WO2018180204A1 - 安定性改良型免疫グロブリン結合性ペプチド - Google Patents
安定性改良型免疫グロブリン結合性ペプチド Download PDFInfo
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- WO2018180204A1 WO2018180204A1 PCT/JP2018/008050 JP2018008050W WO2018180204A1 WO 2018180204 A1 WO2018180204 A1 WO 2018180204A1 JP 2018008050 W JP2018008050 W JP 2018008050W WO 2018180204 A1 WO2018180204 A1 WO 2018180204A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/34—Regenerating or reactivating
- B01J20/345—Regenerating or reactivating using a particular desorbing compound or mixture
- B01J20/3475—Regenerating or reactivating using a particular desorbing compound or mixture in the liquid phase
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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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- the present invention relates to an immunoglobulin-binding peptide with improved chemical stability against an alkaline solution, an affinity separation matrix having the peptide as a ligand, a method for producing an antibody or antibody fragment using the affinity separation matrix, and the peptide encoding the peptide
- the present invention relates to DNA, a vector containing the DNA, and a transformant transformed with the vector.
- a protein A affinity separation matrix (hereinafter referred to as “SpA”) is used for purifying (capturing) antibody drugs from animal cell cultures at a high purity at a time. (May be abbreviated as ")" (Non-Patent Documents 1 and 2).
- SpA protein A affinity separation matrix
- Monoclonal antibodies are basically developed as antibody drugs, and are produced in large quantities using recombinant cultured cell technology.
- “Monoclonal antibody” refers to an antibody obtained from a clone derived from a single antibody-producing cell.
- Most antibody drugs currently on the market are immunoglobulin G (IgG) subclass in terms of molecular structure.
- SpG protein G
- group G streptococci Streptococcus sp.
- SpG affinity separation matrix product immobilized as a ligand (manufactured by GE Healthcare, product name “Protein-G Sepharose 4 Fast Flow”, Patent Document 1).
- SpG is characterized in that it binds strongly to the Fc region of IgG and binds strongly to IgG of a wider range of animal species than SpA.
- Non-Patent Documents 3 and 4 it has been found that it binds to the Fab region even though it is weak (Non-Patent Documents 3 and 4), and efforts have been made to improve the binding force to the Fab region (Patent Documents 2 to 4, Non-Patent Document 5). .
- the SpA affinity separation matrix is more widely used for purification of antibody drugs, and one of the reasons is that SpA has higher stability to alkaline solution than SpG (non-patented). Reference 1). If the stability against alkali is high, the affinity separation matrix can be regenerated and reused by washing with an aqueous sodium hydroxide solution that has a high cleaning and sterilizing effect and is inexpensive.
- protein engineering techniques such as introducing amino acid mutations. Specifically, amino acid substitution mutations to asparagine residues known to be susceptible to deamidation reactions under alkaline conditions and glycine residues after asparagine residues are effective in improving chemical stability.
- Non-Patent Document 1 For all asparagine residues in SpA, such mutations do not show an improvement effect (Non-patent Documents 1 and 6).
- Non-patent Documents 7 and 8 studies have been made to introduce mutations into asparagine residues (Patent Document 5, Non-Patent Documents 7 and 8), but not all mutations are effective as in SpA. In addition, there is still room for improvement because the alkali stability comparable to that of SpA has not been achieved.
- Novel modified protein G which shows binding to immunoglobulin Fc region and Fab region, and has excellent chemical stability to alkaline solution, and affinity separation matrix having modified protein G as a ligand, and the affinity It is an object of the present invention to provide a method for producing an antibody or antibody fragment using a separation matrix.
- the inventor molecularly designed a variant of the IgG binding domain of SpG, and obtained and obtained the variant from a transformed cell using a protein engineering technique and a genetic engineering technique. A study was conducted to compare the physical properties of the mutants.
- the present invention will be described.
- a modified immunoglobulin-binding peptide according to any one of (1) to (3) below.
- Immunoglobulin-binding having an amino acid sequence in which the amino acid residue at position 8 is substituted with Asp, Glu, His, Ile, Lys, Leu, or Val in any one of SEQ ID NOs: 1 to 5 peptide;
- an immunoglobulin binding having an amino acid sequence in which one or several amino acids are further deleted, substituted and / or added in the region excluding the eighth position.
- Sex peptides (3) an immunoglobulin-binding peptide having an amino acid sequence having 80% or more sequence identity to the amino acid sequence defined in (1) above (provided that the 8th in the amino acid sequence defined in (1) above) The substituted amino acid residue at the position shall not be further mutated in (3)).
- amino acid sites to be deleted, substituted and / or added are the 6th, 7th, 13th, 15th, 18th positions. 19th, 21st, 24th, 28th, 29th, 30th, 31st, 33rd, 35th, 39th, 40th, 42nd and The modified immunoglobulin-binding peptide according to any one of the above [1] to [2], which is one or more sites selected from the group consisting essentially of position 47.
- a modified immunoglobulin-binding peptide multimer comprising a plurality of domains obtained by linking two or more modified immunoglobulin-binding peptides according to any one of [1] to [4] above.
- a method for producing a protein comprising an Fc region and / or Fab region of an immunoglobulin A method comprising contacting the affinity separation matrix according to the above [6] with a liquid sample containing the protein, and separating the protein bound to the affinity separation matrix from the affinity separation matrix.
- the affinity purification chromatographic support on which the modified SpG obtained in the present invention is immobilized has a small decrease in immunoglobulin binding activity due to alkali treatment damage. Therefore, in repeated use, cleaning with a sodium hydroxide aqueous solution at a high concentration or for a long time is possible.
- non-patent document 7 shows almost no improvement in alkali resistance in the SpG mutant mutated to Ala.
