（３）の方法で約２７００株の形質転換体を評価し、その中からＦｃＲ５ａと比較して熱安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。選択した形質転換体を５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地にて培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（５）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 Example 6 Screening of Heat Stabilized Fc Binding Protein (1) By culturing the random mutation library prepared in Example 5 by the method described in Examples 3 (1) to (2), Fc binding protein was obtained. Expressed.
(2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, and further 20-fold with 0.1 M sodium carbonate buffer (pH 10.0). Dilute to Thereafter, the diluted solution was heat-treated at 40 ° C. for 15 minutes, and the pH was returned to near neutral with 1M Tris buffer (pH 7.0).
(3) Example 3 (4) shows the antibody binding activity of the Fc binding protein when the heat treatment of (2) is performed and the antibody binding activity of the Fc binding protein when the heat treatment of (2) is not performed. By dividing the antibody-binding activity of the Fc-binding protein when subjected to heat treatment by the antibody-binding activity of the Fc-binding protein when not subjected to heat treatment, the residual activity was measured by the ELISA method described in 1. Calculated.
(4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing Fc-binding proteins with improved thermal stability compared to FcR5a were selected. The selected transformant was cultured in a 2YT liquid medium containing 50 μg / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
　（４）で選択した形質転換体が発現するＦｃ結合性タンパク質の、ＦｃＲ５ａに対するアミノ酸置換位置および熱処理後の残存活性（％）をまとめたものを表８に示す。配列番号３７に記載のアミノ酸配列のうち、３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、Ｐｈｅ２９Ｉｌｅ（この表記は、配列番号１の２９番目（配列番号３７では４５番目）のフェニルアラニンがイソロイシンに置換されていることを表す、以下同様）、Ｐｈｅ２９Ｌｅｕ、Ｇｌｕ３９Ｇｌｙ、Ｇｌｎ４８Ａｒｇ、Ｔｙｒ５１Ｓｅｒ、Ｐｈｅ６１Ｔｙｒ、Ａｓｐ７７Ｇｌｙ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ９０Ａｒｇ、Ｇｌｎ１１２Ｌｅｕ、Ｖａｌ１１７Ｇｌｕ、Ｌｙｓ１１９Ａｓｎ、Ｌｙｓ１１９Ｇｌｕ、Ｔｈｒ１４０Ｉｌｅ、Ｌｅｕ１４２Ｇｌｎ、Ｐｈｅ１７１Ｓｅｒ、Ｌｅｕ１７５Ａｒｇ、Ａｓｎ１８０ＳｅｒおよびＩｌｅ１８８Ｖａｌのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、ＦｃＲ５ａと比較し熱安定性が向上しているといえる。 Table 8 shows a summary of amino acid substitution positions for FcR5a and the residual activity (%) after heat treatment of the Fc-binding protein expressed by the transformant selected in (4). Among the amino acid sequences described in SEQ ID NO: 37, the amino acid residues from the 33rd glycine to the 208th glutamine, and the 33rd to 208th amino acid residues, Phe29Ile (this notation is SEQ ID NO: 1 represents that phenylalanine at position 29 (position 45 in SEQ ID NO: 37) is substituted with isoleucine, and so on), Phe29Leu, Glu39Gly, Gln48Arg, Tyr51Ser, Phe61Tyr, Asp77Gly, Asp82Glu, Gln90Alg, L Of Lys119Glu, Thr140Ile, Leu142Gln, Phe171Ser, Leu175Arg, Asn180Ser and Ile188Val Fc binding protein amino acid substitutions Zureka has at least one occurs, it can be said that the thermal stability as compared to FcR5a is improved.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　表８に示した、ＦｃＲ５ａからアミノ酸置換されたＦｃ結合性タンパク質のうち、Ｐｈｅ２９ＩｌｅおよびＶａｌ１１７Ｇｌｕのアミノ酸置換が生じたＦｃ結合性タンパク質をＦｃＲ７ａと命名し、ＦｃＲ７ａをコードするポリヌクレオチドを含む発現ベクターをｐＥＴ－ＦｃＲ７ａと命名した。ＦｃＲ７ａのアミノ酸配列を配列番号３９に、ＦｃＲ７ａをコードするポリヌクレオチドの配列を配列番号４０に示す。なお配列番号３９において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ７ａのアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号３９において、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目の位置にそれぞれ存在する。 Of the Fc binding proteins with amino acid substitutions from FcR5a shown in Table 8, the Fc binding protein in which Phe29Ile and Val117Glu amino acid substitutions were named FcR7a, and an expression vector containing a polynucleotide encoding FcR7a was designated as pET -It was named FcR7a. The amino acid sequence of FcR7a is shown in SEQ ID NO: 39, and the sequence of the polynucleotide encoding FcR7a is shown in SEQ ID NO: 40. In SEQ ID NO: 39, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR7a (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. In SEQ ID NO: 39, isoleucine of Phe29Ile is present at the 45th position and glutamic acid of Val117Glu is present at the 133rd position.
　実施例７　改良Ｆｃ結合性タンパク質の作製
　実施例６で判明した、Ｆｃ結合性タンパク質の熱安定性向上に関与するアミノ酸置換をＦｃＲ７ａに集積することで、さらなる安定性向上を図った。置換アミノ酸の集積は、主にＰＣＲを用いて行ない、以下の（ａ）から（ｄ）に示す４種類の改良Ｆｃ結合性タンパク質を作製した。
（ａ）ＦｃＲ７ａに対し、さらにＰｈｅ１７１Ｓｅｒのアミノ酸置換を行なったＦｃＲ８
（ｂ）ＦｃＲ８に対し、さらにＧｌｎ４８Ａｒｇのアミノ酸置換を行なったＦｃＲ９
（ｃ）ＦｃＲ８に対し、さらにＧｌｎ４８ＡｒｇおよびＴｙｒ５１Ｓｅｒのアミノ酸置換を行なったＦｃＲ１０
（ｄ）ＦｃＲ１０に対し、さらにＧｌｎ９０Ａｒｇのアミノ酸置換を行なったＦｃＲ１１
　以下、各改良Ｆｃ結合性タンパク質の作製方法を詳細に説明する。
（ａ）ＦｃＲ８
　実施例６で明らかとなった、熱安定性向上に関与するアミノ酸置換の中から、Ｐｈｅ２９Ｉｌｅ、Ｖａｌ１１７ＧｌｕおよびＰｈｅ１７１Ｓｅｒを選択し、それらの置換をＦｃＲ５ａ（実施例４（ｃ））に集積したＦｃＲ８を作製した。具体的には、ＦｃＲ７ａをコードするポリヌクレオチドに対して、Ｐｈｅ１７１Ｓｅｒを生じさせる変異導入を行なうことにより、ＦｃＲ８を作製した。
（ａ－１）実施例６で取得した、ｐＥＴ－ＦｃＲ７ａを鋳型としてＰＣＲを実施した。当該ＰＣＲにおけるプライマーは、配列番号２３および配列番号４１（５’－ＡＣＣＡＧＣＣＣＡＣＧＧＣＡＧＧＡＡＴＡＧＣＴＧＣＣＧＣＴＧ－３’）に記載の配列からなるオリゴヌクレオチドを用いた。ＰＣＲは、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ８Ｆとした。
（ａ－２）実施例６で取得した、ｐＥＴ－ＦｃＲ７ａを鋳型とし、配列番号４２（５’－ＧＡＣＡＧＣＧＧＣＡＧＣＴＡＴＴＣＣＴＧＣＣＧＴＧＧＧＣＴＧ－３’）および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様に行なった。精製したＰＣＲ産物をｍ８Ｒとした。
（ａ－３）（ａ－１）および（ａ－２）で得られた２種類のＰＣＲ産物（ｍ８Ｆ、ｍ８Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ８Ｆとｍ８Ｒを連結したＰＣＲ産物ｍ８ｐを得た。
（ａ－４）（ａ－３）で得られたＰＣＲ産物ｍ８ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ７ａに１箇所アミノ酸置換を導入したＦｃＲ８をコードするポリヌクレオチドを作製した。
（ａ－５）（ａ－４）で得られたポリヌクレオチドを制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ５ａに対して３箇所（野生型Ｆｃ結合性タンパク質に対して８箇所）アミノ酸置換したポリペプチドである、ＦｃＲ８をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ８を得た。
（ａ－７）ｐＥＴ－ＦｃＲ８のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 Example 7 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 6 involved in improving the thermal stability of Fc binding protein in FcR7a. Accumulation of substituted amino acids was mainly performed using PCR, and four types of improved Fc binding proteins shown in (a) to (d) below were prepared.
(A) FcR8 obtained by further performing amino acid substitution of Phe171Ser on FcR7a
(B) FcR9 obtained by further substituting Gln48Arg amino acid for FcR8
(C) FcR10 obtained by further performing amino acid substitution of Gln48Arg and Tyr51Ser on FcR8
(D) FcR11 obtained by further substituting amino acid substitution of Gln90Arg for FcR10
Hereinafter, a method for producing each improved Fc-binding protein will be described in detail.
(A) FcR8
Phe29Ile, Val117Glu, and Phe171Ser were selected from the amino acid substitutions involved in improving thermal stability, which were revealed in Example 6, and FcR8 in which these substitutions were accumulated in FcR5a (Example 4 (c)) was prepared. did. Specifically, FcR8 was produced by introducing a mutation that causes Phe171Ser into a polynucleotide encoding FcR7a.
(A-1) PCR was performed using pET-FcR7a obtained in Example 6 as a template. As a primer in the PCR, an oligonucleotide having a sequence described in SEQ ID NO: 23 and SEQ ID NO: 41 (5′-ACCAGCCCCACGGCAGGAAATAGCTGCCGCTG-3 ′) was used. In PCR, after preparing a reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction in which the third step for 1 minute was set to 1 cycle were performed by heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m8F.
(A-2) Except that pET-FcR7a obtained in Example 6 was used as a template, and an oligonucleotide consisting of SEQ ID NO: 42 (5′-GACACGCGCAGCATTTCCTGCCCTGGGGCTG-3 ′) and the sequence shown in SEQ ID NO: 24 was used as a PCR primer. , (A-1). The purified PCR product was designated as m8R.
(A-3) Two types of PCR products (m8F, m8R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed, and a PCR product m8p in which m8F and m8R were linked was obtained.
(A-4) PCR was performed using the PCR product m8p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR8 in which an amino acid substitution at one site was introduced into FcR7a was prepared.
(A-5) The polynucleotide obtained in (a-4) was digested with restriction enzymes NcoI and HindIII, and ligated to the expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. This was used to transform E. coli BL21 (DE3) strain.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR8, which is a polypeptide in which amino acids are substituted at 3 positions on FcR5a (8 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected microbial cells (transformants) Plasmid pET-FcR8 containing was obtained.
(A-7) The nucleotide sequence of pET-FcR8 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ８のアミノ酸配列を配列番号４３に、前記ＦｃＲ８をコードするポリヌクレオチドの配列を配列番号４４に示す。なお配列番号４３において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ８のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号４３において、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目の位置にそれぞれ存在する。
（ｂ）ＦｃＲ９
　実施例６で明らかとなった、熱安定性向上に関与するアミノ酸置換の中から、Ｐｈｅ２９Ｉｌｅ、Ｇｌｎ４８Ａｒｇ、Ｖａｌ１１７ＧｌｕおよびＰｈｅ１７１Ｓｅｒを選択し、それらの置換をＦｃＲ５ａ（実施例４（ｃ））に集積したＦｃＲ９を作製した。具体的には、ＦｃＲ８をコードするポリヌクレオチドに対して、Ｇｌｎ４８Ａｒｇを生じさせる変異導入を行なうことにより、ＦｃＲ９を作製した。
（ｂ－１）（ａ）で作製した、ｐＥＴ－ＦｃＲ８を鋳型とし、配列番号２４および配列番号４５（５’－ＧＴＧＡＣＣＣＴＴＡＡＡＴＧＣＣＧＧＧＧＣＧＣＧＴＡＴＡＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ９Ｆとした。
（ｂ－２）（ａ）で作製したｐＥＴ－ＦｃＲ８を鋳型とし、配列番号２３および配列番号４６（５’－ＣＣＧＧＧＣＴＡＴＡＣＧＣＧＣＣＣＣＧＧＣＡＴＴＴＡＡＧＧＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ９Ｒとした。
（ｂ－３）（ｂ－１）および（ｂ－２）で得られた２種類のＰＣＲ産物（ｍ９Ｆ、ｍ９Ｒ）を混合後、（ａ－３）と同様の方法にてＰＣＲを行ない、ｍ９Ｆとｍ９Ｒを連結した。得られたＰＣＲ産物をｍ９ｐとした。
（ｂ－４）（ｂ－３）で得られたＰＣＲ産物ｍ９ｐを鋳型とし、配列番号２１および配列番号２２に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－４）と同様の方法でＰＣＲを行なった。これによりＦｃＲ９をコードするポリヌクレオチドを作製した。
（ｂ－５）（ｂ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｂ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ５ａに対して４箇所（野生型Ｆｃ結合性タンパク質に対して９箇所）アミノ酸置換したポリペプチドである、ＦｃＲ９をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ９を得た。
（ｂ－７）ｐＥＴ－ＦｃＲ９のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR8 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 43, and the sequence of the polynucleotide encoding the FcR8 is shown in SEQ ID NO: 44. In SEQ ID NO: 43, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR8 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. In SEQ ID NO: 43, isoleucine of Phe29Ile is present at the 45th position, glutamic acid of Val117Glu is located at the 133rd position, and serine of Phe171Ser is present at the 187th position.
(B) FcR9
Phe29Ile, Gln48Arg, Val117Glu and Phe171Ser were selected from the amino acid substitutions involved in improving thermal stability, which were revealed in Example 6, and FcR9 integrated in FcR5a (Example 4 (c)) was selected. Was made. Specifically, FcR9 was produced by introducing a mutation that caused Gln48Arg to the polynucleotide encoding FcR8.
(B-1) except that pET-FcR8 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 45 (5′-GTGACCCTTAAATGCCGGGGCGCGTAGTAGC-3 ′) was used as a PCR primer. PCR was performed in the same manner as in (a-1). The purified PCR product was designated as m9F.
(B-2) except that pET-FcR8 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 46 (5′-CCGGGCTATACGCGCCCCCGGCATTAAGGG-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m9R.
(B-3) After mixing the two types of PCR products (m9F, m9R) obtained in (b-1) and (b-2), PCR was performed in the same manner as in (a-3), and m9F And m9R were linked. The obtained PCR product was designated as m9p.
(B-4) A method similar to (a-4), using the PCR product m9p obtained in (b-3) as a template and an oligonucleotide consisting of the sequences of SEQ ID NO: 21 and SEQ ID NO: 22 as a PCR primer PCR was performed. This produced a polynucleotide encoding FcR9.
(B-5) After purifying the polynucleotide obtained in (b-4), digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR9, which is a polypeptide in which amino acids are substituted at 4 positions (9 positions relative to a wild-type Fc binding protein) with respect to FcR5a by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR9 containing was obtained.
(B-7) The nucleotide sequence of pET-FcR9 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ９のアミノ酸配列を配列番号４７に、前記ＦｃＲ９をコードするポリヌクレオチドの配列を配列番号４８に示す。なお、配列番号４７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ９のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号４７において、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目の位置にそれぞれ存在する。
（ｃ）ＦｃＲ１０
　実施例６で明らかとなった、熱安定性向上に関与するアミノ酸置換の中から、Ｐｈｅ２９Ｉｌｅ、Ｇｌｎ４８Ａｒｇ、Ｔｙｒ５１Ｓｅｒ、Ｖａｌ１１７ＧｌｕおよびＰｈｅ１７１Ｓｅｒを選択し、それらの置換をＦｃＲ５ａ（実施例４（ｃ））に集積したＦｃＲ１０を作製した。具体的には、ＦｃＲ８をコードするポリヌクレオチドに対して、Ｇｌｎ４８ＡｒｇおよびＴｙｒ５１Ｓｅｒを生じさせる変異導入を行なうことにより、ＦｃＲ１０を作製した。
（ｃ－１）（ａ）で作製した、ｐＥＴ－ＦｃＲ８を鋳型とし、配列番号２２および配列番号４９（５’－ＴＧＣＣＧＧＧＧＣＧＣＧＴＣＴＡＧＣＣＣＧＧＡＡＧＡＴＡＡＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１０Ｆとした。
（ｃ－２）（ａ）で作製したｐＥＴ－ＦｃＲ８を鋳型とし、配列番号２１および配列番号５０（５’－ＧＣＴＡＧＡＣＧＣＧＣＣＣＣＧＧＣＡＴＴＴＡＡＧＧＧＴＣＡＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１０Ｒとした。
（ｃ－３）（ｃ－１）および（ｃ－２）で得られた２種類のＰＣＲ産物（ｍ１０Ｆ、ｍ１０Ｒ）を混合後、（ａ－３）と同様の方法にてＰＣＲを行ない、ｍ１０Ｆとｍ１０Ｒを連結した。得られたＰＣＲ産物をｍ１０ｐとした。
（ｃ－４）（ｃ－３）で得られたＰＣＲ産物ｍ１０ｐを鋳型とし、配列番号２１および配列番号２２に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－４）と同様の方法でＰＣＲを行なった。これによりＦｃＲ１０をコードするポリヌクレオチドを作製した。
（ｃ－５）（ｃ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｃ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ５ａに対して５箇所（野生型Ｆｃ結合性タンパク質に対して１０箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１０をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１０を得た。
（ｃ－７）ｐＥＴ－ＦｃＲ１０のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR9 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 47, and the sequence of the polynucleotide encoding the FcR9 is shown in SEQ ID NO: 48. In SEQ ID NO: 47, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. There is an amino acid sequence of FcR9 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine (Gly). ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. In SEQ ID NO: 47, isoleucine of Phe29Ile is present at the 45th position, arginine of Gln48Arg is present at the 64th position, glutamic acid of Val117Glu is present at the 133rd position, and serine of Phe171Ser is present at the 187th position.
(C) FcR10
Phe29Ile, Gln48Arg, Tyr51Ser, Val117Glu and Phe171Ser were selected from the amino acid substitutions involved in the improvement of thermal stability, which were revealed in Example 6, and these substitutions were accumulated in FcR5a (Example 4 (c)). FcR10 was prepared. Specifically, FcR10 was produced by introducing a mutation that caused Gln48Arg and Tyr51Ser into a polynucleotide encoding FcR8.
(C-1) except that pET-FcR8 prepared in (a) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 22 and SEQ ID NO: 49 (5′-TGCCGGGGCGCGTCTAGCCCGGAAGATAAC-3 ′) was used as a PCR primer PCR was performed in the same manner as in (a-1). The purified PCR product was designated as m10F.
(C-2) except that pET-FcR8 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 21 and SEQ ID NO: 50 (5′-GCTAGACCGCCCCGGCATTTAAGGGTCAC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m10R.
(C-3) After mixing the two kinds of PCR products (m10F, m10R) obtained in (c-1) and (c-2), PCR was performed in the same manner as in (a-3), and m10F And m10R were linked. The obtained PCR product was designated as m10p.
(C-4) The same method as (a-4), using the PCR product m10p obtained in (c-3) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR10.
(C-5) After purifying the polynucleotide obtained in (c-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(C-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR10, which is a polypeptide obtained by substituting amino acids for 5 positions in FcR5a (10 positions with respect to wild-type Fc binding protein) by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR10 containing was obtained.
(C-7) The nucleotide sequence of pET-FcR10 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１０のアミノ酸配列を配列番号５１に、前記ＦｃＲ１０をコードするポリヌクレオチドの配列を配列番号５２に示す。なお、配列番号５１において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１０のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号５１において、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｓｅｒのセリンは６７番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目の位置にそれぞれ存在する。
（ｄ）ＦｃＲ１１
　実施例６で明らかとなった、熱安定性向上に関与するアミノ酸置換の中から、Ｐｈｅ２９Ｉｌｅ、Ｇｌｎ４８Ａｒｇ、Ｔｙｒ５１Ｓｅｒ、Ｇｌｎ９０Ａｒｇ、Ｖａｌ１１７ＧｌｕおよびＰｈｅ１７１Ｓｅｒを選択し、それらの置換をＦｃＲ５ａ（実施例４（ｃ））に集積したＦｃＲ１１を作製した。具体的には、ＦｃＲ１０をコードするポリヌクレオチドに対して、Ｇｌｎ９０Ａｒｇを生じさせる変異導入を行なうことにより、ＦｃＲ１１を作製した。
（ｄ－１）（ｃ）で作製した、ｐＥＴ－ＦｃＲ１０を鋳型とし、配列番号２２および配列番号５３（５’－ＧＧＣＧＡＡＴＡＴＣＧＴＴＧＣＣＧＧＡＣＣＡＧＣＣＴＧＡＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１１Ｆとした。
（ｄ－２）（ｃ）で作製したｐＥＴ－ＦｃＲ１０を鋳型とし、配列番号２１および配列番号５４（５’－ＧＧＴＧＣＴＣＡＧＧＣＴＧＧＴＣＣＧＧＣＡＡＣＧＡＴＡＴＴＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１１Ｒとした。
（ｄ－３）（ｄ－１）および（ｄ－２）で得られた２種類のＰＣＲ産物（ｍ１１Ｆ、ｍ１１Ｒ）を混合後、（ａ－３）と同様の方法にてＰＣＲを行ない、ｍ１１Ｆとｍ１１Ｒを連結した。得られたＰＣＲ産物をｍ１１ｐとした。
（ｄ－４）（ｄ－３）で得られたＰＣＲ産物ｍ１１ｐを鋳型とし、配列番号２１および配列番号２２に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、（ａ－４）と同様の方法でＰＣＲを行なった。これによりＦｃＲ１１をコードするポリヌクレオチドを作製した。
（ｄ－５）（ｄ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｄ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ５ａに対して６箇所（野生型Ｆｃ結合性タンパク質に対して１１箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１１をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１１を得た。
（ｄ－７）ｐＥＴ－ＦｃＲ１１のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR10 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 51, and the sequence of the polynucleotide encoding the FcR10 is shown in SEQ ID NO: 52. In SEQ ID NO: 51, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. There is an amino acid sequence of FcR10 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the 209th to 210th glycine (Gly). ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. Further, in SEQ ID NO: 51, isoleucine of Phe29Ile is located at position 45, arginine of Gln48Arg is located at position 64, serine of Tyr51Ser is located at position 67, glutamic acid of Val117Glu is located at position 133, and serine of Phe171Ser is located at position 187.
(D) FcR11
Phe29Ile, Gln48Arg, Tyr51Ser, Gln90Arg, Val117Glu, and Phe171Ser were selected from the amino acid substitutions involved in improving the thermal stability, which were revealed in Example 6, and these substitutions were FcR5a (Example 4 (c)). FcR11 accumulated in the above was prepared. Specifically, FcR11 was produced by introducing a mutation that caused Gln90Arg to the polynucleotide encoding FcR10.
(D-1) except that pET-FcR10 prepared in (c) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 22 and SEQ ID NO: 53 (5′-GGCGAAATACGTTGCCGGACCAGCCTGGAGC-3 ′) was used as a PCR primer. PCR was performed in the same manner as in (a-1). The purified PCR product was designated as m11F.
(D-2) Except that pET-FcR10 prepared in (c) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 21 and SEQ ID NO: 54 (5′-GGTGCTCAGGCTGGTCCGCACAGAATTTC-3 ′) was used as a PCR primer, PCR was performed in the same manner as (a-1). The purified PCR product was designated as m11R.
(D-3) After mixing the two types of PCR products (m11F, m11R) obtained in (d-1) and (d-2), PCR was performed in the same manner as in (a-3). And m11R were linked. The obtained PCR product was designated as m11p.
(D-4) A method similar to (a-4), using the PCR product m11p obtained in (d-3) as a template and an oligonucleotide comprising the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22 as PCR primers PCR was performed. This produced a polynucleotide encoding FcR11.
(D-5) After purifying the polynucleotide obtained in (d-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(D-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR11, which is a polypeptide in which amino acids are substituted at 6 positions on FcR5a (11 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected microbial cells (transformants) Plasmid pET-FcR11 containing was obtained.
(D-7) The nucleotide sequence of pET-FcR11 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１１のアミノ酸配列を配列番号５５に、前記ＦｃＲ１１をコードするポリヌクレオチドの配列を配列番号５６に示す。なお、配列番号５５において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１１のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号５５において、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｓｅｒのセリンは６７番目、Ｇｌｎ９０Ａｒｇのアルギニンは１０６番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目の位置にそれぞれ存在する。 The amino acid sequence of FcR11 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 55, and the sequence of the polynucleotide encoding the FcR11 is shown in SEQ ID NO: 56. In SEQ ID NO: 55, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, from the 33rd glycine (Gly) to the 208th glutamine (Gln) is the amino acid sequence of FcR11 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. In SEQ ID NO: 55, isoleucine of Phe29Ile is at position 45, arginine of Gln48Arg is position 64, serine of Tyr51Ser is position 67, arginine of Gln90Arg is position 106, glutamic acid of Val117Glu is position 133, and serine of Phe171Ser is position 187. Each exists.
　実施例８　Ｆｃ結合性タンパク質の酸安定性評価
（１）実施例１で作製した野生型Ｆｃ結合性タンパク質、実施例６で選択したＦｃ結合性タンパク質（ＦｃＲ７ａ）、および実施例７で作製したＦｃ結合性タンパク質（ＦｃＲ８、ＦｃＲ９、ＦｃＲ１０、ＦｃＲ１１）を発現する形質転換体を、それぞれ５０μｇ／ｍＬのカナマイシンを含む３ｍＬの２ＹＴ液体培地に接種し、３７℃で一晩、好気的に振とう培養することで前培養を行なった。
（２）５０μｇ／ｍＬのカナマイシンを添加した２０ｍＬの２ＹＴ液体培地（ペプトン１６ｇ／Ｌ、酵母エキス１０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）に前培養液を２００μＬ接種し、３７℃で好気的に振とう培養を行なった。
（３）培養開始１．５時間後、培養温度を２０℃に変更して３０分間振とう培養した。その後、終濃度０．０１ｍＭとなるようＩＰＴＧを添加し、引き続き２０℃で一晩、好気的に振とう培養した。
（４）培養終了後、遠心分離により集菌し、ＢｕｇＢｕｓｔｅｒ　Ｐｒｏｔｅｉｎ　ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（タカラバイオ製）を用いてタンパク質抽出液を調製した。
（５）（４）で調製したタンパク質抽出液中の野生型Ｆｃ結合性タンパク質、ＦｃＲ７ａ、ＦｃＲ８、ＦｃＲ９、ＦｃＲ１０およびＦｃＲ１１の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法を用いて測定した。この時、市販のＦｃγＲＩＩＩａの細胞外領域（Ｒ＆Ｄテクノロジーズ製：４３２５－ＦＣ－０５０）を用いて検量線を作製し、濃度測定を行なった。
（６）各Ｆｃ結合性タンパク質の濃度が３０μｇ／ｍＬになるよう純水で希釈後、前記希釈した溶液１００μＬと０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）２００μＬとを混合し、３０℃で２時間静置した。
（７）グリシン塩酸緩衝液（ｐＨ３．０）による酸処理を行なった後のタンパク質の抗体結合活性と、前記酸処理を行なわなかったときのタンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法によって測定した。その後、酸処理を行なった場合の抗体結合活性を、酸処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出した。 Example 8 Evaluation of acid stability of Fc binding protein (1) Wild type Fc binding protein prepared in Example 1, Fc binding protein selected in Example 6 (FcR7a), and Fc prepared in Example 7 Transformants expressing binding proteins (FcR8, FcR9, FcR10, FcR11) are inoculated into 3 mL of 2YT liquid medium each containing 50 μg / mL kanamycin and cultured aerobically at 37 ° C. overnight. Thus, preculture was performed.
(2) Inoculate 200 μL of the precultured solution in 20 mL of 2YT liquid medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) supplemented with 50 μg / mL kanamycin, and aerobically at 37 ° C. Shaking culture was performed.
(3) 1.5 hours after the start of culture, the culture temperature was changed to 20 ° C., and the culture was shaken for 30 minutes. Thereafter, IPTG was added to a final concentration of 0.01 mM, followed by aerobic shaking culture at 20 ° C. overnight.
(4) After culturing, the cells were collected by centrifugation, and a protein extract was prepared using BugBuster Protein extraction kit (Takara Bio).
(5) Antibody binding activities of the wild-type Fc-binding proteins, FcR7a, FcR8, FcR9, FcR10, and FcR11 in the protein extract prepared in (4) were determined using the ELISA method described in Example 3 (4). It was measured. At this time, a calibration curve was prepared using the extracellular region of commercially available FcγRIIIa (R & D Technologies: 4325-FC-050), and the concentration was measured.
(6) After diluting with pure water so that the concentration of each Fc-binding protein becomes 30 μg / mL, 100 μL of the diluted solution and 200 μL of 0.1 M glycine hydrochloride buffer (pH 3.0) are mixed, and 30 ° C. And left for 2 hours.
(7) Example 3 (4) shows the antibody binding activity of the protein after acid treatment with glycine hydrochloride buffer (pH 3.0) and the antibody binding activity of the protein without acid treatment. It was measured by the described ELISA method. Thereafter, the residual activity was calculated by dividing the antibody binding activity when acid treatment was performed by the antibody binding activity when acid treatment was not performed.