- Patent Document 8 reports data indicating that the SpG mutant mutated to Gly and Thr exhibits high alkali resistance, but in the present invention, an improvement in alkali resistance exceeding those was observed.
- the modified immunoglobulin-binding peptide according to the present invention is one of the following (1) to (3).
- Immunoglobulin-binding having an amino acid sequence in which the amino acid residue at position 8 is substituted with Asp, Glu, His, Ile, Lys, Leu, or Val in any one of SEQ ID NOs: 1 to 5 peptide;
- an immunoglobulin binding having an amino acid sequence in which one or several amino acids are further deleted, substituted and / or added in the region excluding the eighth position.
- Sex peptides (3) an immunoglobulin-binding peptide having an amino acid sequence having 80% or more sequence identity to the amino acid sequence defined in (1) above (provided that the 8th in the amino acid sequence defined in (1) above) The substituted amino acid residue at the position shall not be further mutated in (3)).
- Immunoglobulin is a glycoprotein produced by B cells of lymphocytes and has a function of recognizing and binding molecules such as specific proteins.
- An immunoglobulin has a function of specifically binding to a specific molecule called an antigen, and a function of detoxifying and removing a factor having an antigen in cooperation with other biomolecules and cells.
- Immunoglobulin is generally called “antibody”, which is a name that focuses on such a function. All immunoglobulins basically have the same molecular structure, and each has a basic structure of a four-chain “Y” -shaped structure composed of two light chain and heavy chain polypeptides.
- Immunoglobulin G is a monomeric immunoglobulin and is composed of two heavy chains ( ⁇ chains) and two light chains, and has two antigen-binding sites.
- the place corresponding to the vertical bar of the lower half of the “Y” of immunoglobulin is called the Fc region, and the “V” of the upper half is called the Fab region.
- the Fc region has an effector function that induces a reaction after the antibody binds to the antigen, and the Fab region has a function of binding to the antigen.
- the heavy chain Fab region and the Fc region are connected by a hinge part, and the proteolytic enzyme papain contained in papaya decomposes this hinge part and cleaves it into two Fab regions and one Fc region.
- the domain near the tip of the “Y” is called a variable region (V region) because various changes in the amino acid sequence are seen so that it can bind to various antigens.
- variable region of the light chain is called the VL region
- variable region of the heavy chain is called the VH region
- the Fab region and the Fc region other than the V region are regions with relatively little change, and are called constant regions (C regions).
- the constant region of the light chain is referred to as the CL region
- the constant region of the heavy chain is referred to as the CH region.
- the CH region is further divided into three, CH1 to CH3.
- the heavy chain Fab region consists of a VH region and CH1, and the heavy chain Fc region consists of CH2 and CH3.
- the hinge part is located between CH1 and CH2.
- peptide includes all molecules having a polypeptide structure, and includes not only so-called proteins, but also fragments and those in which other peptides are linked by peptide bonds. Shall be.
- a “domain” is a unit of protein conformation, consisting of a sequence of tens to hundreds of amino acid residues, sufficient to express some physicochemical or biochemical function.
- a “variant” of a protein or peptide refers to a protein or peptide in which at least one substitution, addition or deletion is introduced at the amino acid level with respect to the sequence of a wild-type protein or peptide.
- the amino acid of a wild type or a non-mutation type is attached
- the mutated amino acid is attached
- N08A a mutation that replaces Asn at position 8 with Ala.
- Protein G is a protein derived from the cell wall of group G streptococcus (Streptococcus sp.). SpG has the ability to bind to most mammalian immunoglobulin G (IgG) and binds strongly to the Fc region of IgG and also weakly binds to the Fab region of IgG. More specifically, the Fc region binds in a form straddling CH2 and CH3, and the Fab region binds centering on the CH1 region (CH1 ⁇ ), and a part also binds to the CL region (Non-patent Document 3). 4). For example, SpG mutants having enhanced binding ability to the Fab region have been developed (Patent Documents 2 to 4, Non-Patent Document 5), and the present invention can be applied to these mutants.
- the functional domain showing SpG IgG binding is called ⁇ domain (SpG- ⁇ ).
- ⁇ domain There are two types of cases, referred to as ⁇ (B) domain and C domain (see Akerstrom et al., J. Biol. Chem., 1987, 28, 13388-, FIG. 5).
- ⁇ domain it is called a ⁇ domain according to the definition of Fahnestok et al. (Fahnestock et al., J. Bacteriol., 1986167, 870-).
- the immunoglobulin-binding peptide according to the present invention binds to the Fc region and Fab region of immunoglobulin.
- the immunoglobulin to be bound by the peptide of the present invention is not particularly limited as long as it contains an Fc region or Fab region, and may be an immunoglobulin G (IgG) containing the Fab region and the Fc region without deficiency, It may be a derivative of IgG.
- a fragment fragmented only in the Fc region or Fab region of immunoglobulin G a chimeric IgG in which a part of the IgG domain is replaced with an IgG domain of another species, and a molecule in the sugar chain of the Fc region
- examples include IgG with modification, IgG with a drug covalently bound, and a fusion protein to which an Fc region or Fab region is added as a tag.
- the amino acid sequence defined in the immunoglobulin-binding peptide (1) is basically an amino acid sequence having a structure / function as SpG- ⁇ .
- the present invention relates to a peptide having a sequence in which a mutation is introduced into wild type SpG- ⁇ .
- the amino acid sequences before the mutation introduction are wild type SpG- ⁇ 1 (SEQ ID NO: 1) and wild type SpG- ⁇ 2 (SEQ ID NO: 2). ).
- the N-terminus of wild-type SpG- ⁇ 1 is Asp, but the N-terminus of SEQ ID NO: 1 is Thr like other domains. Depending on the literature (for example, in Non-Patent Document 7), this N-terminus is not included in the domain, and basically any amino acid is not different.