　結果を表９に示す。今回評価したＦｃ結合性タンパク質（ＦｃＲ７ａ、ＦｃＲ８、ＦｃＲ９、ＦｃＲ１０、ＦｃＲ１１）は、野生型Ｆｃ結合性タンパク質と比較し残存活性が高かった。このことから、当該改良Ｆｃ結合性タンパク質の酸安定性が野生型に比べて向上していることが確認された。 The results are shown in Table 9. The Fc binding proteins evaluated this time (FcR7a, FcR8, FcR9, FcR10, FcR11) had higher residual activity than the wild type Fc binding proteins. From this, it was confirmed that the acid stability of the improved Fc-binding protein is improved as compared with the wild type.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　実施例９　システインタグを付加したＦｃＲ５ａ（ＦｃＲ５ａＣｙｓ）の作製
（１）実施例４（ｃ）で作製したｐＥＴ－ＦｃＲ５ａを鋳型としてＰＣＲを実施した。当該ＰＣＲにおけるプライマーは、配列番号２１および配列番号５７（５’－ＣＣＣＡＡＧＣＴＴＡＴＣＣＧＣＡＧＧＴＡＴＣＧＴＴＧＣＧＧＣＡＣＣＣＴＴＧＧＧＴＡＡＴＧＧＴＡＡＴＡＴＴＣＡＣＧＧＴＣＴＣＧＣＴＧＣ－３’）に記載の配列からなるオリゴヌクレオチドを用いた。ＰＣＲは、表２に示す組成の反応液を調製後、当該反応液を９８℃で５分熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。
（２）（１）で得られたポリヌクレオチドを精製し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、当該ライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（３）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて、発現ベクターｐＥＴ－ＦｃＲ５ａＣｙｓを抽出した。
（４）ｐＥＴ－ＦｃＲ５ａＣｙｓのヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。発現ベクターｐＥＴ－ＦｃＲ５ａＣｙｓで発現されるポリペプチドのアミノ酸配列を配列番号５８に、当該ポリペプチドをコードするポリヌクレオチドの配列を配列番号５９にそれぞれ示す。なお配列番号５８において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ５ａのアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目のグリシン（Ｇｌｙ）から２１６番目のグリシン（Ｇｌｙ）までがシステインタグ配列である。 Example 9 Preparation of FcR5a (FcR5aCys) Added with Cysteine Tag (1) PCR was performed using pET-FcR5a prepared in Example 4 (c) as a template. As a primer in the PCR, an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 57 (5′-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3 ′) was used. In PCR, after preparing a reaction solution having the composition shown in Table 2, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was repeated 30 times.
(2) The polynucleotide obtained in (1) is purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. Escherichia coli BL21 (DE3) strain was transformed with the ligation product.
(3) The obtained transformant was cultured in an LB medium containing 50 μg / mL kanamycin, and then the expression vector pET-FcR5aCys was extracted using QIAprep Spin Miniprep kit (Qiagen).
(4) The nucleotide sequence of pET-FcR5aCys was analyzed in the same manner as in Example 1 (5). The amino acid sequence of the polypeptide expressed by the expression vector pET-FcR5aCys is shown in SEQ ID NO: 58, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 59, respectively. In SEQ ID NO: 58, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR5a (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th glycine (Gly) to the 216th Up to glycine (Gly) is the cysteine tag sequence.
　実施例１０　システインタグを付加したＦｃＲ９（ＦｃＲ９Ｃｙｓ）の作製
（１）実施例７（ｂ）で作製したｐＥＴ－ＦｃＲ９を鋳型とし、配列番号２１および配列番号５７に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例９（１）と同様の方法でＰＣＲを行なった。
（２）実施例９（２）と同様な方法で大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（３）得られた形質転換体を実施例９（３）と同様な方法で培養後、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて、発現ベクターｐＥＴ－ＦｃＲ９Ｃｙｓを抽出した。
（４）ｐＥＴ－ＦｃＲ９Ｃｙｓのヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 Example 10 Preparation of FcR9 (FcR9Cys) Added with Cysteine Tag (1) Using the pET-FcR9 prepared in Example 7 (b) as a template, an oligonucleotide comprising the sequences described in SEQ ID NO: 21 and SEQ ID NO: 57 was subjected to PCR. PCR was performed in the same manner as in Example 9 (1) except that primers were used.
(2) E. coli BL21 (DE3) strain was transformed by the same method as in Example 9 (2).
(3) After the obtained transformant was cultured in the same manner as in Example 9 (3), the expression vector pET-FcR9Cys was extracted using QIAprep Spin Miniprep kit (Qiagen).
(4) The nucleotide sequence of pET-FcR9Cys was analyzed in the same manner as in Example 1 (5).
　発現ベクターｐＥＴ－ＦｃＲ９Ｃｙｓで発現されるポリペプチドのアミノ酸配列を配列番号６０に、当該ポリペプチドをコードするポリヌクレオチドの配列を配列番号６１に、それぞれ示す。なお配列番号６０において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ９のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目のグリシン（Ｇｌｙ）から２１６番目のグリシン（Ｇｌｙ）までがシステインタグ配列である。 The amino acid sequence of the polypeptide expressed by the expression vector pET-FcR9Cys is shown in SEQ ID NO: 60, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 61, respectively. In SEQ ID NO: 60, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR9 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th glycine (Gly) to the 216th Up to glycine (Gly) is the cysteine tag sequence.
　実施例１１　ＦｃＲ５ａＣｙｓの調製
（１）実施例９で作製したＦｃＲ５ａＣｙｓを発現する形質転換体を２Ｌのバッフルフラスコに入った５０μｇ／ｍＬのカナマイシンを含む４００ｍＬの２ＹＴ液体培地（ペプトン１６ｇ／Ｌ、酵母エキス１０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）に接種し、３７℃で一晩、好気的に振とう培養することで前培養を行なった。
（２）グルコース１０ｇ／Ｌ、酵母エキス２０ｇ／Ｌ、リン酸三ナトリウム十二水和物３ｇ／Ｌ、リン酸水素二ナトリウム十二水和物９ｇ／Ｌ、塩化アンモニウム１ｇ／Ｌおよび硫酸カナマイシン５０ｍｇ／Ｌを含む液体培地１．８Ｌに、（１）の培養液１８０ｍＬを接種し、３Ｌ発酵槽（バイオット製）を用いて本培養を行なった。温度３０℃、ｐＨ６．９から７．１、通気量１ＶＶＭ、溶存酸素濃度３０％飽和濃度の条件に設定し、本培養を開始した。ｐＨの制御には酸として５０％リン酸、アルカリとして１４％アンモニア水をそれぞれ使用し、溶存酸素の制御は撹拌速度を変化させることで制御し、撹拌回転数は下限５００ｒｐｍ、上限１０００ｒｐｍに設定した。培養開始後、グルコース濃度が測定できなくなった時点で、流加培地（グルコース２４８．９ｇ／Ｌ、酵母エキス８３．３ｇ／Ｌ、硫酸マグネシウム七水和物７．２ｇ／Ｌ）を溶存酸素（ＤＯ）により制御しながら加えた。
（３）菌体量の目安として６００ｎｍの吸光度（ＯＤ６００ｎｍ）が約１５０に達したところで培養温度を２５℃に下げ、設定温度に到達したことを確認した後、終濃度が０．５ｍＭになるようＩＰＴＧを添加し、引き続き２５℃で培養を継続した。
（４）培養開始から約４８時間後に培養を停止し、培養液を４℃で８０００ｒｐｍ、２０分間の遠心分離により菌体を回収した。
（５）回収した菌体を２０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）に５ｍＬ／１ｇ（菌体）となるように懸濁し、超音波発生装置（インソネーター２０１Ｍ（商品名）、久保田商事製）を用いて、４℃で約１０分間、約１５０Ｗの出力で菌体を破砕した。菌体破砕液は４℃で２０分間、８０００ｒｐｍの遠心分離を２回行ない、上清を回収した。
（６）（５）で得られた上清を、あらかじめ２０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）で平衡化した１４０ｍＬのＴＯＹＯＰＥＡＲＬ　ＣＭ－６５０Ｍ（東ソー製）を充填したＶＬ３２×２５０カラム（メルクミリポア製）に流速５ｍＬ／分でアプライした。平衡化に用いた緩衝液で洗浄後、０．５Ｍの塩化ナトリウムを含む２０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）で溶出した。
（７）（６）で得られた溶出液を、あらかじめ１５０ｍＭの塩化ナトリウムを含む２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）で平衡化したＩｇＧセファロース（ＧＥヘルスケア製）９０ｍＬを充填したＸＫ２６／２０カラムカラム（ＧＥヘルスケア製）にアプライした。平衡化に用いた緩衝液で洗浄後、０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）で溶出した。なお溶出液は、溶出液量の１／４量の１Ｍトリス塩酸緩衝液（ｐＨ８．０）を加えることでｐＨを中性付近に戻した。 Example 11 Preparation of FcR5aCys (1) 400 mL of 2YT liquid medium containing 50 μg / mL kanamycin (peptone 16 g / L, yeast extract) containing the transformant expressing FcR5aCys prepared in Example 9 in a 2 L baffle flask 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
(2) Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and kanamycin sulfate 50 mg The liquid culture medium 1.8L containing / L was inoculated with 180 mL of the culture solution of (1), and main culture was performed using a 3 L fermenter (product of Biot). The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation. The pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali. The dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. . When the glucose concentration could not be measured after the start of culture, fed-batch medium (glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L) was dissolved in dissolved oxygen (DO ) Was added while controlling.
(3) When the absorbance at 600 nm (OD 600 nm) reaches about 150 as a measure of the amount of bacterial cells, the culture temperature is lowered to 25 ° C., and after confirming that the set temperature has been reached, the final concentration is 0.5 mM. IPTG was added, and the culture was continued at 25 ° C.
(4) The culture was stopped about 48 hours after the start of the culture, and the cells were collected by centrifugation at 8000 rpm for 20 minutes at 4 ° C.
(5) The collected cells are suspended in 20 mM Tris-HCl buffer (pH 7.0) so as to be 5 mL / 1 g (cells), and an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation) ) Was used to disrupt the cells at an output of about 150 W for about 10 minutes at 4 ° C. The cell disruption solution was centrifuged twice at 8000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
(6) The supernatant obtained in (5) is a VL32 × 250 column (Merck Millipore) packed with 140 mL of TOYOPEARL CM-650M (manufactured by Tosoh) equilibrated in advance with 20 mM Tris-HCl buffer (pH 7.0). Manufactured at a flow rate of 5 mL / min. After washing with the buffer used for equilibration, elution was performed with 20 mM Tris-HCl buffer (pH 7.0) containing 0.5 M sodium chloride.
(7) XK26 / filled with 90 mL of IgG Sepharose (manufactured by GE Healthcare) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride in advance. This was applied to a 20 column column (manufactured by GE Healthcare). After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was returned to near neutrality by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0).
　前記精製により、高純度のＦｃＲ５ａＣｙｓを約２０ｍｇ得た。 About 20 mg of high-purity FcR5aCys was obtained by the purification.
　実施例１２　ＦｃＲ５ａ固定化ゲルの作製と抗体分離
（１）２ｍＬの分離剤用親水性ビニルポリマー（東ソー製：トヨパール）の表面の水酸基をヨードアセチル基で活性化後、実施例１１で調製したＦｃＲ５ａＣｙｓを４ｍｇ反応させることにより、ＦｃＲ５ａ固定化ゲルを得た。
（２）（１）で調製したＦｃＲ５ａ固定化ゲル０．５ｍＬをφ４．６ｍｍ×７５ｍｍのステンレスカラムに充填した。
（３）ＦｃＲ５ａ固定化ゲルを充填したカラムをＡＫＴＡ　Ｅｘｐｌｏｒｅｒ（ＧＥヘルスケア製）につなげ、２０ｍＭの酢酸緩衝液（ｐＨ４．６）で平衡化した。
（４）２０ｍＭの酢酸緩衝液（ｐＨ４．６）で０．５ｍｇ／ｍＬに希釈したモノクローナル抗体（リツキサン、全薬工業製）を流速０．２ｍＬ／ｍｉｎにて０．４ｍＬアプライした。
（５）流速０．２ｍＬ／ｍｉｎのまま平衡化緩衝液で２５分洗浄後、２０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）によるｐＨグラジエント（２５分で２０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）が１００％となるグラジエント）で吸着したモノクローナル抗体を溶出した。 Example 12 Preparation of FcR5a Immobilized Gel and Antibody Separation (1) FcR5aCys prepared in Example 11 after activating the hydroxyl group on the surface of 2 mL of hydrophilic vinyl polymer for separation agent (Tosoh Pearl: Toyopearl) with iodoacetyl group Was reacted to obtain an FcR5a-immobilized gel.
(2) 0.5 mL of FcR5a-immobilized gel prepared in (1) was packed in a stainless steel column of φ4.6 mm × 75 mm.
(3) A column packed with FcR5a-immobilized gel was connected to AKTA Explorer (manufactured by GE Healthcare) and equilibrated with 20 mM acetate buffer (pH 4.6).
(4) A monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) diluted to 0.5 mg / mL with 20 mM acetate buffer (pH 4.6) was applied at a flow rate of 0.2 mL / min.
(5) After washing with equilibration buffer for 25 minutes with a flow rate of 0.2 mL / min, pH gradient with 20 mM glycine hydrochloride buffer (pH 3.0) (20 mM glycine hydrochloride buffer (pH 3.0) over 25 minutes) The adsorbed monoclonal antibody was eluted with a gradient of 100%.
　結果（溶出パターン）を図２に示す。モノクローナル抗体はＦｃＲ５ａと相互作用するため、ゲルろ過クロマトグラフイーのような単一のピークではなく、複数のピークに分離された。 The results (elution pattern) are shown in FIG. Since the monoclonal antibody interacts with FcR5a, it was separated into multiple peaks rather than a single peak as in gel filtration chromatography.
　実施例１３　ＦｃＲ５ａ固定化ゲルで分離した抗体のＡＤＣＣ（抗体依存性細胞傷害作用）活性測定
（１）実施例１２に記載の溶出条件でモノクローナル抗体を分離し、図２に記載の溶出パターン中のフラクションＡ（ＦｒＡ）およびフラクションＢ（ＦｒＢ）の部分を分取した。
（２）分取したＦｒＡおよびＦｒＢを限外ろ過膜（メルクミリポア社）で濃縮しながら、ＰＢＳ（Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ）（ｐＨ７.４）に緩衝液を交換した。
（３）濃縮、緩衝液交換したＦｒＡおよびＦｒＢに含まれる抗体、ならびに分離前のモノクローナル抗体の濃度を２８０ｎｍの吸光度で測定した。
（４）以下に示す方法で、ＦｒＡおよびＦｒＢに含まれる抗体が有するＡＤＣＣ活性を測定した。
（４－１）１．４ｍＬのＬｏｗ　ＩｇＧ　Ｓｅｒｕｍと３３．６ｍＬのＲＰＭＩ１６４０培地とを混合して調製したＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒを用いて、ＦｒＡおよびＦｒＢに含まれる抗体ならびに分離前のモノクローナル抗体を３μｇ／ｍＬから１／３希釈で８段階の希釈系列を調製した。
（４－２）Ｒａｊｉ細胞をＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒにて約５×１０５ｃｅｌｌｓ／ｍＬに調製し、９６ｗｅｌｌプレート（３９１７：コーニング社）に２５μＬ／ｗｅｌｌで加えた。
（４－３）Ｒａｊｉ細胞を加えたｗｅｌｌに（４－１）で調製したフラクションＡ、フラクションＢ、分離前のモノクローナル抗体、ブランク（ＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒのみ）を２５μＬ／ｗｅｌｌ加えた。
（４－４）Ｅｆｆｅｃｔｏｒ細胞（プロメガ製）をＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒにて約３．０×１０５ｃｅｌｌｓ／ｍＬに調製し、Ｒａｊｉ細胞および抗体を加えたｗｅｌｌに２５μＬ／ｗｅｌｌで加えた。その後、ＣＯ２インキュベーター（５％ＣＯ２、３７℃）にて６時間静置した。
（４－５）９６ｗｅｌｌプレートを室温で５分から３０分静置した後、Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ　Ｒｅａｇｅｎｔ（プロメガ製）を７５μＬ／ｗｅｌｌで加えた。室温で３０分反応させたのち、ＧｌｏＭａｘ　Ｍｕｌｔｉ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ（プロメガ製）で発光を測定した。 Example 13 Measurement of ADCC (Antibody Dependent Cytotoxic Activity) Activity of Antibody Separated on FcR5a Immobilized Gel (1) Monoclonal antibodies were separated under the elution conditions described in Example 12, and the elution pattern shown in FIG. Fraction A (FrA) and fraction B (FrB) were fractionated.
(2) While concentrating the fractionated FrA and FrB with an ultrafiltration membrane (Merck Millipore), the buffer solution was exchanged with PBS (Phosphate Buffered Saline) (pH 7.4).
(3) The concentration of the antibody contained in the concentrated and buffer exchanged FrA and FrB, and the monoclonal antibody before separation was measured by absorbance at 280 nm.
(4) The ADCC activity of the antibodies contained in FrA and FrB was measured by the following method.
(4-1) Using ADCC Assay Buffer prepared by mixing 1.4 mL of Low IgG Serum and 33.6 mL of RPMI1640 medium, the antibody contained in FrA and FrB and the monoclonal antibody before separation were 3 μg / mL. 8 dilution series were prepared with 1/3 dilution.
(4-2) Raji cells were prepared to about 5 × 10 5 cells / mL with ADCC Assay Buffer and added to a 96-well plate (3917: Corning) at 25 μL / well.
(4-3) Fraction A, fraction B prepared in (4-1), monoclonal antibody before separation, and blank (ADCC Assay Buffer only) 25 μL / well were added to the well to which Raji cells were added.
(4-4) Effector cells (manufactured by Promega) were prepared to about 3.0 × 10 5 cells / mL with ADCC Assay Buffer, and added to wells containing Raji cells and antibodies at 25 μL / well. Then, it was left still for 6 hours in a CO 2 incubator (5% CO 2 , 37 ° C.).
(4-5) After allowing the 96-well plate to stand at room temperature for 5 to 30 minutes, Luciferase Assay Reagent (manufactured by Promega) was added at 75 μL / well. After reacting at room temperature for 30 minutes, luminescence was measured with a GloMax Multi Detection System (manufactured by Promega).
　実施例１２に記載の溶出条件で分取したＦｒＡおよびＦｒＢならびに分離前のモノクローナル抗体の発光強度を比較した結果を図３に示す。なお図３の結果は、測定した発光強度からブランクの発光強度を引いた値を示しており、発光強度が高いほど、ＡＤＣＣ活性が高いことを意味している。 FIG. 3 shows the results of comparing the luminescence intensity of FrA and FrB fractionated under the elution conditions described in Example 12 and the monoclonal antibody before separation. The result of FIG. 3 shows a value obtained by subtracting the emission intensity of the blank from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
　ＦｒＡは分離前のモノクローナル抗体とほぼ同程度の発光強度であることからＡＤＣＣ活性はほぼ同等といえる。一方、ＦｒＢは分離前のモノクローナル抗体と比べて約３．２倍、ＦｒＡに比べても２．５倍に向上していた。つまり、ＦｒＢは分離前のモノクローナル抗体およびＦｒＡと比べてＡＤＣＣ活性が高いことが分かる。 Since FrA has almost the same luminescence intensity as that of the monoclonal antibody before separation, it can be said that the ADCC activity is almost the same. On the other hand, FrB was improved by about 3.2 times compared with the monoclonal antibody before separation and 2.5 times compared with FrA. That is, it can be seen that FrB has higher ADCC activity than the monoclonal antibody and FrA before separation.
　実施例１４　ＦｃＲ５ａ固定化ゲルで分離した抗体の糖鎖解析
（１）実施例１３（１）で分取したＦｒＡおよびＦｒＢ、ならびに分離前のモノクローナル抗体を１００℃、１０分の熱処理により変性後、グリコアミダーゼＡ／ペプシンおよびプロナーゼで順次処理し、ゲルろ過法による精製操作を経て糖鎖画分を取得した。
（２）（１）で得られた糖鎖をエバポレーターにて濃縮・乾燥後、酢酸溶媒下、２－アミノピリジン、次いでジメチルアミンボランを順次作用させて蛍光ラベル化糖鎖とし、ゲルろ過法により精製した。
（３）（２）で得られた蛍光ラベル化糖鎖を陰イオン交換カラム（ＴＳＫｇｅｌ　ＤＥＡＥ－５ＰＷ、φ７．５ｍｍ×７．５ｃｍ：東ソー製）にて、中性糖鎖画分とモノシアリル化糖鎖画分に分離した。
（４）（３）で得られた中性糖鎖画分とモノシアリル化糖鎖画分をＯＤＳカラムを用いて、個々の糖鎖に単離した。ＭＡＬＤＩ－ＴＯＦ－ＭＳ分析により単離した糖鎖の分子量情報を取得後、ＯＤＳカラムクロマトグラフのリテンションタイムと照らし合わせて糖鎖構造を帰属した。 Example 14 Sugar chain analysis of antibody separated by FcR5a-immobilized gel (1) FrA and FrB fractionated in Example 13 (1) and the monoclonal antibody before separation were denatured by heat treatment at 100 ° C. for 10 minutes, Glycoamidase A / pepsin and pronase were sequentially treated, and a sugar chain fraction was obtained through purification by gel filtration.
(2) After concentrating and drying the sugar chain obtained in (1) with an evaporator, 2-aminopyridine and then dimethylamine borane are successively acted on in an acetic acid solvent to form a fluorescent labeled sugar chain. Purified.
(3) Using the anion exchange column (TSKgel DEAE-5PW, φ7.5 mm × 7.5 cm: manufactured by Tosoh), the neutral sugar chain fraction and the monosialylated sugar were obtained by using the fluorescently labeled sugar chain obtained in (2). Separated into chain fractions.
(4) The neutral sugar chain fraction and monosialylated sugar chain fraction obtained in (3) were isolated into individual sugar chains using an ODS column. After obtaining the molecular weight information of the sugar chain isolated by MALDI-TOF-MS analysis, the sugar chain structure was assigned in comparison with the retention time of the ODS column chromatograph.
　帰属した糖鎖構造（Ｎ１からＮ６、Ｍ１、Ｍ２およびＤ１）を図４に、中性糖鎖の組成比を表１０に、モノシアリル化およびジシアリル化糖鎖の組成比を表１１にそれぞれ示す。糖鎖構造Ｎ４＋Ｎ４’およびＮ６を有した抗体は、分離前およびＦｒＡと比較してＦｒＢで増加していた。一方、Ｎ１、Ｎ２＋Ｎ３’、Ｎ３およびＮ５を有した抗体は、分離前およびＦｒＡと比較してＦｒＢで減少していた。すなわちＮ４＋Ｎ４’およびＮ６糖鎖を持つ抗体はＦｃＲ５ａと強く結合し、Ｎ１、Ｎ２＋Ｎ３’、Ｎ３およびＮ５を有した抗体はＦｃＲ５ａとの結合が弱いことがわかる。またＭ１、Ｍ２およびＤ１を有した抗体は、分離前およびＦｒＡと比較してＦｒＢで増加していた。すなわちＭ１、Ｍ２およびＤ１糖鎖を持つ抗体はＦｃＲ５ａと強く結合することがわかる。 The assigned sugar chain structures (N1 to N6, M1, M2 and D1) are shown in FIG. 4, the composition ratio of neutral sugar chains is shown in Table 10, and the composition ratio of monosialylated and disialylated sugar chains is shown in Table 11. Antibodies with sugar chain structures N4 + N4 'and N6 increased with FrB before separation and compared to FrA. On the other hand, antibodies with N1, N2 + N3 ', N3 and N5 were reduced in FrB before separation and compared to FrA. That is, it can be seen that antibodies having N4 + N4 ′ and N6 sugar chains strongly bind to FcR5a, and antibodies having N1, N2 + N3 ′, N3 and N5 have weak binding to FcR5a. In addition, antibodies with M1, M2 and D1 increased in FrB before separation and compared to FrA. That is, it can be seen that antibodies having M1, M2 and D1 sugar chains strongly bind to FcR5a.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
　前記結果と実施例１３の結果とを合わせると、分離前およびＦｒＡと比較してＦｒＢで増加した糖鎖構造を有する抗体はＡＤＣＣ活性が高いことがわかる。すなわち、ＦｃＲ５ａ固定化ゲルは、抗体が有する糖鎖構造の違いを識別でき、かつ前記識別に基づきＡＤＣＣ活性の高い抗体を分離することができることがわかる。 When the above results and the results of Example 13 are combined, it can be seen that an antibody having a sugar chain structure increased by FrB before separation and FrA has higher ADCC activity. That is, it can be seen that the FcR5a-immobilized gel can identify the difference in the sugar chain structure of the antibody and can separate the antibody with high ADCC activity based on the identification.
　実施例１５　ＦｃＲ９固定化ゲルの作製と抗体分離
（１）実施例１０で作製したＦｃＲ９Ｃｙｓを発現する形質転換体を用いて、実施例１１
（１）から（４）と同様な方法で培養を行なった。
（２）実施例１１と同様な方法で精製し、高純度のＦｃＲ９Ｃｙｓを約１０ｍｇ得た。
（３）実施例１２（１）と同様な方法でＦｃＲ９Ｃｙｓ固定化ゲルを得た後、当該ゲル０．５ｍＬをφ４．０ｍｍ×４０ｍｍのステンレスカラムに充填した。
（４）ＦｃＲ９固定化ゲルを充填したカラムを高速液体クロマトグラフィー装置（東ソー製）につなげ、２０ｍＭの酢酸緩衝液（ｐＨ４．５）で平衡化した。
（５）ＰＢＳ（Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ）（ｐＨ７.４）で４．０ｍｇ／ｍＬに希釈したモノクローナル抗体（リツキサン、全薬工業製）を流速０．３ｍＬ／ｍｉｎにて０．１５ｍＬアプライした。
（６）流速０．３ｍＬ／ｍｉｎのまま平衡化緩衝液で２分洗浄後、１０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）によるｐＨグラジエント（３８分で１０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）が１００％となるグラジエント）で吸着したモノクローナル抗体を溶出した。 Example 15 Preparation of FcR9-immobilized gel and antibody separation (1) Using the transformant expressing FcR9Cys prepared in Example 10, Example 11
Incubation was carried out in the same manner as (1) to (4).
(2) Purification was performed in the same manner as in Example 11 to obtain about 10 mg of high-purity FcR9Cys.
(3) After obtaining an FcR9Cys-immobilized gel in the same manner as in Example 12 (1), 0.5 mL of the gel was packed into a stainless steel column of φ4.0 mm × 40 mm.
(4) A column packed with FcR9-immobilized gel was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation) and equilibrated with 20 mM acetate buffer (pH 4.5).
(5) A monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) diluted to 4.0 mg / mL with PBS (Phosphate Buffered Saline) (pH 7.4) was applied at 0.15 mL at a flow rate of 0.3 mL / min.
(6) After washing with equilibration buffer for 2 minutes with a flow rate of 0.3 mL / min, pH gradient with 10 mM glycine hydrochloride buffer (pH 3.0) (10 mM glycine hydrochloride buffer (pH 3.0) over 38 minutes) The adsorbed monoclonal antibody was eluted with a gradient of 100%.
　結果（溶出パターン）を図５に示す。モノクローナル抗体はＦｃＲ９と相互作用するため、ゲルろ過クロマトグラフィーのような単一のピークではなく、複数のピークに分離された。 The results (elution pattern) are shown in FIG. Since the monoclonal antibody interacts with FcR9, it was separated into multiple peaks rather than a single peak as in gel filtration chromatography.
　実施例１６　ＦｃＲ９固定化ゲルで分離した抗体のＡＤＣＣ活性測定
（１）実施例１５の溶出条件でモノクローナル抗体を分離し、図５に記載の溶出パターン中のフラクションＡ（ＦｒＡ）、フラクションＢ（ＦｒＢ）およびフラクションＣ（ＦｒＣ）の部分を分取した。
（２）ＦｒＡ、ＦｒＢおよびＦｒＣに含まれる抗体、ならびに分離前のモノクローナル抗体の濃度を２８０ｎｍの吸光度で測定し、実施例１３（４）と同様な方法でＡＤＣＣ活性を測定した。 Example 16 ADCC activity measurement of antibody separated on FcR9-immobilized gel (1) Monoclonal antibodies were separated under the elution conditions of Example 15, and fractions A (FrA) and B (FrB) in the elution pattern shown in FIG. ) And fraction C (FrC).
(2) The concentrations of the antibody contained in FrA, FrB and FrC, and the monoclonal antibody before separation were measured by absorbance at 280 nm, and ADCC activity was measured by the same method as in Example 13 (4).
　結果を図６に示す。なお図６の結果は、測定した発光強度からブランクの発光強度を引いた値を示しており、発光強度が高いほど、ＡＤＣＣ活性が高いことを意味している。 The results are shown in FIG. The result of FIG. 6 shows a value obtained by subtracting the blank emission intensity from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
　ＦｒＡおよびＦｒＢは分離前のモノクローナル抗体よりＡＤＣＣ活性がやや低いといえる。一方、ＦｒＣは分離前のモノクローナル抗体と比べてＡＤＣＣ活性が約１．６倍に向上していた。つまり溶出の遅いＦｒＣは溶出の早いＦｒＡおよびＦｒＢならびに分離前のモノクローナル抗体と比べてＡＤＣＣ活性が高いことがわかる。また実施例１４より、本発明のＦｃ結合性タンパク質を固定化したゲルは、抗体が有する糖鎖構造の違いを識別できることから、ＦｒＣに含まれるＦｃＲ９と強く結合する抗体は、高いＡＤＣＣ活性を有した糖鎖構造を有する抗体であることが示唆される。 It can be said that FrA and FrB have slightly lower ADCC activity than the monoclonal antibody before separation. On the other hand, FrC improved ADCC activity by about 1.6 times compared with the monoclonal antibody before separation. That is, it can be seen that FrC that is slowly eluted has higher ADCC activity than FrA and FrB that are eluted quickly and the monoclonal antibody before separation. Further, from Example 14, the gel immobilized with the Fc-binding protein of the present invention can distinguish the difference in the sugar chain structure of the antibody. Therefore, an antibody that strongly binds to FcR9 contained in FrC has high ADCC activity. It is suggested that the antibody has a sugar chain structure.
　実施例１７　ＦｃＲ９への変異導入およびライブラリーの作製
　実施例７（ｂ）で作製したＦｃＲ９をコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例７（ｂ）で作製した発現ベクターｐＥＴ－ＦｃＲ９を用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、ｐＥＴ－ＦｃＲ９を鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをプライマーとした他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。この反応によりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入された。
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養後、プレート上に形成したコロニーをランダム変異ライブラリーとした。 Example 17 Mutation Introduction to FcR9 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR9 prepared in Example 7 (b) by error-prone PCR.
(1) Error-prone PCR was performed using the expression vector pET-FcR9 prepared in Example 7 (b) as a template. In error-prone PCR, pET-FcR9 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared except that oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 were used as primers. Was subjected to a heat treatment at 95 ° C. for 2 minutes, 35 cycles of a reaction comprising a first step at 95 ° C. for 30 seconds, a second step at 50 ° C. for 30 seconds, and a third step at 72 ° C. for 90 seconds were performed for 35 cycles. The sample was heat treated at 72 ° C. for 7 minutes. By this reaction, the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
(2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
　実施例１８　アルカリ安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例１７で作製したランダム変異ライブラリーを実施例３（１）から（２）に記載の方法で培養することでＦｃ結合性タンパク質を発現させた。
（２）培養後、遠心操作によって得られた、Ｆｃ結合性タンパク質を含む培養上清を純水にて１０倍に希釈し、等量の６０ｍＭの水酸化ナトリウム溶液と混合し、３０℃で１．５時間静置することでアルカリ処理した。その後、４倍量の１Ｍトリス緩衝液（ｐＨ７．０）でｐＨを中性付近に戻した。
（３）（２）に記載のアルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（２）に記載のアルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法にてそれぞれ測定し、アルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性を、アルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４）（３）の方法で約２７００株の形質転換体を評価し、その中からＦｃＲ９と比較して安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。選択した形質転換体を５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地にて培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（５）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 Example 18 Screening of Alkaline Stabilized Fc Binding Protein (1) The random mutation library prepared in Example 17 was cultured by the method described in Examples 3 (1) to (2) to obtain Fc binding protein. Expressed.
(2) After culturing, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 10-fold with pure water, mixed with an equal volume of 60 mM sodium hydroxide solution, and 1 at 30 ° C. . Alkali treatment by standing for 5 hours. Thereafter, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
(3) The antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is performed, and the antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is not performed, The antibody binding activity of the Fc binding protein when measured by the ELISA method described in Example 3 (4) and subjected to alkali treatment, and the antibody binding activity of the Fc binding protein without alkali treatment. The residual activity was calculated by dividing by.
(4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing an Fc-binding protein with improved stability compared with FcR9 were selected. The selected transformant was cultured in a 2YT liquid medium containing 50 μg / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
　（４）で選択した形質転換体が発現するＦｃ結合性タンパク質の、ＦｃＲ９に対するアミノ酸置換位置およびアルカリ処理後の残存活性（％）をまとめたものを表１２に示す。配列番号３７に記載のアミノ酸配列のうち、３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、Ｍｅｔ１８Ｉｌｅ（この表記は、配列番号１の１８番目（配列番号３７では３４番目）のメチオニンがイソロイシンに置換されていることを表す、以下同様）、Ｇｌｕ２１Ｌｙｓ、Ｇｌｕ２１Ｇｌｙ、Ｌｅｕ２３Ｍｅｔ、Ｇｌｎ３３Ｐｒｏ、Ｌｙｓ４６Ｇｌｕ、Ｐｈｅ６１Ｔｙｒ、Ｇｌｕ６４Ｇｌｙ、Ｓｅｒ６５Ａｒｇ、Ｓｅｒ６８Ｐｒｏ、Ａｓｐ７７Ｖａｌ、Ａｓｐ７７Ｇｌｕ、Ｖａｌ８１Ｍｅｔ、Ａｓｐ８２Ａｌａ、Ｇｌｎ１０１Ｌｅｕ、Ｇｌｕ１０３Ｖａｌ、Ｈｉｓ１０５Ａｒｇ、Ｇｌｕ１２０Ｖａｌ、Ｓｅｒ１７８ＡｒｇおよびＡｓｎ１８０Ｌｙｓのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、ＦｃＲ９と比較しアルカリ安定性が向上しているといえる。 Table 12 shows a summary of amino acid substitution positions for FcR9 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4). Of the amino acid sequence set forth in SEQ ID NO: 37, the amino acid residue from the 33rd glycine to the 208th glutamine, and the 33rd to 208th amino acid residues, Met18Ile (this notation is SEQ ID NO: 1 represents that methionine at 18th position (34th in SEQ ID NO: 37) is substituted with isoleucine, the same applies to the following), Glu21Lys, Glu21Gly, Leu23Met, Gln33Pro, Lys46Glu, Phe61Tyr, Glu64Gly, Ser65Arg, Ser68Pro, Asp77V , Val81Met, Asp82Ala, Gln101Leu, Glu103Val, His105Arg, Glu120Val, Ser178Arg and Asn180Ly Fc binding proteins or amino acid substitutions are at least one occurs, it can be said that the alkaline stability as compared to FcR9 is improved.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　実施例１９　改良Ｆｃ結合性タンパク質の作製
　実施例１８で判明した、Ｆｃ結合性タンパク質のアルカリ安定性向上に関与するアミノ酸置換をＦｃＲ９に集積することで、さらなる安定性向上を図った。置換アミノ酸の集積は、主にＰＣＲを用いて行ない、以下の（ａ）および（ｂ）に示す２種類の改良Ｆｃ結合性タンパク質を作製した。
（ａ）ＦｃＲ９に対し、さらにＧｌｕ２１Ｇｌｙ、Ｌｅｕ２３ＭｅｔおよびＳｅｒ１７８Ａｒｇのアミノ酸置換を行なったＦｃＲ１２
（ｂ）ＦｃＲ９に対し、さらにＧｌｕ２１Ｇｌｙ、Ｌｅｕ２３Ｍｅｔ、Ｓｅｒ６８ＰｒｏおよびＳｅｒ１７８Ａｒｇのアミノ酸置換を行なったＦｃＲ１３
　以下、各改良Ｆｃ結合性タンパク質の作製方法を詳細に説明する。
（ａ）ＦｃＲ１２
　実施例１８で明らかとなった、アルカリ安定性向上に関与するアミノ酸置換の中から、Ｇｌｕ２１Ｇｌｙ、Ｌｅｕ２３ＭｅｔおよびＳｅｒ１７８Ａｒｇを選択し、それらの置換をＦｃＲ９（実施例７（ｂ））に集積したＦｃＲ１２を作製した。具体的には、実施例１８で得られたＳｅｒ１７８Ａｒｇの変異を含んだポリヌクレオチドに対して、Ｇｌｕ２１ＧｌｙおよびＬｅｕ２３Ｍｅｔを生じさせる変異導入を行なうことにより、ＦｃＲ１２
を作製した。　　　　
（ａ－１）実施例１８で取得した、ＦｃＲ９にＳｅｒ１７８Ａｒｇの変異を含んだＦｃ結合性タンパク質をコードするポリヌクレオチドを鋳型としてＰＣＲを実施した。当該ＰＣＲにおけるプライマーは、配列番号２４および配列番号６２（５’－ＣＴＡＧＣＣＡＴＧＧＧＣＡＴＧＣＧＴＡＣＣＧＧＡＧＡＴＡＴＧＣＣＧＡＡＡＧＣＧＧＡＧ－３’）に記載の配列からなるオリゴヌクレオチドを用いた。ＰＣＲは、鋳型とプライマー以外は表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ１２ｐとした。
（ａ－２）（ａ－１）で得られたｍ１２ｐを制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ－３）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ９に対して３箇所（野生型Ｆｃ結合性タンパク質に対して１２箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１２をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１２を得た。
（ａ－４）ｐＥＴ－ＦｃＲ１２のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 Example 19 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 18 involved in improving the alkali stability of Fc binding protein in FcR9. Accumulation of substituted amino acids was mainly performed using PCR, and two types of improved Fc-binding proteins shown in (a) and (b) below were prepared.
(A) FcR12 in which amino acid substitution of Glu21Gly, Leu23Met and Ser178Arg was further performed on FcR9
(B) FcR13 obtained by further performing amino acid substitution of Glu21Gly, Leu23Met, Ser68Pro and Ser178Arg to FcR9
Hereinafter, a method for producing each improved Fc-binding protein will be described in detail.
(A) FcR12
Glu21Gly, Leu23Met and Ser178Arg were selected from the amino acid substitutions involved in improving alkali stability, which were revealed in Example 18, and FcR12 in which these substitutions were accumulated in FcR9 (Example 7 (b)) was prepared. did. Specifically, FcR12 is obtained by introducing a mutation that causes Glu21Gly and Leu23Met to the polynucleotide containing the Ser178Arg mutation obtained in Example 18.
Was made.
(A-1) PCR was carried out using the polynucleotide obtained in Example 18 encoding Fc-binding protein containing Ser178Arg mutation in FcR9 as a template. As primers for the PCR, oligonucleotides having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 62 (5′-CTAGCCATGGGGCATGCGTACCGAGATATGCCGAAAGCGGGAG-3 ′) were used. In PCR, after preparing a reaction solution having the composition shown in Table 7 except for the template and primer, the reaction solution was heat-treated at 98 ° C. for 5 minutes, the first step at 98 ° C. for 10 seconds, and the second step at 55 ° C. for 5 seconds. Step, 30 cycles of the third step of 1 minute at 72 ° C. were performed for 30 cycles, and finally heat treatment was performed at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m12p.
(A-2) m12p obtained in (a-1) was digested with restriction enzymes NcoI and HindIII, and ligated to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII. This was used to transform E. coli BL21 (DE3) strain.
(A-3) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR12, which is a polypeptide in which amino acid substitution has been performed at three positions on FcR9 (12 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR12 containing was obtained.
(A-4) The nucleotide sequence of pET-FcR12 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１２のアミノ酸配列を配列番号６３に、前記ＦｃＲ１２をコードするポリヌクレオチドの配列を配列番号６４に示す。なお配列番号６３において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１２のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号６３において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。
（ｂ）ＦｃＲ１３
　実施例１８で明らかとなった、アルカリ安定性向上に関与するアミノ酸置換の中から、Ｇｌｕ２１Ｇｌｙ、Ｌｅｕ２３Ｍｅｔ、Ｓｅｒ６８ＰｒｏおよびＳｅｒ１７８Ａｒｇを選択し、それらの置換をＦｃＲ９（実施例７（ｂ））に集積したＦｃＲ１３を作製した。具体的には、ＦｃＲ１２をコードするポリヌクレオチドに対して、Ｓｅｒ６８Ｐｒｏを生じさせる変異導入を行なうことにより、ＦｃＲ１３を作製した。
（ｂ－１）（ａ）で作製した、ｐＥＴ－ＦｃＲ１２を鋳型とし、配列番号２４および配列番号６５（５’－ＣＡＣＡＡＴＧＡＡＡＧＣＣＴＧＡＴＴＣＣＣＡＧＣＣＡＧＧＣＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ１３Ｆとした。
（ｂ－２）（ａ）で作製したｐＥＴ－ＦｃＲ１２を鋳型とし、配列番号６２および配列番号６６（５’－ＧＴＡＧＣＴＧＣＴＣＧＣＣＴＧＧＣＴＧＧＧＡＡＴＣＡＧＧＣＴ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｂ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１３Ｒとした。
（ｂ－３）（ｂ－１）および（ｂ－２）で得られた２種類のＰＣＲ産物（ｍ１３Ｆ、ｍ１３Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行ない、最後に７２℃で５分間熱処理するＰＣＲを行ない、ｍ１３Ｆとｍ１３Ｒを連結した。得られたＰＣＲ産物をｍ１３ｐとした。
（ｂ－４）（ｂ－３）で得られたＰＣＲ産物ｍ１３ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、ＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１２に１箇所アミノ酸置換を導入したＦｃＲ１３をコードするポリヌクレオチドを作製した。
（ｂ－５）（ｂ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｂ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ９に対して４箇所（野生型Ｆｃ結合性タンパク質に対して１３箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１３をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１３を得た。
（ｂ－７）ｐＥＴ－ＦｃＲ１３のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR12 to which a signal sequence and a polyhistidine tag are added is shown in SEQ ID NO: 63, and the sequence of the polynucleotide encoding the FcR12 is shown in SEQ ID NO: 64. In SEQ ID NO: 63, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR12 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. In SEQ ID NO: 63, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, Phe75Lu. Leucine is at position 91, Asn92Ser is at position 108, Val117Glu is at glutamic acid 133, Glu121Gly at 137th is glycine, Phe171Ser is at position 187, and Ser178Arg is at position 194.
(B) FcR13
Glu21Gly, Leu23Met, Ser68Pro and Ser178Arg were selected from the amino acid substitutions involved in improving the alkali stability, which were revealed in Example 18, and these substitutions were accumulated in FcR9 (Example 7 (b)). Was made. Specifically, FcR13 was produced by introducing a mutation that caused Ser68Pro to the polynucleotide encoding FcR12.
(B-1) except that pET-FcR12 prepared in (a) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 24 and SEQ ID NO: 65 (5′-CACAATGAAAGCCCTGATTCCCACGCCAGCGCG-3 ′) was used as a PCR primer. After preparing a reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and 72 ° C. for 1 minute. The reaction with the third step of 1 cycle as 30 cycles was performed by heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m13F.
(B-2) except that pET-FcR12 prepared in (a) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 66 (5′-GTAGCTGCTCGCCCTGCTGGGAATCAGGCT-3 ′) was used as a PCR primer, PCR was performed in the same manner as (b-1). The purified PCR product was designated as m13R.
(B-3) Two types of PCR products (m13F, m13R) obtained in (b-1) and (b-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. Finally, PCR was performed by heat treatment at 72 ° C. for 5 minutes, and m13F and m13R were linked. The obtained PCR product was designated as m13p.
(B-4) PCR was performed using the PCR product m13p obtained in (b-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR13 in which an amino acid substitution at one position was introduced into FcR12 was prepared.
(B-5) After purifying the polynucleotide obtained in (b-4), digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR13, which is a polypeptide in which amino acids are substituted at 4 positions (13 positions with respect to a wild-type Fc-binding protein) with respect to FcR9 by extracting a plasmid from the collected microbial cells (transformants) Plasmid pET-FcR13 containing was obtained.
(B-7) The nucleotide sequence of pET-FcR13 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１３のアミノ酸配列を配列番号６７に、前記ＦｃＲ１３をコードするポリヌクレオチドの配列を配列番号６８に示す。なお、配列番号６７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１３のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号６７において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。 The amino acid sequence of FcR13 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 67, and the sequence of the polynucleotide encoding the FcR13 is shown in SEQ ID NO: 68. In SEQ ID NO: 67, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, the amino acid sequence of FcR13 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the region from the 17th to the 192nd region of SEQ ID NO: 1), the 209th to the 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. In SEQ ID NO: 67, Glu21Gly glycine is 37th, Leu23Met methionine is 39th, Val27Glu glutamate is 43rd, Phe29Ile isoleucine is 45th, Tyr35Asn asparagine is 51st, Gln48Arg arginine is 64th, Ser68Pro Proline at position 84, Phe75Leu leucine at position 91, Asn92Ser serine at position 108, Val117Glu glutamic acid at position 133, Glu121Gly glycine at position 137, Phe171Ser serine at position 187, and Ser178Arg arginine at position 194. Exists.
　実施例２０　Ｆｃ結合性タンパク質のアルカリ安定性評価
（１）実施例４（ｃ）で作製したＦｃ結合性タンパク質（ＦｃＲ５ａ）、実施例７（ｂ）で作製したＦｃ結合性タンパク質（ＦｃＲ９）、および実施例１９で作製したＦｃ結合性タンパク質（ＦｃＲ１２、ＦｃＲ１３）を発現する形質転換体を、実施例８の（１）から（４）に記載の方法で培養し、タンパク質を抽出することでＦｃＲ５ａ、ＦｃＲ９、ＦｃＲ１２およびＦｃＲ１３を調製した。
（２）（１）で調製したタンパク質抽出液中のＦｃＲ５ａ、ＦｃＲ９、ＦｃＲ１２およびＦｃＲ１３の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法を用いて測定した。この時、精製し定量したＦｃＲ９を用いて検量線を作製し、濃度測定を行なった。
（３）各Ｆｃ結合性タンパク質の濃度が３０μｇ／ｍＬになるよう純水で希釈後、前記希釈した溶液５０μＬと４０ｍＭの水酸化ナトリウム溶液５０μＬとを混合し、３０℃で２時間静置することでアルカリ処理した。その後、１Ｍトリス塩酸緩衝液（ｐＨ７．０）を４倍量加えることで中和し、Ｆｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法によって測定した。
（４）アルカリ処理を行なった場合の抗体結合活性をアルカリ処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出しアルカリ安定性を評価した。 Example 20 Evaluation of Alkali Stability of Fc Binding Protein (1) Fc binding protein (FcR5a) prepared in Example 4 (c), Fc binding protein (FcR9) prepared in Example 7 (b), and The transformant expressing the Fc binding protein (FcR12, FcR13) prepared in Example 19 was cultured by the method described in Example 8 (1) to (4), and FcR5a, FcR9, FcR12 and FcR13 were prepared.
(2) The antibody binding activity of FcR5a, FcR9, FcR12 and FcR13 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR9 purified and quantified, and the concentration was measured.
(3) After diluting with pure water so that the concentration of each Fc-binding protein is 30 μg / mL, 50 μL of the diluted solution and 50 μL of 40 mM sodium hydroxide solution are mixed and allowed to stand at 30 ° C. for 2 hours. Treated with alkali. Thereafter, the solution was neutralized by adding 4 volumes of 1M Tris-HCl buffer (pH 7.0), and the antibody binding activity of the Fc binding protein was measured by the ELISA method described in Example 3 (4).
(4) The residual activity was calculated by dividing the antibody binding activity when the alkali treatment was performed by the antibody binding activity when the alkali treatment was not performed, and the alkali stability was evaluated.
　結果を表１３に示す。実施例１９で作製したＦｃＲ１２、ＦｃＲ１３はＦｃＲ５ａ、ＦｃＲ９と比較し残存活性が高いことから、ＦｃＲ１２およびＦｃＲ１３のアルカリ安定性がＦｃＲ５ａ、ＦｃＲ９に比べて向上していることが確認された。 The results are shown in Table 13. Since FcR12 and FcR13 prepared in Example 19 had higher residual activity compared to FcR5a and FcR9, it was confirmed that the alkali stability of FcR12 and FcR13 was improved compared to FcR5a and FcR9.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
　実施例２１　ＦｃＲ１３への変異導入およびライブラリーの作製
　実施例１９（ｂ）で作製したＦｃＲ１３をコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例１９（ｂ）で作製した発現ベクターｐＥＴ－ＦｃＲ１３を用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、ｐＥＴ－ＦｃＲ１３を鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをプライマーとした他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。この反応によりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入された。
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養後、プレート上に形成したコロニーをランダム変異ライブラリーとした。 Example 21 Mutation Introduction to FcR13 and Library Preparation Mutation was randomly introduced into the polynucleotide part encoding FcR13 prepared in Example 19 (b) by error-prone PCR.
(1) Error-prone PCR was performed using the expression vector pET-FcR13 prepared in Example 19 (b) as a template. In error-prone PCR, a reaction solution similar to the composition shown in Table 3 was prepared except that pET-FcR13 was used as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 were used as primers. Was subjected to a heat treatment at 95 ° C. for 2 minutes, 35 cycles of a reaction comprising a first step at 95 ° C. for 30 seconds, a second step at 50 ° C. for 30 seconds, and a third step at 72 ° C. for 90 seconds were performed for 35 cycles. The sample was heat treated at 72 ° C. for 7 minutes. By this reaction, the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
(2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
　実施例２２　アルカリ安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例２１で作製したランダム変異ライブラリーを実施例３（１）から（２）に記載の方法で培養することでＦｃ結合性タンパク質を発現させた。
（２）培養後、遠心操作によって得られた、Ｆｃ結合性タンパク質を含む培養上清を以下に示す方法でアルカリ処理した。なおアルカリ処理後は、４倍量の１Ｍトリス緩衝液（ｐＨ７．０）でｐＨを中性付近に戻した。
（ｉ）純水にて５倍に希釈し、等量の８０ｍＭの水酸化ナトリウム溶液と混合した後、３０℃で２時間静置
（ｉｉ）純水にて２０倍に希釈し、等量の６０ｍＭの水酸化ナトリウム溶液と混合した後、３０℃で２時間静置
（３）（２）に記載のアルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（２）に記載のアルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法にてそれぞれ測定した。その後、アルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性を、アルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４）（３）の方法で約２７００株の形質転換体を評価し、その中からＦｃＲ１３と比較して安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。選択した形質転換体を５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地にて培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（５）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 Example 22 Screening of alkali-stabilized Fc-binding protein (1) The random mutation library prepared in Example 21 was cultured by the method described in Examples 3 (1) to (2) to obtain an Fc-binding protein. Expressed.
(2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation was treated with alkali by the method shown below. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1M Tris buffer (pH 7.0).
(I) Dilute 5 times with pure water and mix with an equal amount of 80 mM sodium hydroxide solution, then stand at 30 ° C. for 2 hours (ii) Dilute 20 times with pure water, After mixing with 60 mM sodium hydroxide solution, left at 30 ° C. for 2 hours (3) The antibody binding activity of the Fc-binding protein when subjected to alkali treatment as described in (2), and as described in (2) The antibody binding activity of the Fc binding protein when not subjected to alkali treatment was measured by the ELISA method described in Example 3 (4). Thereafter, the residual activity was calculated by dividing the antibody binding activity of the Fc binding protein when the alkali treatment was performed by the antibody binding activity of the Fc binding protein when the alkali treatment was not performed.
(4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing an Fc-binding protein with improved stability compared to FcR13 were selected. The selected transformant was cultured in a 2YT liquid medium containing 50 μg / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
　（４）で選択した形質転換体が発現するＦｃ結合性タンパク質の、ＦｃＲ１３に対するアミノ酸置換位置およびアルカリ処理後の残存活性（％）をまとめたものを表１４（アルカリ処理は（ｉ）の条件）および表１５（アルカリ処理は（ｉｉ）の条件）に示す。配列番号３７に記載のアミノ酸配列のうち、３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基（配列番号１の１７番目から１９２番目に該当）を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、Ｍｅｔ１８Ｌｙｓ（この表記は、配列番号１の１８番目（配列番号３７では３４番目）のメチオニンがリジンに置換されていることを表す、以下同様）、Ｍｅｔ１８Ｔｈｒ、Ｌｅｕ（Ｍｅｔ）２３Ａｒｇ（この表記は配列番号１番の２３番目（配列番号３７では３９番目）のロイシンが一度メチオニンに置換されさらにアルギニンに置換されたことを表す、以下同様）、Ｌｙｓ４６Ｉｌｅ、Ｇｌｎ（Ａｒｇ）４８Ｔｒｐ、Ｔｙｒ５１Ｈｉｓ、Ｔｙｒ５１Ａｓｎ、Ｇｌｕ５４Ａｓｐ、Ｇｌｕ５４Ｇｌｙ、Ａｓｎ５６Ｓｅｒ、Ａｓｎ５６Ｉｌｅ、Ｐｈｅ６１Ｌｅｕ、Ｐｈｅ６１Ｔｙｒ、Ｇｌｕ６４Ｇｌｙ、Ｉｌｅ６７Ｌｅｕ、Ｓｅｒ６９Ａｓｎ、Ａｌａ７１Ｔｈｒ、Ｔｙｒ７４Ｐｈｅ、Ｐｈｅ（Ｌｅｕ）７５Ａｒｇ、Ａｌａ７８Ｇｌｕ、Ｖａｌ８１Ｇｌｕ、Ａｓｐ８２Ｇｌｕ、Ｇｌｕ８６Ａｓｐ、Ｇｌｎ９０Ｌｅｕ、Ｌｅｕ９３Ｇｌｎ、Ｐｒｏ１１４Ｌｅｕ、Ｌｙｓ１１９Ａｓｎ、Ｌｙｓ１１９Ｔｙｒ、Ｈｉｓ１２５Ｇｌｎ、Ｓｅｒ１３０Ｔｈｒ、Ｌｙｓ１３８Ａｒｇ、Ｇｌｎ１４３Ｈｉｓ、Ｇｌｙ１４７Ｖａｌ、Ｌｙｓ１４９Ｍｅｔ、Ｐｈｅ１５１Ｔｙｒ、Ｈｉｓ１５３Ｔｙｒ、Ｔｙｒ１５８Ｐｈｅ、Ｌｙｓ１６１Ａｒｇ、Ｓｅｒ１６９Ｇｌｙ、Ａｓｎ１８０Ｓｅｒ、Ｔｈｒ１８５Ａｌａ、Ａｓｎ１８７Ｉｌｅ、Ａｓｎ１８７ＬｙｓおよびＴｈｒ１９１Ａｌａのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、ＦｃＲ１３と比較しアルカリ安定性が向上しているといえる。 Table 14 summarizes the amino acid substitution positions for FcR13 and the residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4) (alkaline treatment is the conditions of (i)). And Table 15 (alkali treatment is the condition (ii)). Of the amino acid sequence described in SEQ ID NO: 37, the amino acid residue from the 33rd glycine to the 208th glutamine (corresponding to the 17th to 192nd of SEQ ID NO: 1), and the 33rd to 208th In amino acid residues, Met18Lys (this notation indicates that the 18th methionine of SEQ ID NO: 1 (34th in SEQ ID NO: 37) is substituted with lysine, the same applies hereinafter), Met18Thr, Leu (Met) 23Arg ( This notation shows that the 23rd leucine of SEQ ID NO: 1 (39th in SEQ ID NO: 37) was once substituted with methionine and further substituted with arginine, and so on), Lys46Ile, Gln (Arg) 48Trp, Tyr51His, Tyr51Asn, Glu54Asp, Glu54Gly, As 56Ser, Asn56Ile, Phe61Leu, Phe61Tyr, Glu64Gly, Ile67Leu, Ser69Asn, Ala71Thr, Tyr74Phe, Phe (Leu) 75Arg, Ala78Glu, Val81Glu, Asp82Glu, Glu86Asp, Gln90Leu, Leu93Gln, Pro114Leu, Lys119Asn, Lys119Tyr, His125Gln, Ser130Thr, Lys138Arg, Gln143His, Gly147Val, Lys149Met, Phe151Tyr, His153Tyr, Tyr158Phe, Lys161Arg, Ser169Gly, Asn180Ser, Thr185Ala, Asn187Ile, Asn187Lys and Thr1 Fc binding proteins or amino acid substitutions 1Ala has at least one occurs, it can be said that the alkaline stability as compared to FcR13 is improved.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
　表１５に示した、ＦｃＲ１３からアミノ酸置換されたＦｃ結合性タンパク質のうち、Ｇｌｙ１４７Ｖａｌのアミノ酸置換が生じたＦｃ結合性タンパク質をＦｃＲ１４と命名し、ＦｃＲ１４をコードするポリヌクレオチドを含む発現ベクターをｐＥＴ－ＦｃＲ１４と命名した。ＦｃＲ１４のアミノ酸配列を配列番号６９に、ＦｃＲ１４をコードするポリヌクレオチドの配列を配列番号７０に示す。なお配列番号６９において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１４のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号６９において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｇｌｙ１４７Ｖａｌのバリンは１６３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。 Of the Fc binding proteins with amino acid substitutions from FcR13 shown in Table 15, the Fc binding protein in which amino acid substitution of Gly147Val occurred was named FcR14, and an expression vector containing a polynucleotide encoding FcR14 was designated as pET-FcR14. Named. The amino acid sequence of FcR14 is shown in SEQ ID NO: 69, and the sequence of the polynucleotide encoding FcR14 is shown in SEQ ID NO: 70. In SEQ ID NO: 69, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. From the 33rd glycine (Gly) to the 208th glutamine (Gln), the amino acid sequence of FcR14 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), 209th to 210th glycine (Gly) Is a linker sequence, and histidines (His) from 211 to 216 are tag sequences. Also, in SEQ ID NO: 69, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, Ser68Pro. Proline 84th, Phe75Leu leucine 91st, Asn92Ser serine 108th, Val117Glu glutamic acid 133rd, Glu121Gly glycine 137th, Gly147Val valine 163rd, Phe171Ser serine 187th and Ser178Arg Exists at the 194th position.