- WO 97/26930 and JP-A 2003-88381 such as mutant SpG- ⁇ 2 (SEQ ID NO: 3) are known. And the amino acid sequences described in the above. Further, the SpG- ⁇ mutant having an improved Fab binding force described in Patent Documents 2 to 4 is also preferable as an application target of the present invention. For example, SpG- having an amino acid sequence represented by any of SEQ ID NOs: 4 to 5 is preferable. Further variants of ⁇ variants are preferred as Fab binding peptides.
- the modified immunoglobulin-binding peptide (1) in the present invention has an amino acid sequence in which the amino acid residue at the eighth position described above is substituted.
- position 8 is Asn.
- the phrase “having a (specific) amino acid sequence” means that the peptide only needs to contain the specified amino acid sequence, and the function of the peptide is maintained. To do.
- sequences other than the amino acid sequence specified in the peptide include a histidine tag, a linker sequence for immobilization, and a cross-linked structure such as an —SS— bond.
- the position corresponding to position 8 of the amino acid sequences of SEQ ID NOs: 1 to 5 on the basis of sequence identity. Is easily possible.
- the position can be specified using the alignment function of GENETYX (https://www.genetyx.co.jp/), which is genetic information processing software.
- the type of the amino acid after the mutation that is, the amino acid that replaces Asn at the 8th position in the amino acid sequences of SEQ ID NOs: 1 to 5 is not particularly limited, including substitution with a non-protein constituent amino acid or a non-natural amino acid,
- natural amino acids can be preferably used, and from the viewpoint of improving alkali resistance, Asp, Glu, His, Ile, Lys, Leu or Val is preferable. Further, Val, Leu or Lys is more preferable, and Lys is more preferable.
- Natural amino acids are classified into neutral amino acids; acidic amino acids of Asp and Glu; basic amino acids of Lys, Arg, and His.
- Neutral amino acids are classified as aliphatic amino acids; Pro imino acids; Phe, Tyr, Trp aromatic amino acids. Aliphatic amino acids are classified into small amino acids of Gly and Ala; branched amino acids of Val, Leu and Ile; hydroxy amino acids of Ser and Thr; sulfur amino acids of Cys and Met; and acid amide amino acids of Asn and Gln. Further, since Tyr has a phenolic hydroxyl group, it may be classified not only as an aromatic amino acid but also as a hydroxy amino acid.
- natural amino acids can be selected from Gly, Ala, Val, Leu, Ile, Trp, Cys, Met, Pro, Phe and highly hydrophobic nonpolar amino acids; Asn, Gln, Ser, Thr, Tyr. It can also be classified into neutral polar amino acids; acidic polar amino acids of Asp and Glu; basic polar amino acids of Lys, Arg and His.
- the amino acid substituted in the present invention is preferably a branched amino acid, an acidic amino acid or a basic amino acid, and more preferably a branched amino acid or a basic amino acid.
- modified immunoglobulin-binding peptide (2) of the present invention in the amino acid sequence of (1) above, one or several amino acid residues are deleted, substituted and substituted in the region excluding the eighth position.
- the range of “one to several” means that the modified immunoglobulin-binding peptide having a deletion or the like has a high binding force to immunoglobulin.
- the range of “1 to several” can be, for example, 30 or less, preferably 20 or less, more preferably 10 or less, still more preferably 7 or less, even more preferably 5 or less, especially Preferably, it can be 3 or less, 1 or more, 2 or less, and 1 or so.
- examples of the positions for deletion, substitution and / or addition of amino acid residues include the N-terminus and / or C-terminus. These sites are particularly preferred as deletion and / or addition sites.
- An embodiment of the amino acid sequence to be added includes adding an amino acid sequence containing Lys or Cys useful for immobilizing the peptide to a matrix to the C-terminal side.
- the position of the amino acid residue after mutagenesis corresponding to the position of the amino acid residue before mutagenesis is the amino acid before and after mutagenesis. It can be easily searched by performing alignment analysis of sequences. Such an alignment analysis technique is widely known to those skilled in the art as described for the modified immunoglobulin-binding peptide (1).
- the sequence identity is preferably 80% or more or 85% or more, more preferably 90% or more, 95% or more or 98% or more, and 99.5 % Or more or 99.8% or more is even more preferable.
- the sequence identity is measured using Clustal (http://www.clustal.org/omega/), which is an amino acid sequence multiple alignment program, as described for the modified immunoglobulin-binding peptide (1). be able to.
- the substituted amino acid residue at position 8 in the amino acid sequence defined in (1) above is not further mutated in (3). Even when the number of amino acids in the amino acid sequence before mutagenesis is different, it is easy for those skilled in the art to identify the position corresponding to the 8th position of SEQ ID NO: 1 when the sequence identity is 80% or more. Is possible.
- the present invention is a technique for creating a mutant characterized in that the chemical stability with respect to an alkaline aqueous solution is improved by introducing an amino acid substitution mutation as compared with that before the mutagenesis.
- the modified immunoglobulin-binding peptide (1) is less damaged by alkali even when treated with an alkaline aqueous solution, and maintains the immunoglobulin binding performance at a high level.
- the modified immunoglobulin-binding peptides (2) and (3) are also excellent in chemical stability.
- the chemical stability of the modified immunoglobulin-binding peptides (2) and (3) to an alkaline aqueous solution is the chemical stability of the immunoglobulin-binding peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 5. It is preferably superior to stability, and is equivalent to or better than the chemical stability of the modified immunoglobulin-binding peptide (1) having the same mutation at position 8 of SEQ ID NOs: 1 to 5. Is more preferable.
- the binding activity of the modified immunoglobulin-binding peptides (2) and (3) to the Fc region and / or Fab region is also the binding property of the immunoglobulin-binding peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 5. It is preferably superior, and more preferably equivalent or superior to the binding property of the modified immunoglobulin-binding peptide (1).
- Alkaline aqueous solution refers to alkalinity that can achieve the purpose of cleaning or sterilization. More specifically, an aqueous solution of sodium hydroxide of 0.01M to 1.0M or 0.01N to 1.0N is applicable, but not limited thereto.
- the lower limit of the concentration is preferably 10 mM, more preferably 15 mM, more preferably 20 mM, and even more preferably 25 mM.
- the upper limit of the concentration of sodium hydroxide is preferably 1.0M, more preferably 0.5M, even more preferably 0.3M, still more preferably 0.2M, and even more preferably 0.1M.
- the alkaline aqueous solution is not necessarily a sodium hydroxide aqueous solution, but the pH is preferably 12 or more and 14 or less. Regarding the lower limit of pH, 12.0 or more is preferable, and 12.5 or more is more preferable. Regarding the upper limit of the pH, it is preferably 14 or less, more preferably 13.5 or less, and even more preferably 13.0 or less.
- “Chemical stability” refers to the property that a peptide retains the function of the peptide against chemical modifications such as chemical changes of amino acid residues and chemical modifications such as amide bond transfer and cleavage.
- function retention of a peptide refers to binding activity to immunoglobulin.
- immunoglobulin binding activity refers to the proportion of a polypeptide that retains affinity for an antibody or fragment thereof without undergoing chemical denaturation. That is, the higher the “chemical stability” is, the smaller the degree to which the binding activity to the immunoglobulin decreases after the immersion treatment in the alkaline aqueous solution.
- the term “alkali resistance” is also synonymous with “chemical stability under alkaline conditions”.
- the time for immersing the peptide in the alkali is not particularly limited because the damage to the peptide varies greatly depending on the concentration of the alkali and the temperature at the time of immersion.
- the concentration of sodium hydroxide is 15 mM and the temperature during immersion is room temperature
- the lower limit of the time for immersion in alkali is preferably 30 minutes, more preferably 1 hour, more preferably 2 hours, and more than 4 hours.
- 10 hours is more preferable, and 20 hours is more preferable, but there is no particular limitation.
- the binding activity to an antibody or a fragment thereof can be tested with a biosensor such as a Biacore system (GE Healthcare) using the surface plasmon resonance principle, but is not limited thereto.
- a biosensor such as a Biacore system (GE Healthcare) using the surface plasmon resonance principle, but is not limited thereto.
- the temperature is a constant temperature of 20 ° C. or more and 40 ° C. or less
- the pH when the bonding state is seen is a neutral condition of 5 or more and 8 or less.
- the buffer component include, but are not limited to, phosphoric acid, tris, bistris, and the like when neutral.
- the sodium chloride concentration of the buffer solution is not particularly limited, but is preferably about 0 M or more and 0.15 M or less.
- an affinity constant (K A ) or dissociation constant (K D ) can be used as a parameter indicating that it is bound to an antibody or a fragment thereof (Nagata et al., “Real-time analysis of biological substance interaction” Law “, Springer Fairlark Tokyo, 1998, page 41).
- the affinity constant for the antibody or fragment thereof of the peptide of the present invention is determined by immobilizing human IgG or a fragment thereof on a sensor chip using the Biacore system, under the conditions of temperature 25 ° C. and pH 7.4. It can be obtained by an experimental system in which a body is added to a flow path.
- the protein according to the present invention has an affinity constant (K A ) for human immunoglobulin of 1 ⁇ 10 5 (M ⁇ 1 ) or more, more preferably 1 ⁇ 10 6 (M ⁇ 1 ) or more.
- K A affinity constant
- the affinity constant varies depending on the type of immunoglobulin and the number of domains of the immunoglobulin-binding peptide, and is not limited to this.
- K A and the K D is inappropriate. This is because, even when the ratio of molecules capable of binding to the antibody or fragment thereof is changed by alkali treatment, if the binding ability of one peptide molecule to the antibody or fragment thereof does not change, no change is seen as a parameter.
- the antibody or fragment thereof is immobilized on a sensor chip, and the binding signal when the same concentration of antibody or fragment thereof is added before and after chemical treatment of the peptide.
- a binding parameter in units of a resonance unit (RU) indicating the size of the binding response, which is the size or the theoretical maximum binding capacity (R max ), but is not limited thereto.
- the magnitude of the binding signal may be compared by immobilizing the peptide and adding the same concentration of antibody or fragment thereof before and after alkali treatment of the immobilized chip.
- the residual binding activity is a comparison before and after alkali treatment, it can be basically expressed as a ratio (percentage) in which the binding activity before alkali treatment is the denominator and the binding activity after alkali treatment is the molecule.
- the numerical value is not particularly limited as long as it is higher than that of the peptide in which the mutation of the present invention treated with alkali under the same conditions is not introduced, but the ratio is preferably 10% or more, and more preferably 20% or more. Is more preferably 30% or more, still more preferably 40% or more, and even more preferably 50% or more.
- the sample to be compared is different only in that it does not contain the mutation of the present invention, the other amino acid sequences are the same, and the alkali treatment and residual binding activity are measured. It is to make all of the conditions together.
- an appropriate treatment such as neutralizing the pH by neutralization with an acid after the alkali treatment is necessary.
- Protein G is a protein containing two or three immunoglobulin binding domains arranged in tandem.
- the modified immunoglobulin-binding peptide according to the present invention also includes, as one embodiment, two or more, preferably three or more, more preferably, the modified immunoglobulin-binding peptide that is a monomer or a single domain. It may be a multimer of multiple domains linked by 4 or more, more preferably 5 or more. The upper limit of the number of domains connected is, for example, 10, preferably 8, and more preferably 6.