　実施例２３　改良Ｆｃ結合性タンパク質の作製
　実施例２２で明らかとなった、Ｆｃ結合性タンパク質のアルカリ安定性向上に関与するアミノ酸置換の中から、Ｔｙｒ５１ＨｉｓおよびＧｌｕ５４Ａｓｐを選択し、それらの置換をＦｃＲ１４に集積することで、改良Ｆｃ結合性タンパク質（ＦｃＲ１６）を作製した。以下、作製方法を詳細に説明する。
（１）実施例２２で得られた、ｐＥＴ－ＦｃＲ１４を鋳型とし、配列番号２４および配列番号７１（５’－ＴＧＣＣＧＧＧＧＣＧＣＧＣＡＴＡＧＣＣＣＧＧＡＴＧＡＴＡＡＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ１６Ｆとした。
（２）実施例２２で得られたｐＥＴ－ＦｃＲ１４を鋳型とし、配列番号２３および配列番号７２（５’－ＧＧＴＧＣＴＧＴＴＡＴＣＡＴＣＣＧＧＧＣＴＡＴＧＣＧＣＧＣＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１６Ｒとした。
（３）（１）および（２）で得られた２種類のＰＣＲ産物（ｍ１６Ｆ、ｍ１６Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行ない最後に７２℃で５分間熱処理するＰＣＲを行ない、ｍ１６Ｆとｍ１６Ｒを連結したＰＣＲ産物ｍ１６ｐを得た。
（４）（３）で得られたＰＣＲ産物ｍ１６ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、ＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない最後に７２℃で５分間熱処理するＰＣＲを行なった。これによりＦｃＲ１４に２箇所アミノ酸置換を導入したＦｃＲ１６をコードするポリヌクレオチドを作製した。
（５）（４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ１４に対して２箇所（野生型Ｆｃ結合性タンパク質に対して１６箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１６をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１６を得た。
（７）ｐＥＴ－ＦｃＲ１６のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 Example 23 Production of Improved Fc Binding Protein Tyr51His and Glu54Asp were selected from among the amino acid substitutions involved in improving the alkali stability of Fc binding protein revealed in Example 22, and these substitutions were changed to FcR14. By accumulating, an improved Fc binding protein (FcR16) was produced. Hereinafter, the production method will be described in detail.
(1) Except that pET-FcR14 obtained in Example 22 was used as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 24 and SEQ ID NO: 71 (5′-TGCCGGGGCGCGCATAGCCCGGATGATAAC-3 ′) was used as a PCR primer, After preparing the reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C for 5 minutes, first step at 98 ° C for 10 seconds, second step at 55 ° C for 5 seconds, and 72 ° C for 1 minute. The reaction with the third step as one cycle was performed for 30 cycles, and finally, heat treatment was performed at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m16F.
(2) Except that pET-FcR14 obtained in Example 22 was used as a template, and an oligonucleotide having the sequence described in SEQ ID NO: 23 and SEQ ID NO: 72 (5′-GGTGCTGTTTATCATCCGGGCTATGCGCGCC-3 ′) was used as a PCR primer, PCR was performed in the same manner as in 1). The purified PCR product was designated as m16R.
(3) The two types of PCR products (m16F and m16R) obtained in (1) and (2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. Finally, PCR was performed by heat treatment at 72 ° C. for 5 minutes to obtain a PCR product m16p in which m16F and m16R were ligated.
(4) PCR was performed using the PCR product m16p obtained in (3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. 30 cycles of the reaction with the third step of 1 minute as one cycle were performed, and finally PCR was performed by heat treatment at 72 ° C. for 5 minutes. As a result, a polynucleotide encoding FcR16 in which two amino acid substitutions were introduced into FcR14 was prepared.
(5) After purification of the polynucleotide obtained in (4), digestion with restriction enzymes NcoI and HindIII, ligation to an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with restriction enzymes NcoI and HindIII, This was used to transform E. coli BL21 (DE3) strain.
(6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR16, which is a polypeptide in which amino acid substitution has been performed at two positions on FcR14 (16 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected cells (transformants) Plasmid pET-FcR16 containing was obtained.
(7) The nucleotide sequence of pET-FcR16 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１６のアミノ酸配列を配列番号７３に、前記ＦｃＲ１６をコードするポリヌクレオチドの配列を配列番号７４に示す。なお、配列番号７３において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１３のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号７３において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｈｉｓのヒスチジンは６７番目、Ｇｌｕ５４Ａｓｐのアスパラギン酸は７０番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｇｌｙ１４７Ｖａｌのバリンは１６３番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。 The amino acid sequence of FcR16 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 73, and the sequence of the polynucleotide encoding the FcR16 is shown in SEQ ID NO: 74. In SEQ ID NO: 73, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, the amino acid sequence of FcR13 from the 33rd glycine (Gly) to the 208th glutamine (Gln) (corresponding to the region from the 17th to the 192nd region of SEQ ID NO: 1), the 209th to the 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. In SEQ ID NO: 73, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, and Tyr51His. Histidine 67th, Glu54Asp aspartic acid 70th, Ser68Pro proline 84th, Phe75Leu leucine 91th, Asn92Ser serine 108th, Val117Glu glutamic acid 133rd, Glu121Gly glycine 137th, Gly147Val Valine is 163rd, Phe171Ser serine is 187th, Ser178Arg Argy Emissions are present respectively in 194th position.
　実施例２４　Ｆｃ結合性タンパク質のアルカリ安定性評価
（１）実施例１９（ｂ）で作製したＦｃ結合性タンパク質（ＦｃＲ１３）、実施例２２で取得したＦｃ結合性タンパク質（ＦｃＲ１４）、および実施例２３で作製したＦｃ結合性タンパク質（ＦｃＲ１６）を発現する形質転換体を、実施例８の（１）から（４）に記載の方法で培養し、タンパク質を抽出することでＦｃＲ１３、ＦｃＲ１４およびＦｃＲ１６を調製した。
（２）（１）で調製したタンパク質抽出液中のＦｃＲ１３、ＦｃＲ１４およびＦｃＲ１６の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法を用いて測定した。この時、精製し定量したＦｃＲ９を用いて検量線を作製し、濃度測定を行なった。
（３）各Ｆｃ結合性タンパク質の濃度が１０μｇ／ｍＬになるよう純水で希釈後、前記希釈した溶液５０μＬと６０ｍＭの水酸化ナトリウム溶液５０μＬとを混合し、３０℃で２時間静置することでアルカリ処理した。その後、１Ｍトリス塩酸緩衝液（ｐＨ７．０）を４倍量加えることで中和し、Ｆｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法によって測定した。
（４）アルカリ処理を行なった場合の抗体結合活性をアルカリ処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出しアルカリ安定性を評価した。 Example 24 Alkali Stability Evaluation of Fc Binding Protein (1) Fc binding protein (FcR13) prepared in Example 19 (b), Fc binding protein (FcR14) obtained in Example 22, and Example 23 FcR13, FcR14, and FcR16 were prepared by culturing the transformant expressing Fc-binding protein (FcR16) prepared in step 1 by culturing by the method described in (1) to (4) of Example 8 and extracting the protein. did.
(2) The antibody binding activity of FcR13, FcR14 and FcR16 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR9 purified and quantified, and the concentration was measured.
(3) After diluting with pure water so that the concentration of each Fc-binding protein becomes 10 μg / mL, 50 μL of the diluted solution and 50 μL of 60 mM sodium hydroxide solution are mixed and allowed to stand at 30 ° C. for 2 hours. Treated with alkali. Thereafter, the solution was neutralized by adding 4 volumes of 1M Tris-HCl buffer (pH 7.0), and the antibody binding activity of the Fc binding protein was measured by the ELISA method described in Example 3 (4).
(4) The residual activity was calculated by dividing the antibody binding activity when the alkali treatment was performed by the antibody binding activity when the alkali treatment was not performed, and the alkali stability was evaluated.
　結果を表１６に示す。実施例２２で作製したＦｃＲ１４、ＦｃＲ１６はＦｃＲ１３と比較し残存活性が高いことから、ＦｃＲ１３に比べてアルカリ安定性が向上していることが確認された。 The results are shown in Table 16. Since FcR14 and FcR16 produced in Example 22 had higher residual activity than FcR13, it was confirmed that the alkali stability was improved compared to FcR13.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
　実施例２５　ＦｃＲ１６への変異導入およびライブラリーの作製
　実施例２３で作製したＦｃＲ１６をコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例２３で作製した発現ベクターｐＥＴ－ＦｃＲ１６を用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、ｐＥＴ－ＦｃＲ１６を鋳型とし、配列番号２３および２４に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。この反応によりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入された。
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養後、プレート上に形成したコロニーをランダム変異ライブラリーとした。 Example 25 Mutation Introduction to FcR16 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR16 prepared in Example 23 by error-prone PCR.
(1) Error-prone PCR was performed using the expression vector pET-FcR16 prepared in Example 23 as a template. In error-prone PCR, pET-FcR16 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared, except that oligonucleotides having the sequences shown in SEQ ID NOs: 23 and 24 were used as primers. Heat treatment at 95 ° C. for 2 minutes, 35 cycles of reaction with 95 ° C. for 30 seconds for the first step, 50 ° C. for 30 seconds for the second step, 72 ° C. for 90 seconds for the third step for 35 cycles, and finally The heat treatment was performed at 72 ° C. for 7 minutes. By this reaction, the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
(2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into Escherichia coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
　実施例２６　アルカリ安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例２５で作製したランダム変異ライブラリーを実施例３（１）から（２）に記載の方法で培養することでＦｃ結合性タンパク質を発現させた。
（２）培養後、遠心操作によって得られた、Ｆｃ結合性タンパク質を含む培養上清を純水にて２０倍に希釈し、等量の８０ｍＭの水酸化ナトリウム溶液と混合した後、３０℃で２時間静置することでアルカリ処理した。アルカリ処理後は、４倍量の１Ｍトリス緩衝液（ｐＨ７．０）でｐＨを中性付近に戻した。
（３）（２）に記載のアルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（２）に記載のアルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法にてそれぞれ測定した。その後、アルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性を、アルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４）（３）の方法で約２７００株の形質転換体を評価し、その中からＦｃＲ１６と比較して安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。選択した形質転換体を５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地にて培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（５）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 Example 26 Screening of alkali-stabilized Fc-binding protein (1) The random mutation library prepared in Example 25 was cultured by the method described in Examples 3 (1) to (2) to obtain an Fc-binding protein. Expressed.
(2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, mixed with an equal amount of 80 mM sodium hydroxide solution, and then at 30 ° C. The alkali treatment was carried out by leaving still for 2 hours. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
(3) The antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is performed, and the antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is not performed, Each was measured by the ELISA method described in Example 3 (4). Thereafter, the residual activity was calculated by dividing the antibody binding activity of the Fc binding protein when the alkali treatment was performed by the antibody binding activity of the Fc binding protein when the alkali treatment was not performed.
(4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing an Fc-binding protein with improved stability compared to FcR16 were selected from the transformants. The selected transformant was cultured in a 2YT liquid medium containing 50 μg / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
　（４）で選択した形質転換体が発現するＦｃ結合性タンパク質の、ＦｃＲ１３に対するアミノ酸置換位置およびアルカリ処理後の残存活性（％）をまとめたものを表１７に示す。配列番号３７に記載のアミノ酸配列のうち、３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基（配列番号１の１７番目から１９２番目に該当）を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、Ａｌａ７８Ｓｅｒ（この表記は、配列番号１の７８番目（配列番号３７では９４番目）のアラニンがセリンに置換されていることを表す、以下同様）、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｇｌｎ１０１Ａｒｇ、Ｔｈｒ１４０Ｉｌｅ、Ｇｌｎ１４３Ｈｉｓ、Ｔｙｒ１５８Ｈｉｓ、Ｌｙｓ１６１Ａｒｇ、Ｌｙｓ１６５Ｇｌｕ、Ｔｈｒ１８５Ａｌａ、Ａｓｎ１８７Ａｓｐ、Ａｓｎ１８７Ｔｙrのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、ＦｃＲ１６と比較しアルカリ安定性が向上しているといえる。 Table 17 shows a summary of amino acid substitution positions for FcR13 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4). Of the amino acid sequence described in SEQ ID NO: 37, the amino acid residue from the 33rd glycine to the 208th glutamine (corresponding to the 17th to 192nd of SEQ ID NO: 1), and the 33rd to 208th In the amino acid residue, Ala78Ser (this notation indicates that the 78th alanine in SEQ ID NO: 1 (94th in SEQ ID NO: 37) is substituted with serine, the same applies hereinafter), Asp82Glu, Gln101Leu, Gln101Arg, Thr140Ile, An Fc-binding protein in which at least one amino acid substitution of any one of Gln143His, Tyr158His, Lys161Arg, Lys165Glu, Thr185Ala, Asn187Asp, Asn187Tyr has occurred is more stable than FcR16. It can be said that the nature has improved.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
　実施例２７　改良Ｆｃ結合性タンパク質の作製
　実施例２６で判明した、Ｆｃ結合性タンパク質のアルカリ安定性向上に関与するアミノ酸置換をＦｃＲ１６に集積することで、さらなる安定性向上を図った。置換アミノ酸の集積は、主にＰＣＲを用いて行ない、以下の（ａ）から（ｃ）に示す３種類の改良Ｆｃ結合性タンパク質を作製した。
（ａ）ＦｃＲ１６に対し、さらにＴｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕのアミノ酸置換を行なったＦｃＲ１９
（ｂ）ＦｃＲ１６に対し、さらにＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕのアミノ酸置換を行なったＦｃＲ２１
（ｃ）ＦｃＲ１６に対し、さらにＡｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８Ｈｉｓ、Ｌｙｓ１６５Ｇｌｕ、Ｔｈｒ１８５ＡｌａおよびＡｓｎ１８７Ａｓｐのアミノ酸置換を行なったＦｃＲ２４
以下、各改良Ｆｃ結合性タンパク質の作製方法を詳細に説明する。
（ａ）ＦｃＲ１９
　実施例２６で明らかとなった、アルカリ安定性向上に関与するアミノ酸置換の中から、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕを選択し、それらの置換をＦｃＲ１６（実施例２３）に集積したＦｃＲ１９を作製した。具体的には、実施例２６で得られたＴｈｒ１４０ＩｌｅおよびＴｙｒ１５８Ｈｉｓの変異を含んだポリヌクレオチドに対して、Ｌｙｓ１６５Ｇｌｕを生じさせる変異導入を行なうことにより、ＦｃＲ１９を作製した。　　　
（ａ－１）実施例２６で取得した、ＦｃＲ１６にＴｈｒ１４０ＩｌｅおよびＴｙｒ１５８Ｈｉｓの変異を含んだＦｃ結合性タンパク質をコードするポリヌクレオチドを鋳型とし、配列番号２４および配列番号７５（５’－ＡＴＴＣＣＣＡＡＡＧＣＧＡＣＧＣＴＧＧＡＧＧＡＣＡＧＣＧＧＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ１９Ｆとした。
（ａ－２）実施例２６で取得した、ＦｃＲ１６にＴｈｒ１４０ＩｌｅおよびＴｙｒ１５８Ｈｉｓの変異を含んだＦｃ結合性タンパク質をコードするポリヌクレオチドを鋳型とし、配列番号２３および配列番号７６（５’－ＡＴＡＧＣＴＧＣＣＧＣＴＧＴＣＣＴＣＣＡＧＣＧＴＣＧＣＴＴＴ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ１９Ｒとした。
（ａ－３）（ａ－１）および（ａ－２）で得られた２種類のＰＣＲ産物（ｍ１９Ｆ、ｍ１９Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ１９Ｆとｍ１９Ｒを連結したＰＣＲ産物ｍ１９ｐを得た。
（ａ－４）（ａ－３）で得られたＰＣＲ産物ｍ１９ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６に３箇所アミノ酸置換を導入したＦｃＲ１９をコードするポリヌクレオチドを作製した。
（ａ－５）（ａ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ１６に対して３箇所（野生型Ｆｃ結合性タンパク質に対して１９箇所）アミノ酸置換したポリペプチドである、ＦｃＲ１９をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ１９を得た。
（ａ－７）ｐＥＴ－ＦｃＲ１９のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 Example 27 Production of Improved Fc Binding Protein The stability was further improved by accumulating amino acid substitutions found in Example 26 involved in improving the alkali stability of Fc binding protein in FcR16. Accumulation of substituted amino acids was mainly performed using PCR, and three types of improved Fc-binding proteins shown in (a) to (c) below were prepared.
(A) FcR19 in which Thr140Ile, Tyr158His and Lys165Glu were further substituted for FcR16
(B) FcR21 in which Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu were further substituted for FcR16
(C) FcR24 in which Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, Lys165Glu, Thr185Ala, and Asn187Asp were further substituted for FcR16
Hereinafter, a method for producing each improved Fc-binding protein will be described in detail.
(A) FcR19
Thr140Ile, Tyr158His and Lys165Glu were selected from the amino acid substitutions involved in improving alkali stability, which were revealed in Example 26, and FcR19 in which these substitutions were accumulated in FcR16 (Example 23) was prepared. Specifically, FcR19 was produced by carrying out mutation introduction | transduction which produces Lys165Glu with respect to the polynucleotide containing the mutation of Thr140Ile and Tyr158His obtained in Example 26.
(A-1) SEQ ID NO: 24 and SEQ ID NO: 75 (5′-ATTCCCAAAGCGACCGCTGGAGGACACGCGGC-3 ′), using as a template the polynucleotide obtained in Example 26 that encodes an Fc-binding protein containing Thr140Ile and Tyr158His mutations in FcR16 The reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, and then the first step at 98 ° C. for 10 seconds. The reaction was performed by 30 cycles of the second step at 55 ° C. for 5 seconds and the third step at 72 ° C. for 1 minute for 30 cycles, and finally by heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m19F.
(A-2) SEQ ID NO: 23 and SEQ ID NO: 76 (5′-ATAGCTGCCGCTGTCTCCCCACGGTCGCTTT-3 ′) using, as a template, a polynucleotide encoding an Fc-binding protein containing Thr140Ile and Tyr158His mutations in FcR16 obtained in Example 26 PCR was performed in the same manner as (a-1) except that the oligonucleotide having the sequence described in (1) was used as a PCR primer. The purified PCR product was designated as m19R.
(A-3) Two types of PCR products (m19F and m19R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was carried out to obtain a PCR product m19p in which m19F and m19R were linked.
(A-4) PCR was performed using the PCR product m19p obtained in (a-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR19 having three amino acid substitutions introduced into FcR16 was prepared.
(A-5) After purifying the polynucleotide obtained in (a-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. A polynucleotide encoding FcR19, which is a polypeptide in which amino acid substitution has been performed on FcR16 at three positions (19 positions relative to a wild-type Fc-binding protein) by extracting a plasmid from the collected bacterial cells (transformants) Plasmid pET-FcR19 containing was obtained.
(A-7) The nucleotide sequence of pET-FcR19 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ１９のアミノ酸配列を配列番号７７に、前記ＦｃＲ１９をコードするポリヌクレオチドの配列を配列番号７８に示す。なお、配列番号７７において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ１９のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号７３において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｈｉｓのヒスチジンは６７番目、Ｇｌｕ５４Ａｓｐのアスパラギン酸は７０番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｔｈｒ１４０Ｉｌｅのイソロイシンは１５６番目、Ｇｌｙ１４７Ｖａｌのバリンは１６３番目、Ｔｙｒ１５８Ｈｉｓのヒスチジンは１７４番目、Ｌｙｓ１６５Ｇｌｕのグルタミン酸は１８１番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。
（ｂ）ＦｃＲ２１
　実施例２６で得られたＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１ＬｅｕおよびＡｓｎ１８７Ａｓｐの変異を含んだポリヌクレオチドに対して、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕを生じさせる変異導入を行ない、改良Ｆｃ結合性タンパク質を得た。なお前記変異のうちＡｓｎ１８７Ａｓｐは後述の（ｂ－９）の操作で欠失したため、本実験で実際に得られた改良Ｆｃ結合性タンパク質はＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕの置換をＦｃＲ１６（実施例２３）に集積したＦｃ結合性タンパク質（ＦｃＲ２１）である。
（ｂ－１）実施例２６で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１ＬｅｕおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃ結合性タンパク質をコードするポリヌクレオチドを鋳型とし、配列番号２４および配列番号７９（５’－ＡＣＣＧＣＣＣＴＧＣＡＴＡＡＡＧＴＧＡＴＣＴＡＣＣＴＧＣＡＡ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ２１－２Ｆとした。
（ｂ－２）実施例２６で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１ＬｅｕおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃ結合性タンパク質をコードするポリヌクレオチドを鋳型とし、配列番号２３および配列番号８０（５’－ＴＴＧＣＡＧＧＴＡＧＡＴＣＡＣＴＴＴＡＴＧＣＡＧＧＧＣＧＧＴ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｂ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ２１－２Ｒとした。
（ｂ－３）（ｂ－１）および（ｂ－２）で得られた２種類のＰＣＲ産物（ｍ２１－２Ｆ、ｍ２１－２Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ２１－２Ｆとｍ２１－２Ｒを連結したＰＣＲ産物ｍ２１－２ｐを得た。
（ｂ－４）（ｂ－３）で得られたＰＣＲ産物ｍ２１－２ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０ＩｌｅおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－２をコードするポリヌクレオチドを作製した。
（ｂ－５）（ｂ－４）で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０ＩｌｅおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－２をコードするポリヌクレオチドを鋳型とし、配列番号２４および配列番号８１（５’－ＣＡＣＣＡＣＡＡＣＴＣＣＧＡＣＴＴＣＣＡＴＡＴＴＣＣＣＡＡＡ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ２１－１Ｆとした。
（ｂ－６）（ｂ－４）で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０ＩｌｅおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－２をコードするポリヌクレオチドを鋳型とし、配列番号２３および配列番号８２（５’－ＣＡＧＣＧＴＣＧＣＴＴＴＧＧＧＡＡＴＡＴＧＧＡＡＧＴＣＧＧＡ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｂ－５）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ２１－１Ｒとした。
（ｂ－７）（ｂ－５）および（ｂ－６）で得られた２種類のＰＣＲ産物（ｍ２１－１Ｆ、ｍ２１－１Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ２１－１Ｆとｍ２１－１Ｒを連結したＰＣＲ産物ｍ２１－１ｐを得た。
（ｂ－８）（ｂ－７）で得られたＰＣＲ産物ｍ２１－１ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－１をコードするポリヌクレオチドを作製した。
（ｂ－９）（ｂ－８）で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－１をコードするポリヌクレオチドを鋳型とし、配列番号２２および配列番号７５に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ２１Ｆとした（本操作によりＡｓｎ１８７Ａｓｐの変異が欠失している）。
（ｂ－１０）（ｂ－８）で取得した、ＦｃＲ１６にＡｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＡｓｎ１８７Ａｓｐの変異を含んだＦｃＲ２１－１をコードするポリヌクレオチドを鋳型とし、配列番号６２および配列番号７６に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｂ－９）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ２１Ｒとした。
（ｂ－１１）（ｂ－９）および（ｂ－１０）で得られた２種類のＰＣＲ産物（ｍ２１Ｆ、ｍ２１Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ２１Ｆとｍ２１Ｒを連結したＰＣＲ産物ｍ２１ｐを得た。
（ｂ－１２）（ｂ－１１）で得られたＰＣＲ産物ｍ２１ｐを鋳型とし、配列番号６２および配列番号２２に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６に５箇所（Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕ）（野生型Ｆｃ結合性タンパク質に対して２１箇所）アミノ酸置換を導入したＦｃＲ２１をコードするポリヌクレオチドを作製した。
（ｂ－１３）（ａ－１２）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｂ－１４）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ１６に対して５箇所アミノ酸置換したポリペプチドである、ＦｃＲ２１をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ２１を得た。
（ｂ－１５）ｐＥＴ－ＦｃＲ２１のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR19 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 77, and the sequence of the polynucleotide encoding the FcR19 is shown in SEQ ID NO: 78. In SEQ ID NO: 77, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, from the 33rd glycine (Gly) to the 208th glutamine (Gln) amino acid sequence of FcR19 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. In SEQ ID NO: 73, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, and Tyr51His. Histidine 67th, Glu54Asp aspartic acid 70th, Ser68Pro proline 84th, Phe75Leu leucine 91th, Asn92Ser serine 108th, Val117Glu glutamic acid 133rd, Glu121Gly glycine 137th, Thr140Ile Isoleucine is 156th, Gly147Val valine is 163rd, Tyr158His hiss Gin 174 th, glutamic acid Lys165Glu 181 th, serine Phe171Ser arginine of 187th and Ser178Arg are present respectively in 194th position.
(B) FcR21
The polynucleotide containing Asp82Glu, Gln101Leu and Asn187Asp mutations obtained in Example 26 was subjected to mutagenesis to produce Thr140Ile, Tyr158His and Lys165Glu to obtain an improved Fc-binding protein. Of these mutations, Asn187Asp was deleted by the operation (b-9) described later, and the improved Fc-binding protein actually obtained in this experiment was replaced with Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu. Fc-binding protein (FcR21) accumulated in Example 23).
(B-1) Using the polynucleotide encoding Fc-binding protein obtained in Example 26 and containing FspR16 mutations Asp82Glu, Gln101Leu and Asn187Asp as a template, SEQ ID NO: 24 and SEQ ID NO: 79 (5′-ACCGCCCCTGCCATAAAGTGATCTACCTGCAA- 3 ′), except that the reaction mixture having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes, followed by a 10-second reaction at 98 ° C. for 10 seconds. The reaction was carried out by 30 cycles of 1 step, the second step at 55 ° C. for 5 seconds, and the third step at 72 ° C. for 1 minute for 30 cycles, and finally heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m21-2F.
(B-2) SEQ ID NO: 23 and SEQ ID NO: 80 (5′-TTGCAGGGTAGACTACTTTATGCAGGGCGGT-) using as a template a polynucleotide encoding an Fc-binding protein obtained in Example 26 and containing Asp82Glu, Gln101Leu and Asn187Asp mutations in FcR16 PCR was performed in the same manner as (b-1) except that the oligonucleotide having the sequence described in 3 ′) was used as a PCR primer. The purified PCR product was designated as m21-2R.
(B-3) Two types of PCR products (m21-2F, m21-2R) obtained in (b-1) and (b-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed to obtain a PCR product m21-2p in which m21-2F and m21-2R were ligated.
(B-4) PCR was performed using the PCR product m21-2p obtained in (b-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR21-2 containing Asp82Glu, Gln101Leu, Thr140Ile and Asn187Asp mutations in FcR16 was prepared.
(B-5) The polynucleotide encoding FcR21-2 containing the mutations Asp82Glu, Gln101Leu, Thr140Ile, and Asn187Asp obtained in (b-4) in FcR16 as a template, SEQ ID NO: 24 and SEQ ID NO: 81 (5 ′ -CACCACAACTCCGAACTTCCATATTCCCAAA-3 ') except that oligonucleotides having the sequence described in the above were used as PCR primers, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C for 5 minutes, The reaction was carried out by performing 30 cycles of the first step for 2 seconds, the second step for 55 seconds at 55 ° C., and the third step for 1 minute at 72 ° C. for 30 cycles, and finally heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m21-1F.
(B-6) The polynucleotide encoding FcR21-2 containing the mutations of Asp82Glu, Gln101Leu, Thr140Ile and Asn187Asp obtained in (b-4) in FcR16 was used as a template, and SEQ ID NO: 23 and SEQ ID NO: 82 (5 ′ PCR was carried out in the same manner as in (b-5) except that the oligonucleotide consisting of the sequence described in CAGCGTCGCTTTGGGAATATGGAAGTCCGGA-3 ′) was used as a PCR primer. The purified PCR product was designated as m21-1R.
(B-7) Two types of PCR products (m21-1F, m21-1R) obtained in (b-5) and (b-6) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed to obtain a PCR product m21-1p in which m21-1F and m21-1R were ligated.
(B-8) PCR was performed using the PCR product m21-1p obtained in (b-7) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. Thus, a polynucleotide encoding FcR21-1 containing Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, and Asn187Asp mutations in FcR16 was prepared.
(B-9) The polynucleotide encoding FcR21-1 containing the mutations of Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Asn187Asp obtained in (b-8) in FcR16 is used as a template. SEQ ID NO: 22 and SEQ ID NO: 75 The reaction solution having the composition shown in Table 5 was prepared except that the oligonucleotide having the sequence described above was used as a PCR primer, and then the reaction solution was heat-treated at 98 ° C. for 5 minutes, and the first step at 98 ° C. for 10 seconds, 55 The reaction was carried out by performing 30 cycles of the second step of 5 seconds at ° C and the third step of 1 minute at 72 ° C for 30 cycles, and finally heat treating at 72 ° C for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m21F (Asn187Asp mutation was deleted by this operation).
(B-10) A polynucleotide encoding FcR21-1 containing the mutations of Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, and Asn187Asp obtained in (b-8) in FcR16 is used as a template. SEQ ID NO: 62 and SEQ ID NO: 76 PCR was carried out in the same manner as in (b-9) except that oligonucleotides having the sequences described were used as PCR primers. The purified PCR product was designated as m21R.
(B-11) Two types of PCR products (m21F, m21R) obtained in (b-9) and (b-10) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed to obtain a PCR product m21p in which m21F and m21R were linked.
(B-12) PCR was performed using the PCR product m21p obtained in (b-11) as a template and oligonucleotides having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 22 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR21 having 5 amino acid substitutions (Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, and Lys165Glu) (21 sites relative to the wild-type Fc-binding protein) in FcR16 was prepared.
(B-13) After purifying the polynucleotide obtained in (a-12), it is digested with restriction enzymes NcoI and HindIII and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-14) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected bacterial cells (transformants), a plasmid pET-FcR21 containing a polynucleotide encoding FcR21, which is a polypeptide obtained by substituting FcR16 with 5 amino acids, was obtained.