- These multimers may be a homopolymer such as a homodimer or homotrimer that is a conjugate of a single modified immunoglobulin-binding peptide, or may be a conjugate of a plurality of types of modified immunoglobulin-binding peptides. It may be a heteropolymer such as a certain heterodimer or heterotrimer.
- Examples of how the modified immunoglobulin-binding peptide according to the present invention is linked include a method of linking with one or a plurality of amino acid residues, and a method of directly linking without sandwiching amino acid residues, It is not limited to these methods.
- the number of amino acid residues to be linked is not particularly limited, but is preferably 1 to 20 residues, more preferably 15 residues or less, still more preferably 10 residues or less, and even more preferably. Is 5 residues or less, and even more preferably 2 residues or less.
- a sequence linking between ⁇ 1 and ⁇ 2 or between ⁇ 2 and ⁇ 3 of wild-type SpG is used. From another viewpoint, those that do not destabilize the three-dimensional structure of the monomer-modified immunoglobulin-binding peptide are preferable.
- the modified immunoglobulin-binding peptide according to the present invention may have any amino acid sequence defined by the modified immunoglobulin-binding peptides (1) to (3), and other peptides and compounds may be bound. It may be.
- a modified immunoglobulin-binding peptide obtained by the present invention, or a multimer in which two or more of the peptides are linked has different functions as one component.
- nucleic acid such as a DNA aptamer
- a drug such as an antibiotic
- a polymer such as PEG (polyethylene glycol)
- the present invention includes the use of the above-mentioned peptide of the present invention as an affinity ligand characterized by having affinity for immunoglobulin, Fc fragment, Fab fragment and the like.
- an affinity separation matrix characterized in that the ligand is immobilized on a water-insoluble carrier is also included as one embodiment.
- affinity ligand refers to a substance or function that selectively binds and collects a target molecule from a set of molecules based on the affinity between specific molecules represented by the binding between an antigen and an antibody. It is a term referring to a group, and in the present invention, it refers to a peptide that specifically binds to immunoglobulins and fragments thereof.
- the expression “ligand” is also synonymous with “affinity ligand”.
- water-insoluble carrier used in the present invention examples include inorganic carriers such as glass beads and silica gel; organic carriers; and organic-organic and organic-inorganic composite carriers obtained by a combination thereof.
- organic carrier examples include synthetic polymer carriers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and polysaccharide carriers such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran.
- porous cellulose gel GCL2000 Sephacryl S-1000 covalently crosslinked with allyldextran and methylenebisacrylamide
- acrylate-based carrier Toyopearl acrylate-based carrier Toyopearl
- agarose-based crosslinked carrier Sepharose CL4B agarose-based crosslinked carrier Sepharose CL4B
- cellulose examples thereof include Cellufine, which is a cross-linking carrier of the system.
- the water-insoluble carrier in the present invention is not limited to these exemplified carriers.
- the water-insoluble carrier used in the present invention preferably has a large surface area in view of the purpose and method of use of the affinity separation matrix of the present invention, and is preferably a porous material having a large number of pores of an appropriate size.
- the form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected.
- the ligand may be bound to the carrier by a conventional coupling method using an amino group, a carboxy group or a thiol group present in the ligand.
- the carrier is activated by reacting the carrier with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine or sodium periodate, or the surface of the carrier.
- the immobilization method include addition of a reagent having a plurality of functional groups in the molecule such as glutaraldehyde, condensation, and crosslinking.
- a spacer molecule composed of a plurality of atoms may be introduced between the ligand and the carrier, or the ligand may be directly immobilized on the carrier. Therefore, the modified immunoglobulin-binding peptide according to the present invention may be chemically modified for immobilization, or an amino acid residue useful for immobilization may be added.
- amino acids useful for immobilization include amino acids having functional groups useful for immobilization chemical reactions in the side chain, such as Lys containing an amino group in the side chain, and thiol groups in the side chain. Cys containing is mentioned.
- the essence of the present invention is that the immunoglobulin binding property imparted to a peptide in the present invention is similarly imparted to a matrix in which the peptide is immobilized as a ligand, and how it is modified and altered for immobilization. Even within the scope of the present invention.
- proteins containing immunoglobulin Fc region and / or Fab region can be separated and purified by affinity column chromatography purification method.
- These protein purification methods can be achieved by a procedure according to the affinity column chromatography purification method of immunoglobulin (Non-patent Document 1). That is, after preparing a buffer solution containing the protein (pH is near neutral), the solution is contacted by passing it through an affinity column packed with the affinity separation matrix of the present invention, and the protein is selectively used. Adsorb. Next, an appropriate amount of pure buffer is passed through the affinity column, and the inside of the column is washed. At this point, the desired protein is adsorbed to the affinity separation matrix of the present invention in the column.
- the affinity separation matrix in which the peptide of the present invention is immobilized as a ligand is excellent in the ability to adsorb and retain the target protein from the sample addition step to the matrix washing step. Then, an acidic buffer adjusted to an appropriate pH is passed through the column to elute the desired protein, thereby achieving high purity purification. A substance that promotes dissociation from the matrix may be added to the acidic buffer used for elution.
- the protein may be an immunoglobulin itself or an antibody fragment such as an Fc fragment or Fab fragment.
- the affinity separation matrix of the present invention can be reused by washing with a suitable strong acid or strong alkaline buffer that does not completely impair the function of the ligand compound or carrier substrate. It is.
- An appropriate denaturing agent or an organic solvent may be added to the regeneration buffer.
- the affinity separation matrix of the present invention is particularly excellent in chemical stability against an alkaline aqueous solution, it is preferably reused by passing it through a strong alkaline pure buffer and washing.
- the timing of regeneration with a strong alkaline pure buffer solution need not be every time after use, and may be, for example, once every five times or once every ten times.
- the present invention also relates to DNA encoding the modified immunoglobulin-binding peptide according to the present invention.
- the DNA encoding the peptide of the present invention may be any DNA as long as the amino acid sequence obtained by translating the base sequence constitutes the peptide of the present invention.
- Such a base sequence can be obtained by using a commonly used known method, for example, a polymerase chain reaction (hereinafter abbreviated as “PCR”) method. It can also be synthesized by a known chemical synthesis method, and can also be obtained from a DNA library.
- the base sequence may not be the same as the original base sequence as long as the codon may be substituted with a degenerate codon and it encodes the same amino acid when translated.
- Recombinant DNA having one or more of the nucleotide sequences vectors such as plasmids and phages containing the recombinant DNA, and transformants transformed with vectors having the DNA, and introducing the DNA And a cell-free protein synthesis system using the DNA as a template DNA for transcription.
- the modified immunoglobulin-binding peptide according to the present invention can be obtained as a fusion peptide with a known protein having an advantage of assisting protein expression or facilitating purification. That is, a microorganism or cell containing at least one recombinant DNA encoding a fusion peptide containing the modified immunoglobulin-binding peptide according to the present invention can be obtained.
- the protein include maltose binding protein (MBP) and glutathione-S-transferase (GST), but are not limited to these proteins.
- the introduction of site-specific mutations for modifying the DNA encoding the peptide of the present invention can be carried out using recombinant DNA techniques, PCR methods and the like as follows. That is, the introduction of mutations by recombinant DNA technology is performed, for example, when there are appropriate restriction enzyme recognition sequences on both sides of the target site where mutations are desired in the gene encoding the peptide of the present invention.
- the recognition sequence can be cleaved with the restriction enzyme, and after removing the region containing the site desired to be mutated, the cassette mutation method can be used in which a DNA fragment mutated only at the desired site is inserted by chemical synthesis or the like. .
- a DNA encoding a multimeric peptide can also be prepared by linking a DNA encoding the monomeric peptide (one domain) of the present invention in series by the intended number.
- an appropriate restriction enzyme site can be introduced into the DNA sequence, and double-stranded DNA fragmented with the restriction enzyme can be ligated with DNA ligase.
- restriction enzyme site There may be one type of restriction enzyme site, but a plurality of different types of restriction enzyme sites may be introduced.
- the sequence identity between the nucleotide sequences of DNA encoding the peptide is 90% or less, preferably 85% or less, more preferably 80% or less, and even more preferably 75% or less.
- the identity of the base sequence can be determined by a conventional method as in the case of the amino acid sequence.
- the “expression vector” of the present invention includes a base sequence encoding the aforementioned peptide of the present invention or a partial amino acid sequence thereof, and a promoter operable in a host operably linked to the base sequence.
- the gene encoding the peptide of the present invention can be obtained by linking or inserting into a suitable vector, and the vector for inserting the gene is not particularly limited as long as it can autonomously replicate in the host.
- plasmid DNA or phage DNA can be used as a vector.
- vectors such as pQE vectors (Qiagen), pET vectors (Merck), and pGEX vectors (GE Healthcare Bioscience) may be mentioned.
- the transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host cell.
- methods for introducing recombinant DNA into a host include a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, an Agrobacterium infection method, a particle gun method, and a polyethylene glycol method.
- examples of a method for expressing the function of the obtained gene in a host include a method for incorporating the gene obtained in the present invention into a genome (chromosome).
- the host cell is not particularly limited, but for mass production at low cost, Escherichia coli, Bacillus subtilis, Brevibacillus genus, Staphylococcus genus, Streptococcus genus, Streptomyces genus (Streptomyces), Corynebacterium Bacteria (Eubacteria) such as Corynebacterium can be preferably used.
- the modified immunoglobulin-binding peptide according to the present invention is obtained by culturing the above-described transformant in a medium, and the peptide of the present invention in the culture (including the cell periplasm region) or in the culture solution (extracellular). Can be produced by accumulating and collecting the desired peptide from the culture.
- the peptide of the present invention is obtained by culturing the above-described transformed cells in a medium to produce a fusion protein containing the peptide of the present invention in the culture (including the cell periplasm region) or in the culture solution (extracellular). It can be produced by accumulating, collecting the fusion peptide from the culture, cleaving the fusion peptide with an appropriate protease, and collecting the desired peptide.
- the method of culturing the transformant of the present invention in a medium is performed according to a usual method used for host culture.
- the medium used for culturing the obtained transformant is not particularly limited as long as it can produce the peptide of the present invention with high efficiency and high yield.
- carbon sources and nitrogen sources such as glucose, sucrose, glycerol, polypeptone, meat extract, yeast extract, and casamino acid can be used.
- inorganic salts such as potassium salt, sodium salt, phosphate, magnesium salt, manganese salt, zinc salt and iron salt are added as necessary.
- an auxotrophic host cell a nutrient substance required for growth may be added. If necessary, antibiotics such as penicillin, erythromycin, chloramphenicol, neomycin may be added.
- protease inhibitors ie, phenylmethanesulfonylfluoride (PMSF), benzamideline, 4- (2-aminoethyl)- Benzenesulfonyl fluoride (AEBSF), Antipain, Chymostatin, Leupeptin, Pepstatin A, Phosphoramidon, Aprotinin, Ethylenediamine etietic or other inhibitors (EDTA)
- molecular chaperones such as GroEL / ES, Hsp70 / DnaK, Hsp90, Hsp104 / ClpB may be used.
- Such molecular chaperone is allowed to coexist with the peptide of the present invention by a technique such as co-expression or fusion proteinization.
- there are techniques such as adding an additive that promotes correct folding to the medium and culturing at a low temperature, but are not limited thereto. .