(B-15) The nucleotide sequence of pET-FcR21 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ２１のアミノ酸配列を配列番号８３に、前記ＦｃＲ２１をコードするポリヌクレオチドの配列を配列番号８４に示す。なお、配列番号８３において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ２１のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号８３において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｈｉｓのヒスチジンは６７番目、Ｇｌｕ５４Ａｓｐのアスパラギン酸は７０番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｓｐ８２Ｇｌｕのグルタミン酸は９８番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｇｌｎ１０１Ｌｅｕのロイシンは１１７番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｔｈｒ１４０Ｉｌｅのイソロイシンは１５６番目、Ｇｌｙ１４７Ｖａｌのバリンは１６３番目、Ｔｙｒ１５８Ｈｉｓのヒスチジンは１７４番目、Ｌｙｓ１６５Ｇｌｕのグルタミン酸は１８１番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目およびＳｅｒ１７８Ａｒｇのアルギニンは１９４番目の位置にそれぞれ存在する。
（ｃ）ＦｃＲ２４
　実施例２６で明らかとなった、アルカリ安定性向上に関与するアミノ酸置換の中から、Ａｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８Ｈｉｓ、Ｌｙｓ１６５Ｇｌｕ、Ｔｈｒ１８５ＡｌａおよびＡｓｎ１８７Ａｓｐを選択し、それらの置換をＦｃＲ１６（実施例２３）に集積したＦｃＲ２４を作製した。具体的には、ＦｃＲ２１をコードするポリヌクレオチドに対して、Ａｌａ７８Ｓｅｒ、Ｔｈｒ１８５ＡｌａおよびＡｓｎ１８７Ａｓｐを生じさせる変異導入を行なうことにより、ＦｃＲ２４を作製した。
（ｃ－１）（ｂ）で作製した、ｐＥＴ－ＦｃＲ２１を鋳型とし、配列番号２４および配列番号８５（５’－ＡＧＣＡＧＣＴＡＣＣＴＴＡＴＴＧＡＴＴＣＧＧＣＧＡＣＧＧＴＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ２４－２Ｆとした。
（ｃ－２）（ｂ）で作製した、ｐＥＴ－ＦｃＲ２１を鋳型とし、配列番号２３および配列番号８６（５’－ＧＣＴＡＴＣＴＴＣＣＡＣＣＧＴＣＧＣＣＧＡＡＴＣＡＡＴＡＡＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｃ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ２４－２Ｒとした。
（ｃ－３）（ｃ－１）および（ｃ－２）で得られた２種類のＰＣＲ産物（ｍ２４－２Ｆ、ｍ２４－２Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ２１－２Ｆとｍ２１－２Ｒを連結したＰＣＲ産物ｍ２４－２ｐを得た。
（ｃ－４）（ｃ－３）で得られたＰＣＲ産物ｍ２４－２ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６にＡｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕの変異を含んだＦｃＲ２４－２をコードするポリヌクレオチドを作製した。
（ｃ－５）（ｃ－４）で取得した、ＦｃＲ１６にＡｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕの変異を含んだＦｃＲ２４－２をコードするポリヌクレオチドを鋳型とし、配列番号２４および配列番号８７（５’－ＡＡＡＡＡＴＧＴＧＡＧＣＡＧＣＧＡＧＧＣＣＧＴＧＧＡＴＡＴＴ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、表５に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行ない、最後に７２℃で５分間熱処理することで行なった。増幅したＰＣＲ産物をアガロースゲル電気泳動に供し、そのゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　ｋｉｔ（キアゲン製）を用いて精製した。精製したＰＣＲ産物をｍ２４Ｆとした。
（ｃ－６）（ｃ－４）で取得した、ＦｃＲ１６にＡｌａ７８Ｓｅｒ、Ａｓｐ８２Ｇｌｕ、Ｇｌｎ１０１Ｌｅｕ、Ｔｈｒ１４０Ｉｌｅ、Ｔｙｒ１５８ＨｉｓおよびＬｙｓ１６５Ｇｌｕの変異を含んだＦｃＲ２４－２をコードするポリヌクレオチドを鋳型とし、配列番号６２および配列番号８８（５’－ＧＧＴＡＡＴＧＧＴＡＡＴＡＴＣＣＡＣＧＧＣＣＴＣＧＣＴＧＣＴ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、（ｃ－５）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物をｍ２４Ｒとした。
（ｃ－７）（ｃ－５）および（ｃ－６）で得られた２種類のＰＣＲ産物（ｍ２４Ｆ、ｍ２４Ｒ）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、ｍ２４Ｆとｍ２４Ｒを連結したＰＣＲ産物ｍ２４ｐを得た。
（ｃ－８）（ｃ－７）で得られたＰＣＲ産物ｍ２４ｐを鋳型とし、配列番号６２および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃＲ１６に８箇所（野生型Ｆｃ結合性タンパク質に対して２４箇所）アミノ酸置換を導入したＦｃＲ２４をコードするポリヌクレオチドを作製した。
（ｃ－９）（ｃ－８）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｃ－１０）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出することで、ＦｃＲ１６に対して８箇所アミノ酸置換したポリペプチドである、ＦｃＲ２４をコードするポリヌクレオチドを含むプラスミドｐＥＴ－ＦｃＲ２４を得た。
（ｃ－１１）ｐＥＴ－ＦｃＲ２４のヌクレオチド配列の解析を、実施例１（５）と同様の方法で行なった。 The amino acid sequence of FcR21 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 83, and the sequence of a polynucleotide encoding the FcR21 is shown in SEQ ID NO: 84. In SEQ ID NO: 83, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, from the 33rd glycine (Gly) to the 208th glutamine (Gln) amino acid sequence of FcR21 (corresponding to the 17th to 192nd region of SEQ ID NO: 1), the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. Also, in SEQ ID NO: 83, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, and Tyr51His. Histidine 67th, Glu54Asp aspartic acid 70th, Ser68Pro proline 84th, Phe75Leu leucine 91th, Asp82Glu glutamic acid 98th, Asn92Ser serine 108th, Gln101Leu leucine 117th, Val117Glu Glutamic acid is 133th, Glu121Gly glycine is 137th, Thr140Ile isologue Singh 156 th, valine Gly147Val 163 th, histidine Tyr158His 174 th, glutamic acid Lys165Glu 181 th, serine Phe171Ser is arginine 187th and Ser178Arg present respectively 194th position.
(C) FcR24
Among the amino acid substitutions involved in improving the alkali stability revealed in Example 26, Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His, Lys165Glu, Thr185Ala and Asn187Asp were selected, and these substitutions were selected as FcR16 (Example 23). FcR24 accumulated in (1) was prepared. Specifically, FcR24 was prepared by introducing a mutation that causes Ala78Ser, Thr185Ala and Asn187Asp to the polynucleotide encoding FcR21.
(C-1) except that pET-FcR21 prepared in (b) was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 24 and SEQ ID NO: 85 (5′-AGCAGCTCACTTTATTGATTCGGCGACGGGTG-3 ′) was used as a PCR primer. After preparing a reaction solution having the composition shown in Table 5, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and 72 ° C. for 1 minute. The reaction with the third step of 1 cycle as 30 cycles was performed by heat treatment at 72 ° C. for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m24-2F.
(C-2) except that pET-FcR21 prepared in (b) was used as a template, and an oligonucleotide consisting of the sequences described in SEQ ID NO: 23 and SEQ ID NO: 86 (5′-GCTATCTCCCACCGTCGCCGAATCAATAAG-3 ′) was used as a PCR primer. PCR was performed in the same manner as in (c-1). The purified PCR product was designated m24-2R.
(C-3) Two types of PCR products (m24-2F and m24-2R) obtained in (c-1) and (c-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed to obtain a PCR product m24-2p in which m21-2F and m21-2R were ligated.
(C-4) PCR was performed using the PCR product m24-2p obtained in (c-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR24-2 containing Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu mutations in FcR16 was prepared.
(C-5) The polynucleotide obtained by (c-4) and encoding FcR24-2 containing mutations of Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu in FcR16 as a template, SEQ ID NO: 24 and SEQ ID NO: 87 (5′-AAAAATGTGAGCAGCGAGGCCCGTGGATTT-3 ′) except that the PCR primer was used as a PCR primer, a reaction solution having the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98 ° C. for 5 minutes. Perform 30 cycles of the first step at 98 ° C for 10 seconds, the second step at 55 ° C for 5 seconds, and the third step at 72 ° C for 1 minute, and finally heat-treat at 72 ° C for 5 minutes. It was done in. The amplified PCR product was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). The purified PCR product was designated as m24F.
(C-6) The polynucleotide obtained by (c-4) and encoding FcR24-2 containing mutations of Ala78Ser, Asp82Glu, Gln101Leu, Thr140Ile, Tyr158His and Lys165Glu in FcR16 was used as a template, and SEQ ID NO: 62 and SEQ ID NO: PCR was carried out in the same manner as in (c-5), except that an oligonucleotide having the sequence described in No. 88 (5′-GGTAATGGTAATATCCCACGCCCCGCGTGCT-3 ′) was used as a PCR primer. The purified PCR product was designated as m24R.
(C-7) Two types of PCR products (m24F and m24R) obtained in (c-5) and (c-6) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was carried out to obtain a PCR product m24p in which m24F and m24R were linked.
(C-8) PCR was performed using the PCR product m24p obtained in (c-7) as a template and oligonucleotides having the sequences described in SEQ ID NO: 62 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding FcR24 in which amino acid substitution was introduced into FcR16 at 8 positions (24 positions relative to the wild-type Fc binding protein) was prepared.
(C-9) After purifying the polynucleotide obtained in (c-8), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(C-10) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. By extracting a plasmid from the collected bacterial cells (transformants), a plasmid pET-FcR24 containing a polynucleotide encoding FcR24, which is a polypeptide obtained by substituting eight amino acids for FcR16, was obtained.
(C-11) The nucleotide sequence of pET-FcR24 was analyzed in the same manner as in Example 1 (5).
　シグナル配列およびポリヒスチジンタグを付加したＦｃＲ２４のアミノ酸配列を配列番号８９に、前記ＦｃＲ２４をコードするポリヌクレオチドの配列を配列番号９０に示す。なお、配列番号８９において、１番目のメチオニン（Ｍｅｔ）から２６番目のアラニン（Ａｌａ）までがＭａｌＥシグナルペプチドであり、２７番目のリジン（Ｌｙｓ）から３２番目のメチオニン（Ｍｅｔ）までがリンカー配列であり、３３番目のグリシン（Ｇｌｙ）から２０８番目のグルタミン（Ｇｌｎ）までがＦｃＲ２４のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２０９番目から２１０番目までのグリシン（Ｇｌｙ）がリンカー配列であり、２１１番目から２１６番目のヒスチジン（Ｈｉｓ）がタグ配列である。また配列番号８９において、Ｇｌｕ２１Ｇｌｙのグリシンは３７番目、Ｌｅｕ２３Ｍｅｔのメチオニンは３９番目、Ｖａｌ２７Ｇｌｕのグルタミン酸は４３番目、Ｐｈｅ２９Ｉｌｅのイソロイシンは４５番目、Ｔｙｒ３５Ａｓｎのアスパラギンは５１番目、Ｇｌｎ４８Ａｒｇのアルギニンは６４番目、Ｔｙｒ５１Ｈｉｓのヒスチジンは６７番目、Ｇｌｕ５４Ａｓｐのアスパラギン酸は７０番目、Ｓｅｒ６８Ｐｒｏのプロリンは８４番目、Ｐｈｅ７５Ｌｅｕのロイシンは９１番目、Ａｌａ７８Ｓｅｒのセリンは９４番目、Ａｓｐ８２Ｇｌｕのグルタミン酸は９８番目、Ａｓｎ９２Ｓｅｒのセリンは１０８番目、Ｇｌｎ１０１Ｌｅｕのロイシンは１１７番目、Ｖａｌ１１７Ｇｌｕのグルタミン酸は１３３番目、Ｇｌｕ１２１Ｇｌｙのグリシンは１３７番目、Ｔｈｒ１４０Ｉｌｅのイソロイシンは１５６番目、Ｇｌｙ１４７Ｖａｌのバリンは１６３番目、Ｔｙｒ１５８Ｈｉｓのヒスチジンは１７４番目、Ｌｙｓ１６５Ｇｌｕのグルタミン酸は１８１番目、Ｐｈｅ１７１Ｓｅｒのセリンは１８７番目、Ｓｅｒ１７８Ａｒｇのアルギニンは１９４番目、Ｔｈｒ１８５Ａｌａのアラニンは２０１番目およびＡｓｎ１８７Ａｓｐのアスパラギン酸は２０３番目の位置にそれぞれ存在する。 The amino acid sequence of FcR24 added with a signal sequence and a polyhistidine tag is shown in SEQ ID NO: 89, and the sequence of the polynucleotide encoding the FcR24 is shown in SEQ ID NO: 90. In SEQ ID NO: 89, the first methionine (Met) to the 26th alanine (Ala) are MalE signal peptides, and the 27th lysine (Lys) to the 32nd methionine (Met) are linker sequences. Yes, the amino acid sequence of FcR24 (corresponding to the 17th to 192nd region of SEQ ID NO: 1) from the 33rd glycine (Gly) to the 208th glutamine (Gln), and the 209th to 210th glycine (Gly) ) Is a linker sequence, and 211 to 216th histidine (His) is a tag sequence. Also, in SEQ ID NO: 89, Glu21Gly glycine is position 37, Leu23Met methionine is position 39, Val27Glu glutamate is position 43, Phe29Ile isoleucine is position 45, Tyr35Asn asparagine is position 51, Gln48Arg arginine is position 64, and Tyr51His. Histidine 67th, Glu54Asp aspartic acid 70th, Ser68Pro proline 84th, Phe75Leu leucine 91th, Ala78Ser serine 94th, Asp82Glu glutamic acid 98th, Asn92Ser serine 108th, Gln101Leu Leucine is 117th, Val117Glu glutamic acid is 133th, Glu121Gly glycine is 1 7th, Thr140Ile isoleucine is 156th, Gly147Val valine is 163rd, Tyr158His histidine is 174th, Lys165Glu glutamic acid is 181st, Phe171Ser serine is 187th, Ser178Arg arginine is 194th, Arhine is 194th Th and Asn187Asp aspartic acid is present at position 203, respectively.
　実施例２８　Ｆｃ結合性タンパク質のアルカリ安定性評価
（１）実施例２３で作製したＦｃ結合性タンパク質（ＦｃＲ１６）、ならびに実施例２７で取得したＦｃ結合性タンパク質（ＦｃＲ１９、ＦｃＲ２１およびＦｃＲ２４）を発現する形質転換体を、実施例８の（１）から（４）に記載の方法で培養し、タンパク質を抽出することでＦｃＲ１６、ＦｃＲ１９、ＦｃＲ２１およびＦｃＲ２４を調製した。
（２）（１）で調製したタンパク質抽出液中のＦｃＲ１６、ＦｃＲ１９、ＦｃＲ２１およびＦｃＲ２４の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法を用いて測定した。この時、精製し定量したＦｃＲ１３を用いて検量線を作製し、濃度測定を行なった。
（３）各Ｆｃ結合性タンパク質の濃度が１０μｇ／ｍＬになるよう純水で希釈後、前記希釈した溶液５０μＬと８０ｍＭの水酸化ナトリウム溶液５０μＬとを混合し、３０℃で２時間静置することでアルカリ処理した。その後、１Ｍトリス塩酸緩衝液（ｐＨ７．０）を４倍量加えることで中和し、Ｆｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法によって測定した。
（４）アルカリ処理を行なった場合の抗体結合活性をアルカリ処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出しアルカリ安定性を評価した。 Example 28 Evaluation of Alkali Stability of Fc Binding Protein (1) Expression of Fc binding protein (FcR16) prepared in Example 23 and Fc binding proteins (FcR19, FcR21 and FcR24) obtained in Example 27 FcR16, FcR19, FcR21 and FcR24 were prepared by culturing the transformant by the method described in Example 8 (1) to (4) and extracting the protein.
(2) The antibody binding activity of FcR16, FcR19, FcR21 and FcR24 in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR13 purified and quantified, and the concentration was measured.
(3) After diluting with pure water so that the concentration of each Fc-binding protein becomes 10 μg / mL, 50 μL of the diluted solution and 50 μL of 80 mM sodium hydroxide solution are mixed and allowed to stand at 30 ° C. for 2 hours. Treated with alkali. Thereafter, the solution was neutralized by adding 4 volumes of 1M Tris-HCl buffer (pH 7.0), and the antibody binding activity of the Fc binding protein was measured by the ELISA method described in Example 3 (4).
(4) The residual activity was calculated by dividing the antibody binding activity when the alkali treatment was performed by the antibody binding activity when the alkali treatment was not performed, and the alkali stability was evaluated.
　結果を表１８に示す。実施例２７で作製したＦｃＲ１９、ＦｃＲ２１およびＦｃＲ２４はＦｃＲ１６と比較し残存活性が高いことから、ＦｃＲ１６に比べてアルカリ安定性が向上していることが確認された。 The results are shown in Table 18. Since FcR19, FcR21 and FcR24 prepared in Example 27 have higher residual activity than FcR16, it was confirmed that the alkali stability was improved compared to FcR16.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　実施例２９　ＦｃＲ２４への変異導入およびライブラリーの作製
　実施例２７（ｃ）で作製したＦｃＲ２４をコードするポリヌクレオチド部分に、エラープローンＰＣＲによりランダムに変異導入を施した。
（１）鋳型として実施例２７（ｃ）で作製した発現ベクターｐＥＴ－ＦｃＲ２４を用いてエラープローンＰＣＲを行なった。エラープローンＰＣＲは、ｐＥＴ－ＦｃＲ２４を鋳型とし、配列番号２３および２４に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することで行なった。この反応によりＦｃ結合性タンパク質をコードするポリヌクレオチドに良好に変異が導入された。
（２）（１）で得られたＰＣＲ産物を精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ同制限酵素で消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションした。
（３）ライゲーション反応終了後、反応液をエレクトロポレーション法により大腸菌ＢＬ２１（ＤＥ３）株に導入し、５０μｇ／ｍＬのカナマイシンを含むＬＢプレート培地で培養後、プレート上に形成したコロニーをランダム変異ライブラリーとした。 Example 29 Mutation Introduction to FcR24 and Library Preparation Mutation was randomly introduced into the polynucleotide portion encoding FcR24 prepared in Example 27 (c) by error-prone PCR.
(1) Error prone PCR was performed using the expression vector pET-FcR24 prepared in Example 27 (c) as a template. In error-prone PCR, pET-FcR24 was used as a template, and a reaction solution having the same composition as shown in Table 3 was prepared except that oligonucleotides having the sequences shown in SEQ ID NOs: 23 and 24 were used as primers. Heat treatment at 95 ° C. for 2 minutes, 35 cycles of reaction with 95 ° C. for 30 seconds for the first step, 50 ° C. for 30 seconds for the second step, 72 ° C. for 90 seconds for the third step for 35 cycles, and finally The heat treatment was performed at 72 ° C. for 7 minutes. By this reaction, the mutation was successfully introduced into the polynucleotide encoding the Fc binding protein.
(2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into an expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) previously digested with the same restriction enzymes.
(3) After completion of the ligation reaction, the reaction solution was introduced into E. coli BL21 (DE3) strain by electroporation, cultured in LB plate medium containing 50 μg / mL kanamycin, and the colonies formed on the plate were randomly mutated. It was rally.
　実施例３０　アルカリ安定化Ｆｃ結合性タンパク質のスクリーニング
（１）実施例２９で作製したランダム変異ライブラリーを実施例３（１）から（２）に記載の方法で培養することでＦｃ結合性タンパク質を発現させた。
（２）培養後、遠心操作によって得られた、Ｆｃ結合性タンパク質を含む培養上清を純水にて２０倍に希釈し、等量の３００ｍＭの水酸化ナトリウム溶液と混合した後、３０℃で２時間静置することでアルカリ処理した。アルカリ処理後は、４倍量の１Ｍトリス緩衝液（ｐＨ７．０）でｐＨを中性付近に戻した。
（３）（２）に記載のアルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性と、（２）に記載のアルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法にてそれぞれ測定した。その後、アルカリ処理を行なったときのＦｃ結合性タンパク質の抗体結合活性を、アルカリ処理を行なわなかったときのＦｃ結合性タンパク質の抗体結合活性で除することで、残存活性を算出した。
（４）（３）の方法で約２７００株の形質転換体を評価し、その中からＦｃＲ２４と比較して安定性が向上したＦｃ結合性タンパク質を発現する形質転換体を選択した。選択した形質転換体を５０μｇ／ｍＬのカナマイシンを含む２ＹＴ液体培地にて培養し、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターを調製した。
（５）得られた発現ベクターに挿入されたＦｃ結合性タンパク質をコードするポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析し、アミノ酸の変異箇所を特定した。 Example 30 Screening of alkali-stabilized Fc binding protein (1) Random mutation library prepared in Example 29 was cultured by the method described in Example 3 (1) to (2) to thereby obtain Fc binding protein. Expressed.
(2) After culture, the culture supernatant containing Fc-binding protein obtained by centrifugation is diluted 20-fold with pure water, mixed with an equal amount of 300 mM sodium hydroxide solution, and then at 30 ° C. The alkali treatment was carried out by leaving still for 2 hours. After the alkali treatment, the pH was returned to near neutral with 4 volumes of 1 M Tris buffer (pH 7.0).
(3) The antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is performed, and the antibody binding activity of the Fc binding protein when the alkali treatment described in (2) is not performed, Each was measured by the ELISA method described in Example 3 (4). Thereafter, the residual activity was calculated by dividing the antibody binding activity of the Fc binding protein when the alkali treatment was performed by the antibody binding activity of the Fc binding protein when the alkali treatment was not performed.
(4) About 2,700 strains of transformants were evaluated by the method of (3), and transformants expressing an Fc-binding protein with improved stability compared to FcR24 were selected from the transformants. The selected transformant was cultured in a 2YT liquid medium containing 50 μg / mL kanamycin, and an expression vector was prepared using QIAprep Spin Miniprep kit (manufactured by Qiagen).
(5) The nucleotide sequence of the sequence of the polynucleotide region encoding the Fc binding protein inserted into the obtained expression vector was analyzed by the method described in Example 1 (5), and amino acid mutation sites were identified.
　（４）で選択した形質転換体が発現するＦｃ結合性タンパク質の、ＦｃＲ２４に対するアミノ酸置換位置およびアルカリ処理後の残存活性（％）をまとめたものを表１９に示す。配列番号８９に記載のアミノ酸配列のうち、３３番目のグリシンから２０８番目のグルタミンまでのアミノ酸残基（配列番号１の１７番目から１９２番目に該当）を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において、Ｌｙｓ４０Ｇｌｎ（この表記は、配列番号１の４０番目（配列番号３７では５６番目）のリジンがグルタミンに置換されていることを表す、以下同様）、Ｌｙｓ４６Ａｓｎ、Ａｌａ５０Ｔｈｒ、Ａｓｎ５６Ｔｙｒ、Ｈｉｓ６２Ｌｅｕ、Ｓｅｒ６５Ｇｌｙ、Ｔｙｒ７４Ｈｉｓ、Ａｓｐ７７Ｖａｌ、Ｇｌｎ９０Ｌｅｕ、Ｌｙｓ１１９Ｔｈｒ、Ｌｙｓ１１９Ｇｌｕ、Ａｓｐ１２２Ｇｌｕ、Ｈｉｓ１３７Ｇｌｎ、Ｔｈｒ（Ｉｌｅ）１４０Ｍｅｔ（この表記は、配列番号１の１４０番目（配列番号３７では１５６番目）のスレオニンが一度イソロイシンに置換されさらにメチオニンに置換されたことを示す、以下同様）、Ｔｙｒ１４１Ｐｈｅ、Ｔｙｒ（Ｈｉｓ）１５８Ｌｅｕ、Ｌｅｕ１７５Ａｒｇ、Ａｓｎ１８０Ｌｙｓ、Ａｓｎ１８０Ｓｅｒ、Ｉｌｅ１９０Ｖａｌ、Ｔｈｒ１９１Ｉｌｅのいずれかのアミノ酸置換が少なくとも１つ生じているＦｃ結合性タンパク質は、ＦｃＲ２４と比較しアルカリ安定性が向上しているといえる。 Table 19 shows a summary of amino acid substitution positions for FcR24 and residual activity (%) after alkali treatment of the Fc-binding protein expressed by the transformant selected in (4). Of the amino acid sequence set forth in SEQ ID NO: 89, the amino acid residues from the 33rd glycine to the 208th glutamine (corresponding to the 17th to 192nd of SEQ ID NO: 1), and from the 33rd to 208th Among amino acid residues, Lys40Gln (this notation represents that 40th lysine in SEQ ID NO: 1 (56th in SEQ ID NO: 37) is substituted with glutamine, the same applies hereinafter), Lys46Asn, Ala50Thr, Asn56Tyr, His62Leu, Ser65Gly, Tyr74His, Asp77Val, Gln90Leu, Lys119Thr, Lys119Glu, Asp122Glu, His137Gln, Thr (Ile) 140Met (This notation is the 140th position of SEQ ID NO: 1 (156th position of SEQ ID NO: 37) Threonine once substituted with isoleucine and further substituted with methionine, the same applies below), and at least one amino acid substitution of Tyr141Phe, Tyr (His) 158Leu, Leu175Arg, Asn180Lys, Asn180Ser, Ile190Val, Thr191Ile occurs It can be said that the Fc-binding protein has improved alkali stability compared to FcR24.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　実施例３１　Ｔｈｒ１４０またはＴｙｒ１５８アミノ酸置換体の作製
　実施例２６で明らかになったＦｃ結合性タンパク質のアルカリ安定性向上に寄与するアミノ酸置換のうち、配列番号１の１４０番目（配列番号３７では１５６番目）のスレオニン（Ｔｈｒ１４０）がイソロイシン（Ｉｌｅ）に、１５８番目（配列番号３７では１７４番目）のチロシン（Ｔｙｒ１５８）がヒスチジンにそれぞれ置換されることでアルカリ安定性が特に向上した。そこで、Ｔｈｒ１４０およびＴｙｒ１５８の他のアミノ酸への置換の有用性を確認するため、実施例２７（ｃ）で作製したＦｃＲ２４（配列番号８９）のうちＴｈｒ１４０（配列番号８９では１５６番目）またはＴｙｒ１５８（配列番号８９では１７４番目）を他のアミノ酸に置換したＦｃ結合性タンパク質を作製した。
（ａ）Ｔｈｒ１４０アミノ酸置換体の作製
（ａ－１）実施例２７（ｃ）で作製した、ｐＥＴ－ＦｃＲ２４を鋳型とし、配列番号２４および配列番号９１（５’－ＣＣＴＧＣＡＴＡＡＡＧＴＧＮＮＫＴＡＣＣＴＧＣＡＡＡＡＣＧＧ－３’）に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することでＰＣＲを行なった。得られたＰＣＲ産物をＴ１４０ｐ１とした。
（ａ－２）実施例２７（ｃ）で作製した、ｐＥＴ－ＦｃＲ２４を鋳型とし、配列番号２３および配列番号９２（５’－ＣＣＧＴＴＴＴＧＣＡＧＧＴＡＭＮＮＣＡＣＴＴＴＡＴＧＣＡＧＧ－３’）に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することでＰＣＲを行なった。得られたＰＣＲ産物をＴ１４０ｐ２とした。
（ａ－３）（ａ－１）および（ａ－２）で得られた２種類のＰＣＲ産物（Ｔ１４０ｐ１、Ｔ１４０ｐ２）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、Ｔ１４０ｐ１とＴ１４０ｐ２を連結したＰＣＲ産物Ｔ１４０ｐを得た。
（ａ－４）（ａ－３）で得られたＰＣＲ産物Ｔ１４０ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃ結合性タンパク質（ＦｃＲ２４）の１４０番目のアミノ酸が任意のアミノ酸に置換されたＦｃ結合性タンパク質をコードするポリヌクレオチドを得た。得られたポリヌクレオチドをＴ１４０ｐ３とした。
（ａ－５）（ａ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ａ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。 Example 31 Production of Thr140 or Tyr158 Amino Acid Substituted Of the amino acid substitutions that contribute to the improvement of alkali stability of the Fc binding protein revealed in Example 26, the 140th position of SEQ ID NO: 1 (156th position of SEQ ID NO: 37) In particular, threonine (Thr140) was replaced with isoleucine (Ile) and 158th (174th in SEQ ID NO: 37) tyrosine (Tyr158) was replaced with histidine, whereby alkali stability was particularly improved. Therefore, in order to confirm the usefulness of substitution of Thr140 and Tyr158 with other amino acids, Thr140 (156th in SEQ ID NO: 89) or Tyr158 (SEQ ID NO: 89) of FcR24 (SEQ ID NO: 89) prepared in Example 27 (c) Fc-binding protein was prepared by substituting 174th in number 89 with another amino acid.
(A) Preparation of Thr140 amino acid substitution product (a-1) Using pET-FcR24 prepared in Example 27 (c) as a template, SEQ ID NO: 24 and SEQ ID NO: 91 (5′-CCTGCATAAAGTGNKNKTACTCGCCAAAACGG-3 ′) After preparing a reaction solution similar to the composition shown in Table 3 except that the oligonucleotide consisting of the sequence was used as a primer, the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C. for 30 seconds, 50 ° C. PCR was performed by performing 35 cycles of the second step of 30 seconds at 72 ° C and the third step of 90 seconds at 72 ° C for one cycle, and finally heat treating at 72 ° C for 7 minutes. The obtained PCR product was designated as T140p1.