- LB medium tryptone 1%, yeast extract 0.5%, NaCl 1%) or 2 ⁇ YT medium (tryptone 1.6%, yeast extract) 1.0%, NaCl 0.5%) and the like.
- the culture temperature is, for example, 15 to 42 ° C., preferably 20 to 37 ° C., and aerobically stirred for several hours to several days under aeration and stirring conditions, whereby the peptide of the present invention is cultured in cultured cells (in the periplasm region). Collected) or in a culture solution (extracellular). In some cases, the culture may be performed anaerobically by blocking aeration.
- the replacement peptide can be recovered.
- the cells are collected from the culture solution by a method such as centrifugation or filtration, and then the cells are sonicated.
- the peptide accumulated and produced in the cells can be recovered by crushing by a French press method and / or solubilizing by adding a surfactant or the like.
- the purification of the peptide according to the present invention can be carried out by affinity chromatography, cation or anion exchange chromatography, gel filtration chromatography or the like alone or in combination. Confirmation that the obtained purified substance is the target protein can be performed by usual methods such as SDS polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, Western blotting and the like.
- the modified peptides obtained in the following examples are expressed in the form of “domain-introduced mutation”, and the peptides before the substitution mutation are described in the form of “domain-Cont”.
- the wild-type SpG- ⁇ 1 having the amino acid sequence of SEQ ID NO: 1 is “GB1-Cont”
- the SpG- ⁇ 1 mutant into which the mutation N08A in which Asn at position 8 is substituted with Ala in the amino acid sequence of SEQ ID NO: 1 was introduced Is represented as “GB1-N08A”.
- Mutant mutations introduced with a plurality of mutations at the same time are written together using slashes.
- an SpG- ⁇ 1 mutant introduced with mutations N08A and D36V is referred to as “GB1-N08A / D36V”.
- various mutants in which the eighth position is mutated are collectively referred to as “N08X”.
- wild-type SpG- ⁇ 1 may be simply referred to as “SpG- ⁇ 1”.
- a peptide obtained by linking two SpG- ⁇ 1 mutants introduced with mutation N08A is represented as “GB1-N08A.2d”.
- a Cys residue (C) having a functional group for immobilization is introduced at the C-terminus in order to immobilize a peptide on a water-insoluble substrate, one letter of the amino acid introduced after “d” Give the notation.
- the SpG- ⁇ 1 mutant in which the SpG- ⁇ 1 mutant introduced with the mutation N08A is ligated to give Cys to the C-terminus is expressed as “GB1-N08A.2dC”.
- Example 1 Preparation of various GB1-N08X (1) Preparation of expression plasmids of various GB1-N08X Back translation was performed from the amino acid sequences of various GB1-N08X, and DNA sequences encoding the peptides were designed. The coding DNA was subjected to PCR using three kinds of single-stranded oligo DNAs (f31 / f32 / f33), then digested with the restriction enzymes BamHI / EcoRI and treated with the same restriction enzymes pGEX-6P-1. It was incorporated into the multiple cloning site.
- f32 and f33 (10 ⁇ M, lagging) are first extended by overlap PCR, and then extended by f31 (10 ⁇ M, reading) and the previous reaction.
- the double-stranded DNA formed by overlapping PCR with f33 (lagging) becomes a coding DNA sequence having restriction enzyme sites at both ends.
- the overlap PCR of f31 and f32 may be the starting point, but the final product is the same.
- Various oligo DNA sequences corresponding to f31 to f33 of each GB1-N08X were designed so that the reaction proceeds and synthesized by outsourcing to Eurofin.
- DoubleTaq Plus (TOYOBO) as a polymerase
- PCR reaction is performed, agarose electrophoresis is performed, and the double-stranded DNA extracted by cutting out the target band is subjected to restriction enzymes BamHI and EcoRI ( Cut by Takara Bio Inc.).
- plasmid vector pGEX-6P-1 (GE Healthcare Bioscience) was similarly treated with BamHI / EcoRI and then dephosphorylated with dephosphorylation enzyme CIAP (Takara Bio). Then, a ligation reaction using Ligation high (TOYOBO) was performed.
- the DNA encoding the GB1-N08X mutant was prepared using oligo DNAs having common f32 (SEQ ID NO: 6) and f33 (SEQ ID NO: 7), and differing only in the sequence of f31.
- the oligo DNA sequences of f31, f32, and f33, the coding DNA sequence, and the amino acid sequence SEQ ID NOs used are summarized in Table 1 for each type of mutant.
- GB1 # -N08K in which the mutation N08K was introduced into GB1 # -Cont (SEQ ID NO: 4) with enhanced Fab binding force was also prepared.
- This mutant was prepared in the same manner as described above using single-stranded oligo DNAs f31 (SEQ ID NO: 29), f32 (SEQ ID NO: 30), and f33 (SEQ ID NO: 7).
- the coding DNA sequence is shown as SEQ ID NO: 31, and the amino acid sequence is shown as SEQ ID NO: 32 (added to Table 1).
- plasmid vector pGEX-6P-1 transformation of competent cells (Takara Bio Inc. “E. coli HB101”) was performed according to the protocol attached to this competent cell product.
- GST glutathione-S-transferase
- 1d can be produced.
- plasmid DNA was amplified and extracted using a plasmid purification kit (“WizardPlus SV Minipreps DNA Purification System” manufactured by Promega) according to the standard protocol attached to the kit.
- the cells were collected by centrifugation and resuspended in 5 mL of PBS buffer.
- the cells were disrupted by ultrasonic disruption, centrifuged, and fractionated into a supernatant fraction (cell-free extract) and an insoluble fraction.
- GST is expressed as a fusion peptide attached to the N-terminus.