(A-2) Using, as a primer, an oligonucleotide having the sequence described in SEQ ID NO: 23 and SEQ ID NO: 92 (5′-CCGTTTTGCAGGTAMNNCACTTTATGCAGGG-3 ′), which was prepared in Example 27 (c), using pET-FcR24 as a template After preparing a reaction solution having the same composition as shown in Table 3, the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C. for 30 seconds, second step at 50 ° C. for 30 seconds, 72 PCR was carried out by performing 35 cycles of a reaction in which the third step of 90 seconds at 90 ° C. was carried out, and finally heat treating at 72 ° C. for 7 minutes. The obtained PCR product was designated as T140p2.
(A-3) Two types of PCR products (T140p1, T140p2) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed, and a PCR product T140p in which T140p1 and T140p2 were ligated was obtained.
(A-4) PCR was performed using the PCR product T140p obtained in (a-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding an Fc binding protein in which the 140th amino acid of the Fc binding protein (FcR24) was substituted with an arbitrary amino acid was obtained. The obtained polynucleotide was designated as T140p3.
(A-5) After purifying the polynucleotide obtained in (a-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(A-6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin.
　回収した菌体（形質転換体）からプラスミドを抽出し、ポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析したところ、Ｆｃ結合性タンパク質ＦｃＲ２４のＴｈｒ１４０（配列番号８９では１５６番目のイソロイシン）がＡｌａ、Ａｒｇ、Ｇｌｙ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、Ｔｈｒ、ＳｅｒまたはＶａｌに置換されたＦｃ結合性タンパク質を発現する形質転換体を得た。
（ｂ）Ｔｙｒ１５８アミノ酸置換体の作製
（ｂ－１）実施例２７（ｃ）で作製した、ｐＥＴ－ＦｃＲ２４を鋳型とし、配列番号２４および配列番号９３（５’－ＣＡＡＣＴＣＣＧＡＣＴＴＣＮＮＫＡＴＴＣＣＣＡＡＡＧＣＧＡＣ－３’）に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することでＰＣＲを行なった。得られたＰＣＲ産物をＹ１５８ｐ１とした。
（ｂ－２）実施例２７（ｃ）で作製した、ｐＥＴ－ＦｃＲ２４を鋳型とし、配列番号２３および配列番号９４（５’－ＧＴＣＧＣＴＴＴＧＧＧＡＡＴＭＮＮＧＡＡＧＴＣＧＧＡＧＴＴＧ－３’）に記載の配列からなるオリゴヌクレオチドをプライマーとして用いた他は表３に示す組成と同様の反応液を調製後、当該反応液を９５℃で２分間熱処理し、９５℃で３０秒間の第１ステップ、５０℃で３０秒間の第２ステップ、７２℃で９０秒間の第３ステップを１サイクルとする反応を３５サイクル行ない、最後に７２℃で７分間熱処理することでＰＣＲを行なった。得られたＰＣＲ産物をＹ１５８ｐ２とした。
（ｂ－３）（ｂ－１）および（ｂ－２）で得られた２種類のＰＣＲ産物（Ｙ１５８ｐ１、Ｙ１５８ｐ２）を混合し、表６に示す組成の反応液を調製した。当該反応液を９８℃で５分間熱処理後、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を５サイクル行なうＰＣＲを行ない、Ｙ１４０ｐ１とＹ１４０ｐ２を連結したＰＣＲ産物Ｙ１４０ｐを得た。
（ｂ－４）（ｂ－３）で得られたＰＣＲ産物Ｙ１４０ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとしてＰＣＲを行なった。ＰＣＲは、表７に示す組成の反応液を調製後、当該反応液を９８℃で５分間熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル行なった。これによりＦｃ結合性タンパク質（ＦｃＲ２４）の１５８番目のアミノ酸が任意のアミノ酸に置換されたＦｃ結合性タンパク質をコードするポリヌクレオチドを得た。得られたポリヌクレオチドをＹ１５８ｐ３とした。
（ｂ－５）（ｂ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（ｂ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。 A plasmid was extracted from the collected cells (transformants), and the nucleotide sequence of the polynucleotide region was analyzed by the method described in Example 1 (5). As a result, Thr140 (SEQ ID NO: 89) of the Fc-binding protein FcR24 was analyzed. Then, a transformant expressing an Fc-binding protein in which 156th isoleucine) was substituted with Ala, Arg, Gly, Leu, Lys, Phe, Thr, Ser or Val was obtained.
(B) Preparation of Tyr158 amino acid substitution product (b-1) Using pET-FcR24 prepared in Example 27 (c) as a template, SEQ ID NO: 24 and SEQ ID NO: 93 (5′-CAACTCCGAACTTCNKNATCTCCAAAGCGAC-3 ′) After preparing a reaction solution similar to the composition shown in Table 3 except that the oligonucleotide consisting of the sequence was used as a primer, the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C. for 30 seconds, 50 ° C. PCR was performed by performing 35 cycles of the second step of 30 seconds at 72 ° C and the third step of 90 seconds at 72 ° C for one cycle, and finally heat treating at 72 ° C for 7 minutes. The obtained PCR product was designated as Y158p1.
(B-2) An oligonucleotide having the sequence described in SEQ ID NO: 23 and SEQ ID NO: 94 (5′-GTCGCTTTGGGAATNNNGAAGTCGGAGTTG-3 ′) prepared in Example 27 (c) as a template and used as a primer After preparing a reaction solution having the same composition as shown in Table 3, the reaction solution was heat-treated at 95 ° C. for 2 minutes, first step at 95 ° C. for 30 seconds, second step at 50 ° C. for 30 seconds, 72 PCR was carried out by performing 35 cycles of a reaction in which the third step of 90 seconds at 90 ° C. was carried out, and finally heat treating at 72 ° C. for 7 minutes. The obtained PCR product was designated as Y158p2.
(B-3) Two types of PCR products (Y158p1, Y158p2) obtained in (b-1) and (b-2) were mixed to prepare a reaction solution having the composition shown in Table 6. The reaction solution is heat-treated at 98 ° C. for 5 minutes, followed by 5 cycles of reaction in which the first step is 98 ° C. for 10 seconds, the second step is 55 ° C. for 5 seconds, and the third step is 72 ° C. for 1 minute. PCR was performed to obtain a PCR product Y140p in which Y140p1 and Y140p2 were ligated.
(B-4) PCR was performed using the PCR product Y140p obtained in (b-3) as a template and oligonucleotides having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers. In PCR, after preparing a reaction solution having the composition shown in Table 7, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was performed 30 cycles. As a result, a polynucleotide encoding an Fc binding protein in which the 158th amino acid of the Fc binding protein (FcR24) was substituted with an arbitrary amino acid was obtained. The obtained polynucleotide was designated as Y158p3.
(B-5) After purifying the polynucleotide obtained in (b-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Unexamined Patent Publication No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin.
　回収した菌体（形質転換体）からプラスミドを抽出し、ポリヌクレオチド領域の配列を実施例１（５）に記載の方法によりヌクレオチド配列を解析したところ、Ｆｃ結合性タンパク質ＦｃＲ２４のＴｙｒ１５８（配列番号８９では１７４番目のヒスチジン）がＡｌａ、Ａｒｇ、Ａｓｎ、Ｃｙｓ、Ｇｌｎ、Ｇｌｕ、Ｇｌｙ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｍｅｔ、Ｐｈｅ、Ｐｒｏ、Ｓｅｒ、Ｔｈｒ、Ｔｒｐ、ＴｙｒまたはＶａｌに置換されたＦｃ結合性タンパク質を発現する形質転換体を得た。 A plasmid was extracted from the collected cells (transformants), and the nucleotide sequence of the polynucleotide region was analyzed by the method described in Example 1 (5). As a result, Tyr158 (SEQ ID NO: 89) of the Fc-binding protein FcR24 was analyzed. Fc-binding protein in which 174th histidine is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val A transformant expressing was obtained.
　実施例３２　Ｔｈｒ１４０またはＴｙｒ１５８アミノ酸置換体の評価
（１）実施例３１で作製したＦｃ結合性タンパク質を発現する形質転換体を、実施例８の（１）から（４）に記載の方法で培養し、タンパク質を抽出した。
（２）（１）で調製したタンパク質抽出液中のＦｃ結合タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法を用いて測定した。この時、精製し定量したＦｃＲ２４を用いて検量線を作製し、濃度測定を行なった。
（３）各Ｆｃ結合性タンパク質の濃度が１０μｇ／ｍＬになるよう純水で希釈後、前記希釈した溶液５０μＬと、３００ｍＭ（Ｔｈｒ１４０アミノ酸置換体の場合）または３５０ｍＭ（Ｔｙｒ１５８アミノ酸置換体の場合）の水酸化ナトリウム溶液５０μＬとを混合し、３０℃で２時間静置することでアルカリ処理した。その後、１Ｍトリス塩酸緩衝液（ｐＨ７．０）を４倍量加えることで中和し、Ｆｃ結合性タンパク質の抗体結合活性を、実施例３（４）に記載のＥＬＩＳＡ法によって測定した。
（４）アルカリ処理を行なった場合の抗体結合活性をアルカリ処理を行なわなかったときの抗体結合活性で除することで、残存活性を算出しアルカリ安定性を評価した。 Example 32 Evaluation of Thr140 or Tyr158 Amino Acid Substitution (1) The transformant expressing the Fc-binding protein prepared in Example 31 was cultured by the method described in Example 8 (1) to (4). The protein was extracted.
(2) The antibody binding activity of the Fc binding protein in the protein extract prepared in (1) was measured using the ELISA method described in Example 3 (4). At this time, a calibration curve was prepared using FcR24 purified and quantified, and the concentration was measured.
(3) After dilution with pure water so that the concentration of each Fc-binding protein becomes 10 μg / mL, 50 μL of the diluted solution and 300 mM (in the case of Thr140 amino acid substitution product) or 350 mM (in the case of Tyr158 amino acid substitution product) Sodium hydroxide solution (50 μL) was mixed and allowed to stand at 30 ° C. for 2 hours for alkali treatment. Thereafter, the solution was neutralized by adding 4 volumes of 1M Tris-HCl buffer (pH 7.0), and the antibody binding activity of the Fc binding protein was measured by the ELISA method described in Example 3 (4).
(4) The residual activity was calculated by dividing the antibody binding activity when the alkali treatment was performed by the antibody binding activity when the alkali treatment was not performed, and the alkali stability was evaluated.
　得られた結果を表２０（Ｔｈｒ１４０アミノ酸置換体の結果）および表２１（Ｔｙｒ１５８アミノ酸置換体の結果）に示した。なお表２０のうちＩｌｅの結果、および表２１のうちＨｉｓの結果はＦｃＲ２４の結果である。Ｔｈｒ１４０の場合（表２０）は、Ａｌａ、Ａｒｇ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、ＳｅｒまたはＶａｌに置換することで、Ｔｙｒ１５８の場合（表２１）は、Ｃｙｓ、Ｈｉｓ、Ｉｌｅ、Ｌｅｕ、Ｌｙｓ、Ｐｈｅ、ＴｒｐまたはＶａｌに置換することで、それぞれアルカリ安定性が向上することを確認した。 The results obtained are shown in Table 20 (result of Thr140 amino acid substitution product) and Table 21 (Result of Tyr158 amino acid substitution product). In Table 20, the result of Ile and the result of His in Table 21 are the results of FcR24. In the case of Thr140 (Table 20), by replacing with Ala, Arg, Ile, Leu, Lys, Phe, Ser or Val, in the case of Tyr158 (Table 21), Cys, His, Ile, Leu, Lys, Phe. It was confirmed that the alkali stability was improved by substituting with Trp or Val.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
　実施例３３　Ｆｃ結合性タンパク質固定化ゲルの調製
（１）配列番号６０に記載のアミノ酸配列からなるヒトＦｃ結合性タンパク質をコードするポリヌクレオチドを含むプラスミドで大腸菌を形質転換して得られた形質転換体を培養し、得られた菌体から前記Ｆｃ受容体タンパク質を精製することで、不溶性担体に固定化させるリガンドを調製した。なお配列番号６０に記載のアミノ酸配列からなるヒトＦｃ結合性タンパク質は、配列番号５８に記載のアミノ酸配列からなるヒトＦｃ結合性タンパク質において、以下の（ａ）から（ｄ）のアミノ酸置換が生じたタンパク質である。
（ａ）配列番号５８の４５番目のフェニルアラニンがイソロイシンに置換
（ｂ）配列番号５８の６４番目のグルタミンがアルギニンに置換
（ｃ）配列番号５８の１３３番目のバリンがグルタミン酸に置換
（ｄ）配列番号５８の１８７番目のフェニルアラニンがセリンに置換
（２）ビニルポリマーゲル（粒子径１０μｍ、東ソー社製）が有するヒドロキシ基を常法により官能基変換を行なうことで、ヨードアセチル基にて活性化されたビニルポリマーゲルを得た。
（３）得られた前記活性化されたゲルに（１）で調製したリガンドを反応させることにより、ヒトＦｃ結合性タンパク質固定化ゲルを作製した。
（４）得られたゲルをφ４．６×７５ｍｍのステンレスカラムに充填し、分離カラムを作製した。 Example 33 Preparation of Fc-binding protein-immobilized gel (1) Transformation obtained by transforming Escherichia coli with a plasmid containing a polynucleotide encoding a human Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 60 The body was cultured, and the Fc receptor protein was purified from the obtained cells to prepare a ligand to be immobilized on an insoluble carrier. The human Fc binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 60 had the following amino acid substitutions (a) to (d) in the human Fc binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 58. It is a protein.
(A) 45th phenylalanine of SEQ ID NO: 58 is replaced with isoleucine (b) 64th glutamine of SEQ ID NO: 58 is replaced with arginine (c) 133rd valine of SEQ ID NO: 58 is replaced with glutamic acid (d) SEQ ID NO: The 187th phenylalanine in 58 was substituted with serine. (2) The hydroxy group of the vinyl polymer gel (particle size: 10 μm, manufactured by Tosoh Corporation) was activated with an iodoacetyl group by performing functional group conversion by a conventional method. A vinyl polymer gel was obtained.
(3) A human Fc-binding protein-immobilized gel was prepared by reacting the obtained activated gel with the ligand prepared in (1).
(4) The obtained gel was packed into a φ4.6 × 75 mm stainless steel column to produce a separation column.
　比較例１　モノクローナル抗体の分離
（１）市販のモノクローナル抗体（リツキサン、全薬工業社製）をＰＢＳ（Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ）で１ｍｇ／ｍＬに調製し、これをモノクローナル抗体溶液として用いた。
（２）実施例１で作製した分離カラムを２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ５．０）（緩衝液Ａ）で平衡化後、（１）で調製したモノクローナル抗体溶液を５μＬ添加した。
（３）モノクローナル抗体溶液添加後２分間は緩衝液Ａを流し、２分から４０分までの間は、緩衝液Ａ１００％－１０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）（緩衝液Ｂ）０％から、緩衝液Ａ０％－緩衝液Ｂ１００％とするグラジエントにより、カラムに添加したモノクローナル抗体を分離し溶出した。溶出したモノクローナル抗体の検出はＵＶ検出器（２８０ｎｍの吸収）で行なった。 Comparative Example 1 Separation of Monoclonal Antibody (1) A commercially available monoclonal antibody (Rituxan, Zenyaku Kogyo Co., Ltd.) was prepared to 1 mg / mL with PBS (Phosphate Buffered Saline), and this was used as a monoclonal antibody solution.
(2) The separation column prepared in Example 1 was equilibrated with 20 mM sodium acetate buffer (pH 5.0) (buffer A), and 5 μL of the monoclonal antibody solution prepared in (1) was added.
(3) Buffer A is allowed to flow for 2 minutes after the addition of the monoclonal antibody solution, and from 2% to 40 minutes, from Buffer A 100% -10 mM glycine hydrochloride buffer (pH 3.0) (Buffer B) 0% The monoclonal antibody added to the column was separated and eluted with a gradient of Buffer A 0%-Buffer B 100%. The eluted monoclonal antibody was detected with a UV detector (absorption at 280 nm).
　モノクローナル抗体を分離した結果（クロマトグラム）を図１に示す。３つの大きなピークが検出され、溶出時間の早いピークからピーク１、ピーク２、ピーク３とした。また、それぞれのピークの分離度（Ｒｓ値）を下記の式に従って算出したところ、ピーク１とピーク２との分離度は０．６３に、ピーク２とピーク３との分離度は０．６１に、それぞれなった。 The results (chromatogram) of the monoclonal antibody separation are shown in FIG. Three large peaks were detected, and the peak with the earlier elution time was designated as peak 1, peak 2, and peak 3. Moreover, when the separation degree (Rs value) of each peak was calculated according to the following formula, the separation degree between peak 1 and peak 2 was 0.63, and the separation degree between peak 2 and peak 3 was 0.61. , Each became.
　　Ｒｓ値＝１．１８×（溶出時間の遅いピークの溶出時間－溶出時間の早いピークの溶出時間）／（溶出時間の早いピークの半値幅＋溶出時間の遅いピークの半値幅）
　実施例３４　本発明によるモノクローナル抗体の分離（その１）
　緩衝液Ａとして、塩化ナトリウムを５０ｍＭ、１００ｍＭ、２００ｍＭ、５００ｍＭまたは１０００ｍＭ添加した２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ５．０）を用いた他は、比較例１と同様な実験を行なった。結果を図７に示す。溶出時間の早いピークからピーク１、ピーク２、ピーク３とし、比較例１と同様な方法でＲｓ値を算出したところ、表２２に示す結果となった。２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ５．０）に塩化ナトリウム（塩化物イオン）を添加することで、ピーク１とピーク２とのＲｓ値、および／またはピーク２とピーク３とのＲｓ値が向上していることがわかる。特に５０ｍＭから５００ｍＭの塩化ナトリウム（塩化物イオン）を添加した緩衝液では、ピーク１とピーク２とのＲｓ値、およびピーク２とピーク３とのＲｓ値が向上しているため特に好ましいといえる。 Rs value = 1.18 × (elution time of peak with late elution time−elution time of peak with early elution time) / (half width of peak with early elution time + half width of peak with late elution time)
Example 34 Separation of monoclonal antibody according to the present invention (Part 1)
The same experiment as Comparative Example 1 was performed, except that 20 mM sodium acetate buffer (pH 5.0) supplemented with 50 mM, 100 mM, 200 mM, 500 mM, or 1000 mM sodium chloride was used as buffer A. The results are shown in FIG. When the Rs value was calculated in the same manner as in Comparative Example 1 from the peak with the earlier elution time to Peak 1, Peak 2, and Peak 3, the results shown in Table 22 were obtained. By adding sodium chloride (chloride ion) to 20 mM sodium acetate buffer (pH 5.0), the Rs value between peak 1 and peak 2 and / or the Rs value between peak 2 and peak 3 is improved. You can see that In particular, a buffer solution to which 50 mM to 500 mM sodium chloride (chloride ion) is added is particularly preferable because the Rs value between peak 1 and peak 2 and the Rs value between peak 2 and peak 3 are improved.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　実施例３５　本発明によるモノクローナル抗体の分離（その２）
　緩衝液Ａとして、塩化カリウムを１００ｍＭまたは２００ｍＭ添加した２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ５．０）を用いた他は、比較例１と同様な実験を行なった。結果を図８に示す。溶出時間の早いピークからピーク１、ピーク２、ピーク３とし、比較例１と同様な方法でＲｓ値を算出したところ、表２３に示す結果となった。この結果から塩化ナトリウムの代わりに塩化カリウムを用いても、実施例３４の結果と同様、ピーク１とピーク２とのＲｓ値、および／またはピーク２とピーク３とのＲｓ値が向上していることがわかる。 Example 35 Separation of monoclonal antibody according to the present invention (part 2)
The same experiment as in Comparative Example 1 was performed except that 20 mM sodium acetate buffer (pH 5.0) supplemented with 100 mM or 200 mM potassium chloride was used as buffer A. The results are shown in FIG. When the Rs value was calculated in the same manner as in Comparative Example 1 from the peak with the earlier elution time to Peak 1, Peak 2, and Peak 3, the results shown in Table 23 were obtained. From this result, even when potassium chloride was used instead of sodium chloride, the Rs value between peak 1 and peak 2 and / or the Rs value between peak 2 and peak 3 was improved as in the result of Example 34. I understand that.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　実施例３６　本発明によるモノクローナル抗体の分離（その３）
　緩衝液Ａとして、硫酸ナトリウムまたは硫酸アンモニウムを１００ｍＭ添加した２０ｍＭの酢酸ナトリウム緩衝液（ｐＨ５．０）を用いた他は、比較例１と同様な実験を行なった。結果を図９に示す。溶出時間の早いピークからピーク１、ピーク２、ピーク３とし、比較例１と同様な方法でＲｓ値を算出したところ、表２４に示す結果となった。この結果から塩化物イオンの代わりに硫酸イオンを用いても、実施例３４の結果と同様、ピーク１とピーク２とのＲｓ値、またはピーク２とピーク３とのＲｓ値が向上していることがわかる。 Example 36 Separation of monoclonal antibody according to the present invention (part 3)
The same experiment as Comparative Example 1 was performed except that 20 mM sodium acetate buffer (pH 5.0) to which 100 mM sodium sulfate or ammonium sulfate was added as buffer A was used. The results are shown in FIG. When the Rs value was calculated in the same manner as in Comparative Example 1 from the peak with the earlier elution time to Peak 1, Peak 2, and Peak 3, the results shown in Table 24 were obtained. From this result, even when sulfate ion is used instead of chloride ion, the Rs value between peak 1 and peak 2 or the Rs value between peak 2 and peak 3 is improved as in the result of Example 34. I understand.
Figure JPOXMLDOC01-appb-T000024
Figure JPOXMLDOC01-appb-T000024
　実施例３７　１箇所アミノ酸置換したＦｃ結合性タンパク質の作製
　実施例３で明らかになったＦｃ結合性タンパク質の安定性向上に関与するアミノ酸置換のうち、配列番号１の２７番目のバリン（Ｖａｌ）、３５番目のチロシン（Ｔｙｒ）および１２１番目のグルタミン酸（Ｇｌｕ）について、他のアミノ酸に置換したＦｃ結合性タンパク質を、それぞれ下記の方法で作製した。 Example 37 Production of Fc binding protein with amino acid substitution at one position Among the amino acid substitutions involved in improving the stability of Fc binding protein revealed in Example 3, the 27th valine (Val) of SEQ ID NO: 1, Regarding the 35th tyrosine (Tyr) and the 121st glutamic acid (Glu), Fc binding proteins substituted with other amino acids were prepared by the following methods, respectively.
　（Ａ）配列番号１の２７番目のバリン（Ｖａｌ）を他のアミノ酸に置換したＦｃ結合性タンパク質の作製
（Ａ－１）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２４および配列番号９５（５’－ＣＴＧＣＣＧＡＡＡＧＣＧＮＮＫＧＴＧＴＴＴＣＴＧＧＡＡＣＣＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を２７ｐＦとした。
（Ａ－２）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２３および配列番号９６（５’－ＴＴＣＣＡＧＡＡＡＣＡＣＭＮＮＣＧＣＴＴＴＣＧＧＣＡＧＡＴＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を２７ｐＲとした。
（Ａ－３）（Ａ－１）および（Ａ－２）で得られた２種類のＰＣＲ産物（２７ｐＦ、２７ｐＲ）を混合後、実施例４（ａ－３）と同様の方法でＰＣＲを行ない、２７ｐＦと２７ｐＲを連結した。得られたＰＣＲ産物を２７ｐとした。
（Ａ－４）（Ａ－３）で得られたＰＣＲ産物２７ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、実施例４（ａ－４）と同様の方法でＰＣＲを行なった。これにより配列番号１の２７番目のバリンを任意のアミノ酸に置換したＦｃ結合性タンパク質をコードするポリヌクレオチドを作製した。
（Ａ－５）（Ａ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（Ａ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出し、実施例１（５）と同様の方法でヌクレオチド配列解析を行なった。 (A) Preparation of Fc-binding protein in which 27th valine (Val) in SEQ ID NO: 1 is substituted with another amino acid (A-1) Using pET-eFcR prepared in Example 1 as a template, SEQ ID NO: 24 and sequence PCR was performed in the same manner as in Example 4 (a-1) except that an oligonucleotide having the sequence described in No. 95 (5′-CTGCCGAAAGCGNNKGTGTTTCTGGGAACCG-3 ′) was used as a PCR primer. The purified PCR product was 27 pF.
(A-2) Except that pET-eFcR prepared in Example 1 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 96 (5′-TTCCGAAAACACNCNCGCTTTCGGCAGATC-3 ′) was used as a PCR primer, PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 27 pR.
(A-3) After mixing the two PCR products (27pF, 27pR) obtained in (A-1) and (A-2), PCR was performed in the same manner as in Example 4 (a-3). 27pF and 27pR were ligated. The obtained PCR product was designated as 27p.
(A-4) Example 4 (a-4) and the PCR product 27p obtained in (A-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed in the same manner. Thus, a polynucleotide encoding an Fc-binding protein in which the 27th valine of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
(A-5) After purifying the polynucleotide obtained in (A-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(A-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected microbial cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).
　結果、Ｖａｌ２７Ｇｌｙ（Ｖ２７Ｇ）、Ｖａｌ２７Ｌｙｓ（Ｖ２７Ｋ）、Ｖａｌ２７Ｔｈｒ（Ｖ２７Ｔ）、Ｖａｌ２７Ａｌａ（Ｖ２７Ａ）、Ｖａｌ２７Ｔｒｐ（Ｖ２７Ｗ）、またはＶａｌ２７Ａｒｇ（Ｖ２７Ｒ）のアミノ酸置換が生じたＦｃ結合性タンパク質をコードするポリヌクレオチドを得た。 As a result, a polynucleotide encoding an Fc binding protein in which an amino acid substitution of Val27Gly (V27G), Val27Lys (V27K), Val27Thr (V27T), Val27Ala (V27A), Val27Trp (V27W) or Val27Arg (V27R) occurred was obtained. .
　（Ｂ）配列番号１の３５番目のチロシン（Ｔｙｒ）を他のアミノ酸に置換したＦｃ結合性タンパク質の作製
（Ｂ－１）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２４および配列番号９７（５’－ＡＡＣＣＧＣＡＧＴＧＧＮＮＫＣＧＣＧＴＧＣＴＧＧＡＧＡＡＡＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を３５ｐＦとした。
（Ｂ－２）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２３および配列番号９８（５’－ＡＧＣＡＣＧＣＧＭＮＮＣＣＡＣＴＧＣＧＧＴＴＣＣＡＧＡＡＡＣ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を３５ｐＲとした。
（Ｂ－３）（Ｂ－１）および（Ｂ－２）で得られた２種類のＰＣＲ産物（３５ｐＦ、３５ｐＲ）を混合後、実施例４（ａ－３）と同様の方法にてＰＣＲを行ない、３５ｐＦと３５ｐＲを連結した。得られたＰＣＲ産物を３５ｐとした。
（Ｂ－４）（Ｂ－３）で得られたＰＣＲ産物３５ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして、実施例４（ａ－４）と同様の方法でＰＣＲを行なった。これにより配列番号１の３５番目のチロシンを任意のアミノ酸に置換したＦｃ結合性タンパク質をコードするポリヌクレオチドを作製した。
（Ｂ－５）（Ｂ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、これを用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（Ｂ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出し、実施例１（５）と同様の方法でヌクレオチド配列解析を行なった。 (B) Preparation of Fc-binding protein in which 35th tyrosine (Tyr) of SEQ ID NO: 1 is substituted with another amino acid (B-1) Using pET-eFcR prepared in Example 1 as a template, SEQ ID NO: 24 and sequence PCR was performed in the same manner as in Example 4 (a-1) except that the oligonucleotide having the sequence described in No. 97 (5′-AACCGCAGTGGNNKCGCGGTCGTGGAGAAAG-3 ′) was used as a PCR primer. The purified PCR product was 35 pF.
(B-2) Except that pET-eFcR prepared in Example 1 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 98 (5′-AGCACGCGMNNCCACTGCGGTTCCAGAAAC-3 ′) was used as a PCR primer, PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 35 pR.
(B-3) After mixing the two types of PCR products (35pF, 35pR) obtained in (B-1) and (B-2), PCR was performed in the same manner as in Example 4 (a-3). And 35 pF and 35 pR were ligated. The obtained PCR product was 35p.
(B-4) Example 4 (a-4) and the PCR product 35p obtained in (B-3) as a template and oligonucleotides having the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed in the same manner. Thereby, a polynucleotide encoding an Fc-binding protein in which the 35th tyrosine of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
(B-5) After purifying the polynucleotide obtained in (B-4), digested with restriction enzymes NcoI and HindIII, and previously digested with restriction enzymes NcoI and HindIII (Japanese Patent Laid-Open No. 2011-206046) Was used to transform Escherichia coli BL21 (DE3) strain.
(B-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected microbial cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).
　結果、Ｔｙｒ３５Ｃｙｓ（Ｙ３５Ｃ）、Ｔｙｒ３５Ａｓｐ（Ｙ３５Ｄ）、Ｔｙｒ３５Ｐｈｅ（Ｙ３５Ｆ）、Ｔｙｒ３５Ｇｌｙ（Ｙ３５Ｇ）、Ｔｙｒ３５Ｌｙｓ（Ｙ３５Ｋ）、Ｔｙｒ３５Ｌｅｕ（Ｙ３５Ｌ）、Ｔｙｒ３５Ａｓｎ（Ｙ３５Ｎ）、Ｔｙｒ３５Ｐｒｏ（Ｙ３５Ｐ）、Ｔｙｒ３５Ａｒｇ（Ｙ３５Ｒ）、Ｔｙｒ３５Ｓｅｒ（Ｙ３５Ｓ）、Ｔｙｒ３５Ｔｈｒ（Ｙ３５Ｔ）、Ｔｙｒ３５Ｖａｌ（Ｙ３５Ｖ）、またはＴｙｒ３５Ｔｒｐ（Ｙ３５Ｗ）のアミノ酸置換が生じたＦｃ結合性タンパク質をコードするポリヌクレオチドを得た。 As a result, Tyr35Cys (Y35C), Tyr35Asp (Y35D), Tyr35Phe (Y35F), Tyr35Gly (Y35G), Tyr35Lys (Y35K), Tyr35Leu (Y35L), Tyr35Asn (Y35N), Tyr35Tr35Yr, YrN ), Tyr35Thr (Y35T), Tyr35Val (Y35V), or Tyr35Trp (Y35W), a polynucleotide encoding an Fc-binding protein in which an amino acid substitution occurred was obtained.
　（Ｃ）配列番号１の１２１番目のグルタミン酸（Ｇｌｕ）を他のアミノ酸に置換したＦｃ結合性タンパク質の作製
（Ｃ－１）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２４および配列番号９９（５’－ＧＴＧＴＴＣＡＡＡＧＡＧＮＮＫＧＡＴＣＣＧＡＴＴＣＡＴＣＴＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を１２１ｐＦとした。
（Ｃ－２）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型とし、配列番号２３および配列番号１００（５’－ＡＡＴＣＧＧＡＴＣＭＮＮＣＴＣＴＴＴＧＡＡＣＡＣＣＣＡＣＣＧ－３’）に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとした他は、実施例４（ａ－１）と同様の方法でＰＣＲを行なった。精製したＰＣＲ産物を１２１ｐＲとした。
（Ｃ－３）（Ｃ－１）および（Ｃ－２）により得られた２種類のＰＣＲ産物（１２１ｐＦ、１２１ｐＲ）を混合後、実施例４（ａ－３）と同様の方法にてＰＣＲを行ない、１２１ｐＦと１２１ｐＲを連結した。得られたＰＣＲ産物を１２１ｐとした。
（Ｃ－４）（Ｃ－３）で得られたＰＣＲ産物１２１ｐを鋳型とし、配列番号２３および配列番号２４に記載の配列からなるオリゴヌクレオチドをＰＣＲプライマーとして実施例４（ａ－４）と同様のＰＣＲを行なった。これにより配列番号１の１２１番目のグルタミン酸が任意のアミノ酸に置換されたＦｃ結合性タンパク質をコードするポリヌクレオチドを作製した。
（Ｃ－５）（Ｃ－４）で得られたポリヌクレオチドを精製後、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した発現ベクターｐＥＴＭａｌＥ（特開２０１１－２０６０４６号公報）にライゲーションし、当該ライゲーション産物を用いて大腸菌ＢＬ２１（ＤＥ３）株を形質転換した。
（Ｃ－６）得られた形質転換体を５０μｇ／ｍＬのカナマイシンを添加したＬＢ培地で培養した。回収した菌体（形質転換体）からプラスミドを抽出し、実施例１（５）と同様の方法でヌクレオチド配列解析を行なった。 (C) Preparation of an Fc binding protein in which the 121st glutamic acid (Glu) of SEQ ID NO: 1 is substituted with another amino acid (C-1) Using pET-eFcR prepared in Example 1 as a template, SEQ ID NO: 24 and PCR was carried out in the same manner as in Example 4 (a-1) except that an oligonucleotide having the sequence described in No. 99 (5′-GTGTTCAAAGAGNKNKATCCGATTCATCTG-3 ′) was used as a PCR primer. The purified PCR product was 121 pF.
(C-2) except that pET-eFcR prepared in Example 1 was used as a template, and an oligonucleotide having the sequences described in SEQ ID NO: 23 and SEQ ID NO: 100 (5′-AATCGGATCMNNCTCTTTGAACACCCCACCG-3 ′) was used as a PCR primer, PCR was performed in the same manner as in Example 4 (a-1). The purified PCR product was 121 pR.
(C-3) After mixing the two kinds of PCR products (121pF, 121pR) obtained by (C-1) and (C-2), PCR was performed in the same manner as in Example 4 (a-3). In practice, 121pF and 121pR were ligated. The obtained PCR product was designated as 121p.
(C-4) The same as in Example 4 (a-4), using the PCR product 121p obtained in (C-3) as a template and the oligonucleotide consisting of the sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 as PCR primers PCR was performed. Thus, a polynucleotide encoding an Fc binding protein in which the 121st glutamic acid of SEQ ID NO: 1 was substituted with an arbitrary amino acid was prepared.
(C-5) After purifying the polynucleotide obtained in (C-4), digested with restriction enzymes NcoI and HindIII, and digested with restriction enzymes NcoI and HindIII into expression vector pETmalE (Japanese Patent Laid-Open No. 2011-206046) After ligation, the ligation product was used to transform E. coli BL21 (DE3) strain.
(C-6) The obtained transformant was cultured in an LB medium supplemented with 50 μg / mL kanamycin. A plasmid was extracted from the collected microbial cells (transformants), and nucleotide sequence analysis was performed in the same manner as in Example 1 (5).
　結果、Ｇｌｕ１２１Ｌｙｓ（Ｅ１２１Ｋ）、Ｇｌｕ１２１Ｐｒｏ（Ｅ１２１Ｐ）、Ｇｌｕ１２１Ａｒｇ（Ｅ１２１Ｒ）、Ｇｌｕ１２１Ｇｌｙ（Ｅ１２１Ｇ）、Ｇｌｕ１２１Ｈｉｓ（Ｅ１２１Ｈ）、またはＧｌｕ１２１Ｖａｌ（Ｅ１２１Ｖ）のアミノ酸置換が生じたＦｃ結合性タンパク質をコードするポリヌクレオチドを得た。 As a result, a polynucleotide encoding an Fc-binding protein in which an amino acid substitution of Glu121Lys (E121K), Glu121Pro (E121P), Glu121Arg (E121R), Glu121Gly (E121G), Glu121His (E121H), or Glu121Val (E121V) occurred was obtained. .
　実施例３８　１アミノ酸置換Ｆｃ結合性タンパク質の抗体結合活性評価
（１）実施例１で作製した野生型Ｆｃ結合性タンパク質、および実施例３７で作製した１箇所アミノ酸置換したＦｃ結合性タンパク質を発現する形質転換体を、実施例３（１）および（２）と同様の方法でそれぞれ培養を行ない、野生型Ｆｃ結合性タンパク質および１アミノ酸置換したＦｃ結合性タンパク質を発現させた。
（２）発現した１アミノ酸置換したＦｃ結合性タンパク質を実施例３（３）および（４）に記載のＥＬＩＳＡ法にて抗体との結合活性を調べた。 Example 38 Evaluation of Antibody Binding Activity of 1 Amino Acid-Substituted Fc Binding Protein (1) Expressing the wild-type Fc binding protein prepared in Example 1 and the Fc binding protein with one amino acid substitution prepared in Example 37 The transformant was cultured in the same manner as in Example 3 (1) and (2), and wild type Fc binding protein and Fc binding protein substituted with 1 amino acid were expressed.
(2) The binding activity of the expressed Fc binding protein with one amino acid substitution was examined by the ELISA method described in Example 3 (3) and (4).
　結果を図１０に示す。配列番号１の２７番目のバリンをグリシン（Ｖ２７Ｇ）、リジン（Ｖ２７Ｋ）、スレオニン（Ｖ２７Ｔ）、アラニン（Ｖ２７Ａ）、アルギニン（Ｖ２７Ｒ）、に置換することで、野生型Ｆｃ結合性タンパク質と比較し抗体結合活性が向上した。一方、配列番号１の２７番目のＶａｌがトリプトファン（Ｖ２７Ｗ）へ置換すると、野生型Ｆｃ結合性タンパク質と比較し抗体結合活性が低下した。 The results are shown in FIG. By replacing the 27th valine of SEQ ID NO: 1 with glycine (V27G), lysine (V27K), threonine (V27T), alanine (V27A), arginine (V27R), antibody compared to the wild type Fc binding protein The binding activity was improved. On the other hand, when the 27th Val in SEQ ID NO: 1 was substituted with tryptophan (V27W), the antibody binding activity was reduced as compared with the wild-type Fc binding protein.
　配列番号１の３５番目のチロシンを、アスパラギン酸（Ｙ３５Ｄ）、フェニルアラニン（Ｙ３５Ｆ）、グリシン（Ｙ３５Ｇ）、リジン（Ｙ３５Ｋ）、ロイシン（Ｙ３５Ｌ）、アスパラギン（Ｙ３５Ｎ）、プロリン（Ｙ３５Ｐ）、セリン（Ｙ３５Ｓ）、スレオニン（Ｙ３５Ｔ）、バリン（Ｙ３５Ｖ）、トリプトファン（Ｙ３５Ｗ）に置換することで、野生型Ｆｃ結合性タンパク質と比較し抗体結合活性が向上した。中でもＹ３５Ｄ、Ｙ３５Ｇ、Ｙ３５Ｋ、Ｙ３５Ｌ、Ｙ３５Ｎ、Ｙ３５Ｐ、Ｙ３５Ｓ、Ｙ３５Ｔ、Ｙ３５Ｗは、野生型Ｆｃ結合性タンパク質に比較し大幅に抗体結合活性が向上した。一方、配列番号１の３５番目のチロシンを、システイン（Ｙ３５Ｃ）、アルギニン（Ｙ３５Ｒ）に置換した場合は、野生型Ｆｃ結合性タンパク質とほぼ同等の抗体結合活性であった。 The 35th tyrosine of SEQ ID NO: 1 is converted to aspartic acid (Y35D), phenylalanine (Y35F), glycine (Y35G), lysine (Y35K), leucine (Y35L), asparagine (Y35N), proline (Y35P), serine (Y35S). By substituting threonine (Y35T), valine (Y35V), and tryptophan (Y35W), the antibody binding activity was improved as compared with the wild-type Fc-binding protein. Among them, Y35D, Y35G, Y35K, Y35L, Y35N, Y35P, Y35S, Y35T, and Y35W have significantly improved antibody binding activity compared to the wild-type Fc-binding protein. On the other hand, when the 35th tyrosine of SEQ ID NO: 1 was substituted with cysteine (Y35C) or arginine (Y35R), the antibody binding activity was almost equivalent to that of the wild type Fc binding protein.
　配列番号１の１２１番目のグルタミン酸を、リジン（Ｅ１２１Ｋ）、アルギニン（Ｅ１２１Ｒ）、グリシン（Ｅ１２１Ｇ）、ヒスチジン（Ｅ１２１Ｈ）に置換することで、野生型Ｆｃ結合性タンパク質と比較し抗体結合活性が向上した。中でもＥ１２１Ｇは、野生型Ｆｃ結合性タンパク質と比較し大幅に抗体結合活性が向上した。一方、配列番号１の１２１番目のグルタミン酸を、バリン（Ｅ１２１Ｖ）に置換した場合は野生型Ｆｃ結合性タンパク質とほぼ同等の抗体結合活性であり、プロリン（Ｅ１２１Ｐ）に置換した場合は野生型Ｆｃ結合性タンパク質と比較し抗体結合活性が低下した。 By replacing the 121st glutamic acid of SEQ ID NO: 1 with lysine (E121K), arginine (E121R), glycine (E121G), and histidine (E121H), the antibody binding activity was improved compared to the wild-type Fc-binding protein. . Among them, E121G showed significantly improved antibody binding activity as compared with wild-type Fc binding protein. On the other hand, when the glutamic acid at position 121 of SEQ ID NO: 1 was substituted with valine (E121V), the antibody-binding activity was almost equivalent to that of wild-type Fc binding protein, and when substituted with proline (E121P), wild-type Fc binding Antibody binding activity was reduced compared to the sex protein.
　実施例３９　Ｆｃ結合性タンパク質発現ベクターの作製
　発現ベクターｐＴｒｃ９９ａに、配列番号１０１（ＭＫＹＬＬＰＴＡＡＡＧＬＬＬＬＡＡＱＰＡＭＡ）に記載のＰｅｌＢシグナルペプチドのうち６番目のプロリン（Ｐ）がセリン（Ｓ）に置換されたシグナルペプチドをコードするポリヌクレオチドを挿入し、シグナルペプチドを含む発現ベクターを作製した。
（１）配列番号１０２（５’－ＣＡＴＧＡＡＡＴＡＣＣＴＧＣＴＧＴＣＧＡＣＣＧＣＴＧＣＴＧＣＴＧＧＴＣＴＧＣＴＧＣＴＣＣＴＣＧＣＴＧＣＣＣＡＧＣＣＧＧＣＧＡＴＧＧＣ－３’）および配列番号１０３（５’－ＣＡＴＧＧＣＣＡＴＣＧＣＣＧＧＣＴＧＧＧＣＡＧＣＧＡＧＧＡＧＣＡＧＣＡＧＡＣＣＡＧＣＡＧＣＡＧＣＧＧＴＣＧＡＣＡＧＣＡＧＧＴＡＴＴＴ－３’）に記載の配列からなるオリゴヌクレオチドを等量混合し、９５℃に５分間加熱後、１分間で１℃毎に温度を下げ、１５℃に達したところで保持することで二本鎖オリゴヌクレオチドを作製した。
（２）（１）で作製した二本鎖オリゴヌクレオチドを、あらかじめ制限酵素ＮｃｏＩで処理した発現ベクターｐＴｒｃ９９ａにライゲーションし、これを用いて大腸菌ＪＭ１０９株（タカラバイオ製）を形質転換した。
（３）得られた形質転換体を１００μｇ／ｍＬのカルベニシリンを含むＬＢ培地により培養後、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて発現ベクターｐＴｒｃ－ＰｅｌＢＶ３を得た。 Example 39 Preparation of Fc-binding protein expression vector The expression vector pTrc99a encodes a signal peptide in which the sixth proline (P) of the PelB signal peptide described in SEQ ID NO: 101 (MKYLLPTAAAGLLLLAAQPAMA) is substituted with serine (S) An expression vector containing a signal peptide was prepared.
(1) SEQ ID NO: 102 (5′-CATGAAATACCTGCTGTCGCACCGCTGCTGCTGTGTCTGCTGCTCCCTCGCTGCCCAGCCGGCGGATGGCGCGCGCGCTCGC The temperature was lowered every 1 ° C. in 1 minute, and a double-stranded oligonucleotide was prepared by holding when the temperature reached 15 ° C.
(2) The double-stranded oligonucleotide prepared in (1) was ligated to the expression vector pTrc99a previously treated with the restriction enzyme NcoI, and this was used to transform Escherichia coli JM109 strain (Takara Bio).
(3) The obtained transformant was cultured in an LB medium containing 100 μg / mL carbenicillin, and an expression vector pTrc-PelBV3 was obtained using QIAprep Spin Miniprep kit (manufactured by Qiagen).
　実施例４０　システインタグを付加したＦｃ結合性タンパク質（ＦｃＲＣｙｓ）の作製
（１）実施例１で作製したｐＥＴ－ｅＦｃＲを鋳型としてＰＣＲを実施した。当該ＰＣＲにおけるプライマーは、配列番号２１および配列番号５７（５’－ＣＣＣＡＡＧＣＴＴＡＴＣＣＧＣＡＧＧＴＡＴＣＧＴＴＧＣＧＧＣＡＣＣＣＴＴＧＧＧＴＡＡＴＧＧＴＡＡＴＡＴＴＣＡＣＧＧＴＣＴＣＧＣＴＧＣ－３’）に記載の配列からなるオリゴヌクレオチドを用いた。ＰＣＲは、表２に示す組成の反応液を調製後、当該反応液を９８℃で５分熱処理し、９８℃で１０秒間の第１ステップ、５５℃で５秒間の第２ステップ、７２℃で１分間の第３ステップを１サイクルとする反応を３０サイクル繰り返すことで実施した。
（２）（１）で得られたポリヌクレオチドを精製し、制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化後、あらかじめ制限酵素ＮｃｏＩとＨｉｎｄＩＩＩで消化した実施例３９で作製の発現ベクターｐＴｒｃ－ＰｅｌＢＶ３にライゲーションし、当該ライゲーション産物を用いて大腸菌Ｗ３１１０株を形質転換した。
（３）得られた形質転換体を１００μｇ／ｍＬのカルベニシリンを含むＬＢ培地にて培養後、ＱＩＡｐｒｅｐ　Ｓｐｉｎ　Ｍｉｎｉｐｒｅｐ　ｋｉｔ（キアゲン製）を用いて、発現ベクターｐＴｒｃ－ｅＦｃＲＣｙｓを得た。
（４）ｐＴｒｃ－ｅＦｃＲＣｙｓのヌクレオチド配列の解析を、配列番号１０４（５’－ＴＧＴＧＧＴＡＴＧＧＣＴＧＴＧＣＡＧＧ－３’）または配列番号１０５（５’－ＴＣＧＧＣＡＴＧＧＧＧＴＣＡＧＧＴＧ－３’）に記載の配列からなるオリゴヌクレオチドをシーケンス用プライマーに使用した以外は、実施例１（５）と同様の方法で行なった。 Example 40 Preparation of Fc Binding Protein (FcRCys) Added with Cysteine Tag (1) PCR was performed using pET-eFcR prepared in Example 1 as a template. As a primer in the PCR, an oligonucleotide having the sequence described in SEQ ID NO: 21 and SEQ ID NO: 57 (5′-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3 ′) was used. In PCR, after preparing a reaction solution having the composition shown in Table 2, the reaction solution was heat-treated at 98 ° C. for 5 minutes, first step at 98 ° C. for 10 seconds, second step at 55 ° C. for 5 seconds, and at 72 ° C. The reaction with the third step of 1 minute as one cycle was repeated 30 times.
(2) The polynucleotide obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrc-PelBV3 prepared in Example 39 previously digested with restriction enzymes NcoI and HindIII. Escherichia coli W3110 was transformed with the ligation product.
(3) The obtained transformant was cultured in an LB medium containing 100 μg / mL carbenicillin, and then an expression vector pTrc-eFcRCys was obtained using QIAprep Spin Miniprep kit (Qiagen).
(4) Analysis of the nucleotide sequence of pTrc-eFcRCys was carried out by using an oligonucleotide consisting of the sequence described in SEQ ID NO: 104 (5′-TGTGGTTATGGCTGTGCAGG-3 ′) or SEQ ID NO: 105 (5′-TCGGCATGGGGTCAGGGTG-3 ′) as a sequencing primer. The procedure was the same as in Example 1 (5) except that the procedure was used for the above.
　発現ベクターｐＴｒｃ－ｅＦｃＲＣｙｓで発現されるポリペプチドのアミノ酸配列を配列番号１０６に、当該ポリペプチドをコードするポリヌクレオチドの配列を配列番号１０７にそれぞれ示す。なお配列番号１０７において、１番目のメチオニン（Ｍｅｔ）から２２番目のアラニン（Ａｌａ）までが６番目のプロリンがセリンに置換されたＰｅｌＢシグナルペプチドであり、２４番目のグリシン（Ｇｌｙ）から１９９番目のグルタミン（Ｇｌｎ）までがＦｃ結合性タンパク質のアミノ酸配列（配列番号１の１７番目から１９２番目までの領域に相当）、２００番目のグリシン（Ｇｌｙ）から２０７番目のグリシン（Ｇｌｙ）までがシステインタグ配列である。 The amino acid sequence of the polypeptide expressed by the expression vector pTrc-eFcRCys is shown in SEQ ID NO: 106, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 107, respectively. In SEQ ID NO: 107, from the first methionine (Met) to the 22nd alanine (Ala) is a PelB signal peptide in which the 6th proline is substituted with serine, and the 199th from the 24th glycine (Gly). Up to glutamine (Gln) is the amino acid sequence of the Fc binding protein (corresponding to the 17th to 192nd region of SEQ ID NO: 1), and the cysteine tag sequence is from the 200th glycine (Gly) to the 207th glycine (Gly). It is.
　実施例４１　ＦｃＲＣｙｓの調製
（１）実施例４０で作製したＦｃＲＣｙｓを発現する形質転換体を２Ｌのバッフルフラスコに入った１００μｇ／ｍＬのカルベニシリンを含む４００ｍＬの２ＹＴ液体培地（ペプトン１６ｇ／Ｌ、酵母エキス１０ｇ／Ｌ、塩化ナトリウム５ｇ／Ｌ）に接種し、３７℃で一晩、好気的に振とう培養することで前培養を行なった。
（２）グルコース１０ｇ／Ｌ、酵母エキス２０ｇ／Ｌ、リン酸三ナトリウム十二水和物３ｇ／Ｌ、リン酸水素二ナトリウム十二水和物９ｇ／Ｌ、塩化アンモニウム１ｇ／Ｌおよび硫酸カナマイシン５０ｍｇ／Ｌを含む液体培地１．８Ｌに、（１）の培養液１８０ｍＬを接種し、３Ｌ発酵槽（バイオット製）を用いて本培養を行なった。温度３０℃、ｐＨ６．９から７．１、通気量１ＶＶＭ、溶存酸素濃度３０％飽和濃度の条件に設定し、本培養を開始した。ｐＨの制御には酸として５０％リン酸、アルカリとして１４％アンモニア水をそれぞれ使用し、溶存酸素の制御は撹拌速度を変化させることで制御し、撹拌回転数は下限５００ｒｐｍ、上限１０００ｒｐｍに設定した。培養開始後、グルコース濃度が測定できなくなった時点で、流加培地（グルコース２４８．９ｇ／Ｌ、酵母エキス８３．３ｇ／Ｌ、硫酸マグネシウム七水和物７．２ｇ／Ｌ）を溶存酸素（ＤＯ）により制御しながら加えた。
（３）菌体量の目安として６００ｎｍの吸光度（ＯＤ６００ｎｍ）が約１５０に達したところで培養温度を２５℃に下げ、設定温度に到達したことを確認した後、終濃度が０．５ｍＭになるようＩＰＴＧを添加し、引き続き２５℃で培養を継続した。
（４）培養開始から約４８時間後に培養を停止し、培養液を４℃で８０００ｒｐｍ、２０分間の遠心分離により菌体を回収した。
（５）回収した菌体を２０ｍＭのトリス塩酸緩衝液（ｐＨ７．０）に５ｍＬ／１ｇ（菌体）となるように懸濁し、超音波発生装置（インソネーター２０１Ｍ（商品名）、久保田商事製）を用いて、４℃で約１０分間、約１５０Ｗの出力で菌体を破砕した。菌体破砕液は４℃で２０分間、８０００ｒｐｍの遠心分離を２回行ない、上清を回収した。
（６）（５）で得られた上清を、あらかじめ２０ｍＭのリン酸緩衝液（８ｍＭリン酸二水素ナトリウム、１２ｍＭリン酸水素二ナトリウム）（ｐＨ７．０）で平衡化した１４０ｍＬのＴＯＹＯＰＥＡＲＬ　ＣＭ－６５０Ｍ（東ソー製）を充填したＶＬ３２×２５０カラム（メルクミリポア製）に流速５ｍＬ／分でアプライした。平衡化に用いた緩衝液で洗浄後、０．５Ｍの塩化ナトリウムを含む２０ｍＭのリン酸緩衝液（ｐＨ７．０）で溶出した。
（７）（６）で得られた溶出液を、あらかじめ１５０ｍＭの塩化ナトリウムを含む２０ｍＭのトリス塩酸緩衝液（ｐＨ７．４）で平衡化したＩｇＧセファロース（ＧＥヘルスケア製）９０ｍＬを充填したＸＫ２６／２０カラム（ＧＥヘルスケア製）にアプライした。平衡化に用いた緩衝液で洗浄後、０．１Ｍのグリシン塩酸緩衝液（ｐＨ３．０）で溶出した。なお溶出液は、溶出液量の１／４量の１Ｍトリス塩酸緩衝液（ｐＨ８．０）を加えることでｐＨを中性付近に戻した。 Example 41 Preparation of FcRCys (1) 400 mL of 2YT liquid medium (peptone 16 g / L, yeast extract) containing 100 μg / mL carbenicillin in a 2 L baffle flask containing the transformant expressing FcRCys produced in Example 40 10 g / L, sodium chloride 5 g / L), and precultured by aerobic shaking culture at 37 ° C. overnight.
(2) Glucose 10 g / L, yeast extract 20 g / L, trisodium phosphate dodecahydrate 3 g / L, disodium hydrogen phosphate dodecahydrate 9 g / L, ammonium chloride 1 g / L and kanamycin sulfate 50 mg The liquid culture medium 1.8L containing / L was inoculated with 180 mL of the culture solution of (1), and main culture was performed using a 3 L fermenter (manufactured by Biot). The main culture was started under the conditions of a temperature of 30 ° C., a pH of 6.9 to 7.1, an aeration rate of 1 VVM, and a dissolved oxygen concentration of 30% saturation. The pH was controlled by using 50% phosphoric acid as the acid and 14% ammonia water as the alkali. The dissolved oxygen was controlled by changing the stirring speed, and the stirring speed was set at the lower limit of 500 rpm and the upper limit of 1000 rpm. . When the glucose concentration could not be measured after the start of culture, fed-batch medium (glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L) was dissolved in dissolved oxygen (DO ) Was added while controlling.
(3) When the absorbance at 600 nm (OD 600 nm) reaches about 150 as a measure of the amount of bacterial cells, the culture temperature is lowered to 25 ° C., and after confirming that the set temperature has been reached, the final concentration is 0.5 mM. IPTG was added, and the culture was continued at 25 ° C.
(4) The culture was stopped about 48 hours after the start of the culture, and the cells were collected by centrifugation at 8000 rpm for 20 minutes at 4 ° C.
(5) The collected cells are suspended in 20 mM Tris-HCl buffer (pH 7.0) so as to be 5 mL / 1 g (cells), and an ultrasonic generator (Insonator 201M (trade name), manufactured by Kubota Corporation) ) Was used to disrupt the cells at an output of about 150 W for about 10 minutes at 4 ° C. The cell disruption solution was centrifuged twice at 8000 rpm for 20 minutes at 4 ° C., and the supernatant was collected.
(6) The supernatant obtained in (5) was previously equilibrated with 20 mM phosphate buffer (8 mM sodium dihydrogen phosphate, 12 mM disodium hydrogen phosphate) (pH 7.0) in 140 mL of TOYOPEARL CM- This was applied to a VL32 × 250 column (Merck Millipore) packed with 650M (Tosoh) at a flow rate of 5 mL / min. After washing with the buffer used for equilibration, elution was performed with 20 mM phosphate buffer (pH 7.0) containing 0.5 M sodium chloride.
(7) XK26 / filled with 90 mL of IgG Sepharose (manufactured by GE Healthcare) equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride in advance. 20 columns (manufactured by GE Healthcare) were applied. After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine hydrochloride buffer (pH 3.0). The eluate was returned to near neutrality by adding 1/4 volume of 1M Tris-HCl buffer (pH 8.0).
　前記精製により、高純度のＦｃＲＣｙｓを約１２ｍｇ得た。 About 12 mg of high-purity FcRCys was obtained by the purification.
　実施例４２　Ｆｃ結合性タンパク質（ＦｃＲ）固定化ゲルの作製と抗体分離
（１）２ｍＬの分離剤用親水性ビニルポリマー（東ソー製：トヨパール）の表面の水酸基をヨードアセチル基で活性化後、実施例４１で調製したＦｃＲＣｙｓを４ｍｇ反応させることにより、ＦｃＲ固定化ゲルを得た。
（２）（１）で作製したＦｃＲ固定化ゲル０．５ｍＬをφ４．６ｍｍ×７５ｍｍのステンレスカラムに充填してＦｃＲカラムを作製した。
（３）（２）で作製したＦｃＲカラムを高速液体クロマトグラフィー装置（東ソー製）につなげ、２０ｍＭの酢酸緩衝液（ｐＨ４．５）で平衡化した。
（４）ＰＢＳ（Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ）（ｐＨ７.４）で４．０ｍｇ／ｍＬに希釈したモノクローナル抗体（リツキサン、全薬工業製）を流速０．３ｍＬ／ｍｉｎにて０．１５ｍＬアプライした。
（５）流速０．３ｍＬ／ｍｉｎのまま平衡化緩衝液で２分洗浄後、１０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）によるｐＨグラジエント（３８分で１０ｍＭのグリシン塩酸緩衝液（ｐＨ３．０）が１００％となるグラジエント）で吸着したモノクローナル抗体を溶出した。 Example 42 Preparation of Fc Binding Protein (FcR) Immobilized Gel and Antibody Separation (1) After activation of the hydroxyl group on the surface of 2 mL of a hydrophilic vinyl polymer for separation agent (Toyopearl: Toyopearl) with an iodoacetyl group An FcR-immobilized gel was obtained by reacting 4 mg of FcRCys prepared in Example 41.
(2) An FcR column was prepared by filling 0.5 mL of the FcR-immobilized gel prepared in (1) into a φ4.6 mm × 75 mm stainless steel column.
(3) The FcR column prepared in (2) was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation), and equilibrated with 20 mM acetate buffer (pH 4.5).
(4) A monoclonal antibody (Rituxan, manufactured by Zenyaku Kogyo Co., Ltd.) diluted to 4.0 mg / mL with PBS (Phosphate Buffered Saline) (pH 7.4) was applied at 0.15 mL at a flow rate of 0.3 mL / min.
(5) After washing with equilibration buffer for 2 minutes with a flow rate of 0.3 mL / min, pH gradient with 10 mM glycine hydrochloride buffer (pH 3.0) (10 mM glycine hydrochloride buffer (pH 3.0) over 38 minutes) The adsorbed monoclonal antibody was eluted with a gradient of 100%.
　結果（溶出パターン）を図１１に示す。モノクローナル抗体はＦｃＲと相互作用するため、ゲルろ過クロマトグラフィーのような単一のピークではなく、複数のピークに分離された。 The results (elution pattern) are shown in FIG. Since the monoclonal antibody interacts with FcR, it was separated into multiple peaks rather than a single peak as in gel filtration chromatography.
　実施例４３　ＦｃＲ固定化ゲルで分離した抗体の抗体依存性細胞傷害（ＡＤＣＣ）活性測定
（１）実施例４２に記載の溶出条件でモノクローナル抗体を分離し、図２に記載の溶出パターン中のフラクションＡ（ＦｒＡ）およびフラクションＢ（ＦｒＢ）の領域を分取した。
（２）分取したＦｒＡおよびＦｒＢを限外ろ過膜（メルクミリポア製）で濃縮しながらＰＢＳ（１０ｍＭリン酸水素二ナトリウム、１．７６ｍＭリン酸二水素カリウム、１３７ｍＭ塩化ナトリウム、２．７ｍＭ塩化カリウム）（ｐＨ７.４）に緩衝液を交換した。
（３）濃縮、緩衝液交換したＦｒＡおよびＦｒＢに含まれる抗体、ならびに分離前のモノクローナル抗体の濃度を２８０ｎｍの吸光で測定した。
（４）以下に示す方法で、ＦｒＡおよびＦｒＢに含まれる抗体ならびに分離前のモノクローナル抗体が有するＡＤＣＣ活性を測定した。
（４－１）１．４ｍＬのＬｏｗ　ＩｇＧ　Ｓｅｒｕｍと３３．６ｍＬのＲＰＭＩ１６４０培地とを混合して調製したＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒを用いてＦｒＡ、ＦｒＢに含まれるモノクローナル抗体ならびに分離前のモノクローナル抗体を３μｇ／ｍＬから１／３希釈で８段階の希釈系列を調製した。
（４－２）Ｒａｊｉ細胞をＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒにて約５×１０５ｃｅｌｌｓ／ｍＬに調製し、９６ウェルプレート（３９１７：コーニング社）に２５μＬ／ｗｅｌｌで加えた。
（４－３）Ｒａｊｉ細胞を加えたｗｅｌｌに（２）で調整したＦｒＡ、ＦｒＢ、分離前のモノクローナル抗体、ブランクのＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒのみを２５μＬ／ｗｅｌｌ加えた。
（４－４）Ｅｆｆｅｃｔｏｒ細胞（プロメガ社）をＡＤＣＣ　Ａｓｓａｙ　Ｂｕｆｆｅｒにて約３．０×１０５ｃｅｌｌｓ／ｍＬに調製し、Ｒａｊｉ細胞および抗体を加えたｗｅｌｌに２５μＬ／ｗｅｌｌで加えた。その後、ＣＯ２インキュベーター（５％ＣＯ２、３７℃）に６時間静置した。
（４－５）９６穴プレートを室温で５分から３０分間静置した後、Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ　Ｒｅａｇｅｎｔ（プロメガ製）を７５μＬ／ｗｅｌｌで加えた。室温で３０分反応させたのち、ＧｌｏＭａｘ　Ｍｕｌｔｉ　Ｄｅｔｅｃｔｉｏｎ　Ｓｙｓｔｅｍ（プロメガ社）で発光を測定した。 Example 43 Antibody Dependent Cytotoxicity (ADCC) Activity Measurement of Antibody Separated on FcR-Immobilized Gel (1) Monoclonal antibody was separated under the elution conditions described in Example 42, and fractions in the elution pattern described in FIG. The region of A (FrA) and fraction B (FrB) was fractionated.
(2) PBS (10 mM disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride) while concentrating the fractionated FrA and FrB with an ultrafiltration membrane (Merck Millipore) ) The buffer solution was exchanged to pH 7.4.
(3) The concentrations of the antibody contained in the concentrated and buffer exchanged FrA and FrB and the monoclonal antibody before separation were measured by absorbance at 280 nm.
(4) The ADCC activity of the antibody contained in FrA and FrB and the monoclonal antibody before separation was measured by the method described below.
(4-1) Using ADCC Assay Buffer prepared by mixing 1.4 mL of Low IgG Serum and 33.6 mL of RPMI1640 medium, 3 μg / mL of the monoclonal antibody contained in FrA and FrB and the monoclonal antibody before separation 8 dilution series were prepared with 1/3 dilution.
(4-2) Raji cells were prepared to about 5 × 10 5 cells / mL with ADCC Assay Buffer and added to a 96-well plate (3917: Corning) at 25 μL / well.
(4-3) Only 25 μL / well of FrA and FrB prepared in (2), the monoclonal antibody before separation, and blank ADCC Assay Buffer were added to the well to which Raji cells were added.
(4-4) Effector cells (Promega) were prepared to about 3.0 × 10 5 cells / mL using ADCC Assay Buffer, and added to wells containing Raji cells and antibodies at 25 μL / well. Then, it was left still for 6 hours in a CO 2 incubator (5% CO 2 , 37 ° C.).
(4-5) After allowing the 96-well plate to stand at room temperature for 5 to 30 minutes, Luciferase Assay Reagent (manufactured by Promega) was added at 75 μL / well. After reacting at room temperature for 30 minutes, luminescence was measured with GloMax Multi Detection System (Promega).
　実施例４２に記載の溶出条件で分取したＦｒＡおよびＦｒＢならびに分離前のモノクローナル抗体の発光強度を比較した結果を図１２に示す。なお図１２の結果は、測定した発光強度からブランクの発光強度を引いた値を示しており、発光強度が高いほど、ＡＤＣＣ活性が高いことを意味している。 FIG. 12 shows the results of comparing the luminescence intensities of FrA and FrB fractionated under the elution conditions described in Example 42 and the monoclonal antibody before separation. The result of FIG. 12 shows a value obtained by subtracting the blank emission intensity from the measured emission intensity. The higher the emission intensity, the higher the ADCC activity.
　分離前のモノクローナル抗体と比較し、ＦｒＡの発光強度は低下している一方、ＦｒＢの発光強度は約１．４倍に向上していた。つまり、ＦｒＢは分離前のモノクローナル抗体およびＦｃＡと比べてＡＤＣＣ活性が高いことがわかる。またＦｃＲ固定化ゲルからの溶出が遅い（カラムに保持される時間が長い）フラクション（ＦｒＢ）にＡＤＣＣ活性の強い抗体が含まれていることから、ＦｃＲ固定化ゲルはＡＤＣＣ活性の強さに基づいて分離できることがわかる。 Compared with the monoclonal antibody before separation, the emission intensity of FrA was decreased, while the emission intensity of FrB was improved about 1.4 times. That is, FrB has higher ADCC activity than the monoclonal antibody and FcA before separation. In addition, since an antibody with strong ADCC activity is contained in the fraction (FrB), which is eluted slowly from the FcR-immobilized gel (long time held in the column), the FcR-immobilized gel is based on the strength of ADCC activity. Can be separated.
　なお、２０１４年６月２７日に出願された特願２０１４－１３３１８１号、２０１４年７月１７日に出願された特願２０１４－１４７２０６号、２０１４年７月１７日に出願された特願２０１４－１４７２０７号、２０１４年１２月２５日に出願された特願２０１４－２６３４０７号、２０１５年３月１０日に出願された特願２０１５－０４７４６２号、ならびに２０１５年６月５日に出願された特願２０１５－１１５０７８号の明細書、配列表、特許請求の範囲、図面及び要約書の全内容をここに引用し、本発明の明細書の開示として、取り入れるものとする。 Japanese Patent Application No. 2014-133181 filed on June 27, 2014, Japanese Patent Application No. 2014-147206 filed on July 17, 2014, Japanese Patent Application No. 2014-2014 filed on July 17, 2014 No. 147207, Japanese Patent Application No. 2014-263407 filed on December 25, 2014, Japanese Patent Application No. 2015-047462 filed on Mar. 10, 2015, and Japanese Patent Application filed on June 5, 2015 The entire contents of the specification, sequence listing, claims, drawings and abstract of 2015-115078 are hereby incorporated by reference as the disclosure of the specification of the present invention.

Claims (17)

  1. 配列番号３７に記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含み、かつ当該３３番目から２０８番目までのアミノ酸残基において以下の（１）から（８４）のうち少なくともいずれか１つのアミノ酸置換が生じている、Ｆｃ結合性タンパク質。
    （１）配列番号３７の４５番目のフェニルアラニンがイソロイシンまたはロイシンに置換
    （２）配列番号３７の５５番目のグルタミン酸がグリシンに置換
    （３）配列番号３７の６４番目のグルタミンがアルギニンに置換
    （４）配列番号３７の６７番目のチロシンがセリンに置換
    （５）配列番号３７の７７番目のフェニルアラニンがチロシンに置換
    （６）配列番号３７の９３番目のアスパラギン酸がグリシンに置換
    （７）配列番号３７の９８番目のアスパラギン酸がグルタミン酸に置換
    （８）配列番号３７の１０６番目のグルタミンがアルギニンに置換
    （９）配列番号３７の１２８番目のグルタミンがロイシンに置換
    （１０）配列番号３７の１３３番目のバリンがグルタミン酸に置換
    （１１）配列番号３７の１３５番目のリジンがアスパラギンまたはグルタミン酸に置換
    （１２）配列番号３７の１５６番目のスレオニンがイソロイシンに置換
    （１３）配列番号３７の１５８番目のロイシンがグルタミンに置換
    （１４）配列番号３７の１８７番目のフェニルアラニンがセリンに置換
    （１５）配列番号３７の１９１番目のロイシンがアルギニンに置換
    （１６）配列番号３７の１９６番目のアスパラギンがセリンに置換
    （１７）配列番号３７の２０４番目のイソロイシンがバリンに置換
    （１８）配列番号３７の３４番目のメチオニンがイソロイシン、リジンまたはスレオニンに置換
    （１９）配列番号３７の３７番目のグルタミン酸がグリシンまたはリジンに置換
    （２０）配列番号３７の３９番目のロイシンがメチオニンまたはアルギニンに置換
    （２１）配列番号３７の４９番目のグルタミンがプロリンに置換
    （２２）配列番号３７の６２番目のリジンがイソロイシンまたはグルタミン酸に置換
    （２３）配列番号３７の６４番目のグルタミンがトリプトファンに置換
    （２４）配列番号３７の６７番目のチロシンがヒスチジンまたはアスパラギンに置換
    （２５）配列番号３７の７０番目のグルタミン酸がグリシンまたはアスパラギン酸に置換
    （２６）配列番号３７の７２番目のアスパラギンがセリンまたはイソロイシンに置換
    （２７）配列番号３７の７７番目のフェニルアラニンがロイシンに置換
    （２８）配列番号３７の８０番目のグルタミン酸がグリシンに置換
    （２９）配列番号３７の８１番目のセリンがアルギニンに置換
    （３０）配列番号３７の８３番目のイソロイシンがロイシンに置換
    （３１）配列番号３７の８４番目のセリンがプロリンに置換
    （３２）配列番号３７の８５番目のセリンがアスパラギンに置換
    （３３）配列番号３７の８７番目のアラニンがスレオニンに置換
    （３４）配列番号３７の９０番目のチロシンがフェニルアラニンに置換
    （３５）配列番号３７の９１番目のフェニルアラニンがアルギニンに置換
    （３６）配列番号３７の９３番目のアスパラギン酸がバリンまたはグルタミン酸に置換
    （３７）配列番号３７の９４番目のアラニンがグルタミン酸に置換
    （３８）配列番号３７の９７番目のバリンがメチオニンとグルタミン酸に置換
    （３９）配列番号３７の９８番目のアスパラギン酸がアラニンに置換
    （４０）配列番号３７の１０２番目のグルタミン酸がアスパラギン酸に置換
    （４１）配列番号３７の１０６番目のグルタミンがロイシンに置換
    （４２）配列番号３７の１０９番目のロイシンがグルタミンに置換
    （４３）配列番号３７の１１７番目のグルタミンがロイシンに置換
    （４４）配列番号３７の１１９番目のグルタミン酸がバリンに置換
    （４５）配列番号３７の１２１番目のヒスチジンがアルギニンに置換
    （４６）配列番号３７の１３０番目のプロリンがロイシンに置換
    （４７）配列番号３７の１３５番目のリジンがチロシンに置換
    （４８）配列番号３７の１３６番目のグルタミン酸がバリンに置換
    （４９）配列番号３７の１４１番目のヒスチジンがグルタミンに置換
    （５０）配列番号３７の１４６番目のセリンがスレオニンに置換
    （５１）配列番号３７の１５４番目のリジンがアルギニンに置換
    （５２）配列番号３７の１５９番目のグルタミンがヒスチジンに置換
    （５３）配列番号３７の１６３番目のグリシンがバリンに置換
    （５４）配列番号３７の１６５番目のリジンがメチオニンに置換
    （５５）配列番号３７の１６７番目のフェニルアラニンがチロシンに置換
    （５６）配列番号３７の１６９番目のヒスチジンがチロシンに置換
    （５７）配列番号３７の１７４番目のチロシンがフェニルアラニンに置換
    （５８）配列番号３７の１７７番目のリジンがアルギニンに置換
    （５９）配列番号３７の１８５番目のセリンがグリシンに置換
    （６０）配列番号３７の１９４番目のセリンがアルギニンに置換
    （６１）配列番号３７の１９６番目のアスパラギンがリジンに置換
    （６２）配列番号３７の２０１番目のスレオニンがアラニンに置換
    （６３）配列番号３７の２０３番目のアスパラギンがイソロイシンまたはリジンに置換
    （６４）配列番号３７の２０７番目のスレオニンがアラニンに置換
    （６５）配列番号３７の９４番目のアラニンがセリンに置換
    （６６）配列番号３７の９８番目のアスパラギン酸がグルタミン酸に置換
    （６７）配列番号３７の１１７番目のグルタミンがアルギニンに置換
    （６８）配列番号３７の１７４番目のチロシンがヒスチジンに置換
    （６９）配列番号３７の１８１番目のリジンがグルタミン酸に置換
    （７０）配列番号３７の２０３番目のアスパラギンがアスパラギン酸またはチロシンに置換
    （７１）配列番号３７の５６番目のリジンがグルタミンに置換
    （７２）配列番号３７の６２番目のリジンがアスパラギンに置換
    （７３）配列番号３７の６６番目のアラニンがスレオニンに置換
    （７４）配列番号３７の７２番目のアスパラギンがチロシンに置換
    （７５）配列番号３７の７８番目のヒスチジンがロイシンに置換
    （７６）配列番号３７の８１番目のセリンがグリシンに置換
    （７７）配列番号３７の９０番目のチロシンがヒスチジンに置換
    （７８）配列番号３７の１３８番目のアスパラギン酸がグルタミン酸に置換
    （７９）配列番号３７の１５３番目のヒスチジンがグルタミンに置換
    （８０）配列番号３７の１５６番目のスレオニンがアラニン、アルギニン、ロイシン、リジン、フェニルアラニン、セリン、バリンまたはメチオニンに置換
    （８１）配列番号３７の１５７番目のチロシンがフェニルアラニンに置換
    （８２）配列番号３７の１７４番目のチロシンがロイシン、システイン、イソロイシン、リジン、トリプトファンまたはバリンに置換
    （８３）配列番号３７の２０６番目のイソロイシンがバリンに置換
    （８４）配列番号３７の２０７番目のスレオニンがイソロイシンに置換     The amino acid sequence of SEQ ID NO: 37 comprises the amino acid residues from the 33rd to the 208th, and the amino acid residues from the 33rd to the 208th are at least any one of the following (1) to (84) An Fc binding protein in which one amino acid substitution has occurred.
    (1) 45th phenylalanine of SEQ ID NO: 37 is replaced with isoleucine or leucine (2) 55th glutamic acid of SEQ ID NO: 37 is replaced with glycine (3) 64th glutamine of SEQ ID NO: 37 is replaced with arginine (4) The 67th tyrosine of SEQ ID NO: 37 is replaced with serine (5) The 77th phenylalanine of SEQ ID NO: 37 is replaced with tyrosine (6) The 93rd aspartic acid of SEQ ID NO: 37 is replaced with glycine (7) of SEQ ID NO: 37 98th aspartic acid replaced with glutamic acid (8) 106th glutamine of SEQ ID NO: 37 replaced with arginine (9) 128th glutamine of SEQ ID NO: 37 replaced with leucine (10) 133rd valine of SEQ ID NO: 37 Is replaced with glutamic acid (11) The 135th lysine of SEQ ID NO: 37 is asparagine Or substituted with glutamic acid (12) 156th threonine of SEQ ID NO: 37 replaced with isoleucine (13) 158th leucine of SEQ ID NO: 37 replaced with glutamine (14) 187th phenylalanine of SEQ ID NO: 37 replaced with serine (15) 191st leucine of SEQ ID NO: 37 is replaced with arginine (16) 196th asparagine of SEQ ID NO: 37 is replaced with serine (17) 204th isoleucine of SEQ ID NO: 37 is replaced with valine (18) SEQ ID NO: 37th methionine of 37 was replaced with isoleucine, lysine or threonine (19) 37th glutamic acid of SEQ ID NO: 37 was replaced with glycine or lysine (20) 39th leucine of SEQ ID NO: 37 was replaced with methionine or arginine (21 49th glutamine of SEQ ID NO: 37 Replacement with proline (22) Replacement of 62nd lysine of SEQ ID NO: 37 with isoleucine or glutamic acid (23) Replacement of 64th glutamine of SEQ ID NO: 37 with tryptophan (24) Replacement of 67th tyrosine of SEQ ID NO: 37 with histidine or asparagine (25) The 70th glutamic acid of SEQ ID NO: 37 is replaced with glycine or aspartic acid (26) The 72nd asparagine of SEQ ID NO: 37 is replaced with serine or isoleucine (27) The 77th phenylalanine of SEQ ID NO: 37 is leucine (28) The 80th glutamic acid of SEQ ID NO: 37 is replaced with glycine (29) The 81st serine of SEQ ID NO: 37 is replaced with arginine (30) The 83rd isoleucine of SEQ ID NO: 37 is replaced with leucine (31) 84th serine of SEQ ID NO: 37 Replacement with proline (32) Replacement of 85th serine of SEQ ID NO: 37 with asparagine (33) Replacement of 87th alanine of SEQ ID NO: 37 with threonine (34) Replacement of 90th tyrosine of SEQ ID NO: 37 with phenylalanine (35 ) The 91st phenylalanine of SEQ ID NO: 37 is replaced with arginine (36) The 93rd aspartic acid of SEQ ID NO: 37 is replaced with valine or glutamic acid (37) The 94th alanine of SEQ ID NO: 37 is replaced with glutamic acid (38) The 97th valine of No. 37 is substituted with methionine and glutamic acid (39) The 98th aspartic acid of SEQ ID NO: 37 is substituted with alanine (40) The 102nd glutamic acid of SEQ ID NO: 37 is substituted with aspartic acid (41) 37 of 106th glutamine replaced by leucine (42) The 109th leucine in column No. 37 is replaced with glutamine (43) The 117th glutamine in SEQ ID NO: 37 is replaced with leucine (44) The 119th glutamic acid in SEQ ID NO: 37 is replaced with valine (45) 121 of SEQ ID NO: 37 The histidine is replaced with arginine (46) The 130th proline of SEQ ID NO: 37 is replaced with leucine (47) The 135th lysine of SEQ ID NO: 37 is replaced with tyrosine (48) The 136th glutamic acid of SEQ ID NO: 37 is valine (49) The 141st histidine of SEQ ID NO: 37 is replaced with glutamine (50) The 146th serine of SEQ ID NO: 37 is replaced with threonine (51) The 154th lysine of SEQ ID NO: 37 is replaced with arginine (52) The 159th glutamine of SEQ ID NO: 37 was replaced with histidine (53) SEQ ID NO: 3 The 163rd glycine of SEQ ID NO: 37 is replaced with methineine (55) the 167th phenylalanine of SEQ ID NO: 37 is replaced with tyrosine (56) the 169th histidine of SEQ ID NO: 37 (57) 174th tyrosine of SEQ ID NO: 37 replaced with phenylalanine (58) 177th lysine of SEQ ID NO: 37 replaced with arginine (59) 185th serine of SEQ ID NO: 37 replaced with glycine ( 60) 194th serine of SEQ ID NO: 37 is replaced with arginine (61) 196th asparagine of SEQ ID NO: 37 is replaced with lysine (62) 201st threonine of SEQ ID NO: 37 is replaced with alanine (63) SEQ ID NO: 37 The asparagine at position 203 is substituted with isoleucine or lysine (64) 207 threonine of column number 37 is replaced with alanine (65) 94th alanine of SEQ ID NO: 37 is replaced with serine (66) 98th aspartic acid of SEQ ID NO: 37 is replaced with glutamic acid (67) 117th glutamine is replaced with arginine (68) 174th tyrosine of SEQ ID NO: 37 is replaced with histidine (69) 181st lysine of SEQ ID NO: 37 is replaced with glutamic acid (70) 203rd asparagine of SEQ ID NO: 37 is Substitution with aspartic acid or tyrosine (71) Replacement of 56th lysine of SEQ ID NO: 37 with glutamine (72) Replacement of 62nd lysine of SEQ ID NO: 37 with asparagine (73) Replacement of 66th alanine of SEQ ID NO: 37 with threonine Substitution (74) Asparagine at position 72 in SEQ ID NO: 37 replaced with tyrosine 75) 78th histidine of SEQ ID NO: 37 is replaced with leucine (76) 81st serine of SEQ ID NO: 37 is replaced with glycine (77) 90th tyrosine of SEQ ID NO: 37 is replaced with histidine (78) SEQ ID NO: 37 138th aspartic acid is replaced with glutamic acid (79) 153rd histidine of SEQ ID NO: 37 is replaced with glutamine (80) 156th threonine of SEQ ID NO: 37 is alanine, arginine, leucine, lysine, phenylalanine, serine, valine Alternatively, methionine is substituted (81) 157th tyrosine of SEQ ID NO: 37 is replaced with phenylalanine (82) 174th tyrosine of SEQ ID NO: 37 is replaced with leucine, cysteine, isoleucine, lysine, tryptophan or valine (83) SEQ ID NO: 37 The 206th isoro Substituted Singh is 207th threonine substitution valine (84) SEQ ID NO: 37 with isoleucine
  2. 配列番号３９、配列番号４３、配列番号４７、配列番号５１、配列番号５５、配列番号６３、配列番号６７、配列番号６９、配列番号７３、配列番号７７、配列番号８３、配列番号８９のいずれかに記載のアミノ酸配列のうち３３番目から２０８番目までのアミノ酸残基を含む、請求項１に記載のＦｃ結合性タンパク質。 Sequence number 39, sequence number 43, sequence number 47, sequence number 51, sequence number 55, sequence number 63, sequence number 67, sequence number 69, sequence number 73, sequence number 77, sequence number 83, or sequence number 89 The Fc-binding protein according to claim 1, comprising amino acid residues from the 33rd to the 208th in the amino acid sequence described in 1.
  3. 配列番号３９、配列番号４３、配列番号４７、配列番号５１、配列番号５５、配列番号６３、配列番号６７、配列番号６９、配列番号７３、配列番号７７、配列番号８３、配列番号８９のいずれかに記載のアミノ酸配列からなる、請求項２に記載のＦｃ結合性タンパク質。 Sequence number 39, sequence number 43, sequence number 47, sequence number 51, sequence number 55, sequence number 63, sequence number 67, sequence number 69, sequence number 73, sequence number 77, sequence number 83, or sequence number 89 The Fc-binding protein according to claim 2, comprising the amino acid sequence according to claim 2.
  4. 請求項１に記載のＦｃ結合性タンパク質において、さらに以下の（８５）から（８８）のうち少なくともいずれか１つのアミノ酸置換が生じている、Ｆｃ結合性タンパク質。
    （８５）配列番号３７の８２番目のロイシンがヒスチジンまたはアルギニンに置換
    （８６）配列番号３７の１６３番目のグリシンがアスパラギン酸に置換
    （８７）配列番号３７の１７４番目のチロシンがヒスチジンに置換
    （８８）配列番号３７の１９２番目のバリンがフェニルアラニンに置換     The Fc-binding protein according to claim 1, wherein at least one of the following (85) to (88) is substituted.
    (85) 82nd leucine of SEQ ID NO: 37 was replaced with histidine or arginine (86) 163rd glycine of SEQ ID NO: 37 was replaced with aspartic acid (87) 174th tyrosine of SEQ ID NO: 37 was replaced with histidine (88 ) The 192nd valine of SEQ ID NO: 37 is substituted with phenylalanine.
  5. 請求項１から４のいずれかに記載のＦｃ結合性タンパク質を不溶性担体に固定化して得られる吸着剤。 An adsorbent obtained by immobilizing the Fc-binding protein according to any one of claims 1 to 4 on an insoluble carrier.
  6. 請求項５に記載の吸着剤を充填したカラムに平衡化液を添加してカラムを平衡化する工程と、前記平衡化したカラムに抗体を含む溶液を添加して前記抗体を前記担体に吸着させる工程と、前記担体に吸着した抗体を溶出液を用いて溶出させる工程とを含む、抗体の分離方法。 6. A step of adding an equilibration solution to the column packed with the adsorbent according to claim 5 to equilibrate the column; and adding a solution containing an antibody to the equilibrated column to adsorb the antibody to the carrier. A method for separating an antibody, comprising: a step; and a step of eluting the antibody adsorbed on the carrier using an eluent.
  7. 平衡化液が３０ｍＭ以上の塩化物イオンまたは硫酸イオンを含む、請求項６に記載の分離方法。 The separation method according to claim 6, wherein the equilibration liquid contains chloride ions or sulfate ions of 30 mM or more.
  8. 請求項５に記載の吸着剤を用いた、抗体依存性細胞傷害活性の強さに基づき抗体を分離する方法。 A method for separating antibodies based on the strength of antibody-dependent cytotoxic activity using the adsorbent according to claim 5.
  9. 請求項６から８のいずれかに記載の分離方法で得られる抗体。 An antibody obtained by the separation method according to any one of claims 6 to 8.
  10. 請求項５に記載の吸着剤を用いて抗体を分離することで、抗体が有する糖鎖構造の違いを識別する方法。 A method for identifying a difference in sugar chain structure of an antibody by separating the antibody using the adsorbent according to claim 5.
  11. 請求項５に記載の吸着剤を用いた糖鎖の分離方法。 A method for separating sugar chains using the adsorbent according to claim 5.
  12. 請求項１１に記載の分離方法で得られる糖鎖。 A sugar chain obtained by the separation method according to claim 11.
  13. 請求項１から４のいずれかに記載のＦｃ結合性タンパク質をコードするポリヌクレオチド。 A polynucleotide encoding the Fc-binding protein according to any one of claims 1 to 4.
  14. 請求項１３に記載のポリヌクレオチドを含む発現ベクター。 An expression vector comprising the polynucleotide according to claim 13.
  15. 請求項１４に記載の発現ベクターで宿主を形質転換して得られる形質転換体。 A transformant obtained by transforming a host with the expression vector according to claim 14.
  16. 宿主が大腸菌である、請求項１５に記載の形質転換体。 The transformant according to claim 15, wherein the host is Escherichia coli.
  17. 請求項１５または１６に記載の形質転換体を培養することによりＦｃ結合性タンパク質を発現させ、その培養物から発現されたＦｃ結合性タンパク質を回収する、Ｆｃ結合性タンパク質の製造方法。 A method for producing an Fc-binding protein, wherein the Fc-binding protein is expressed by culturing the transformant according to claim 15 or 16, and the Fc-binding protein expressed from the culture is recovered.
PCT/JP2015/068259 2014-06-27 2015-06-24 IMPROVED Fc-BINDING PROTEIN, METHOD FOR PRODUCING SAID PROTEIN, ANTIBODY ADSORBENT USING SAID PROTEIN, AND METHOD FOR SEPARATING ANTIBODY USING SAID ADSORBENT WO2015199154A1 (en)

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WO2018198817A1 (en) * 2017-04-26 2018-11-01 東ソー株式会社 Stable fc binding protein, method for producing said protein, and antibody adsorbent in which said protein is used
WO2019083048A1 (en) 2017-10-27 2019-05-02 東ソー株式会社 Fc-BINDING PROTEIN EXHIBITING IMPROVED ALKALINE RESISTANCE, METHOD FOR PRODUCING SAID PROTEIN, ANTIBODY ADSORBENT USING SAID PROTEIN, AND METHOD FOR SEPARATING ANTIBODY USING SAID ANTIBODY ADSORBENT
WO2019163919A1 (en) * 2018-02-22 2019-08-29 東ソー株式会社 Fc-binding protein having improved acid stability, production method for said protein, and antibody-adsorbing agent using said protein
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JP2020028285A (en) * 2018-02-22 2020-02-27 東ソー株式会社 Fc binding protein with improved acid stability, method for producing protein and antibody adsorbent using protein
JP2020125280A (en) * 2018-08-06 2020-08-20 東ソー株式会社 Antibody-adsorbing agents comprising an immobilized fc-binding protein, and methods for separating antibodies using the same

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