- SDS electrophoresis all of the various cell-free extracts prepared from the respective transformed cell cultures were found to have peptides that were thought to have been induced by IPTG at a molecular weight of about 25,000 or more. I confirmed the band. The molecular weight was almost the same, but the position of the band was different depending on the type of mutant.
- the GST fusion peptide was roughly purified from each cell-free extract containing the GST fusion peptide by affinity chromatography using a GSTrap FF column (GE Healthcare Bioscience) having affinity for GST. Each cell-free extract is added to the GSTRap FF column, and the column is washed with a standard buffer (20 mM NaH 2 PO 4 -Na 2 HPO 4 , 150 mM NaCl, pH 7.4), followed by an elution buffer ( The target GST fusion peptide was eluted with 50 mM Tris-HCl, 20 mM glutathione, pH 8.0).
- this eluate was concentrated using Amicon (Merck Millipore), a centrifugal filter unit, and the elution buffer was used as a standard buffer. A peptide solution substituted with the solution was used.
- the amino acid sequence that can cleave GST with the sequence-specific protease PreScission Protease is between GST and the target peptide.
- GST cleavage reaction was performed using PreScience Protease according to the attached protocol.
- the target peptide was purified by gel filtration chromatography using Superdex 75 10/300 GL column (GE Healthcare Biosciences) from the sample thus cut GST. Each reaction solution was added to a Superdex 75 10/300 GL column equilibrated with a standard buffer, and the target peptide was separated and purified from cleaved GST and PreScission Protease.
- each peptide after GST cleavage obtained in this example has an amino acid sequence in which Gly-Pro-Leu-Gly-Ser derived from the vector pGEX-6P-1 is added to the N-terminal side on the N-terminal side. .
- Example 2 Alkali tolerance evaluation of various GB1-N08X (1) Preparation of Fab fragment / Fc fragment Humanized monoclonal IgG preparation was fragmented into Fab fragment and Fc fragment with papain, and Fc fragment and Fab fragment were separated and purified , Each fragment is simply abbreviated as “Fc” and “Fab”, respectively). Specifically, an anti-human TNF ⁇ monoclonal IgG preparation (generic name “infliximab”, product name “Remicade”, Mitsubishi Tanabe Seiyaku) was added to papain digestion buffer (0.1 M AcOH-AcONa, 2 mM EDTA, 1 mM cysteine, pH 5).
- Papain Agarose from papaya latex papain-immobilized agarose was added, and the mixture was incubated at 37 ° C. for about 8 hours while mixing with a rotator. From the reaction solution separated from papain-immobilized agarose (Fab and Fc coexisting), the Fab was collected in a flow-through fraction by affinity chromatography using KanCapA (Kaneka), and separated and purified from Fc. Fc adsorbed on the column was eluted with 0.05 M acetic acid / sodium acetate buffer (pH 3.5) and immediately neutralized with 0.1 M Tris-HCl buffer (pH 8.0).
- the solution containing Fab and the solution containing Fc were purified by gel filtration chromatography using a Superdex 75 10/300 GL column (GE Healthcare) (standard buffer was used for equilibration and separation). And a solution of Fc was obtained.
- purification of the peptide by chromatography was performed using the AKTAprime plus system.
- Example 2 (2) Observation of various IgG1-N08X immunoglobulin binding responses Various GB1-obtained in Example 1 (2) using biosensor Biacore 3000 (GE Healthcare Bioscience) utilizing surface plasmon resonance The binding response of N08X mutant to Fc was observed.
- Fc obtained in Example 2 (1) was immobilized on a sensor chip, and various GB1-N08X mutants were flowed on the chip to detect the interaction between them.
- Fc was immobilized on the sensor chip CM5 by an amine coupling method using N-hydroxysuccinimide (NHS) and N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
- NHS N-hydroxysuccinimide
- EDC N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride
- Ethanolamine was used for blocking.
- the sensor chip and the immobilization reagent were all manufactured by GE Healthcare Bioscience.
- the Fc solution was diluted about 10 times with an immobilization buffer (10 mM CH 3 COOH—CH 3 COONa, pH 4.5), and immobilized on the sensor chip according to the protocol attached to the Biacore 3000.
- a reference cell serving as a negative control was prepared by performing a process of immobilizing only ethanolamine after activation with EDC / NHS for another flow cell on the chip.
- the amount of immobilized Fc was about 5000 RU. It should be noted that by increasing the immobilization amount to 5000 RU or more and decreasing the flow rate, the dependence on the detection sensitivity and the analyte concentration is improved. That is, in an environment in which mass transport limited is applied, the dependence of the binding response on the affinity decreases, and the dependence on the concentration relatively increases.
- the binding response 1 minute after the addition (resonance unit value of the binding reaction curve) is plotted on the vertical axis and the concentration of the added analyte at that time is plotted on the horizontal axis.
- the binding response is proportional to the analyte concentration to some extent in this concentration range.
- the way of increasing the binding response to the analyte concentration differs depending on the mutant.
- the residual binding activity was calculated after correcting for the concentration, instead of simply evaluating the response ratio before and after the alkali treatment.
- GB1-N08A Non-patent document 7, in the document, the position corresponding to the mutation position of the present invention is defined as the seventh position
- GB1-N08G Non-patent document 8, Document
- the single-residue mutation that was most effective was shown, but a plurality of mutations showing alkali resistance significantly higher than those were found.
- the reason why GB1-N08G did not have high alkali resistance is unclear, but the mutation to Gly basically destabilizes the main chain structure, so the results of this experiment are considered valid.
- Example 1 Comparative Example For GB1-N08A, GB1-N08G, and GB1 # -N08A used as comparative controls in this example, oligo DNAs f31 to f33 having the sequence numbers shown in Table 2 were used and Example 1 (1) was used. An expression plasmid was prepared in the same manner. And the protein sample was prepared by the method similar to Example 1 (2), and the alkali tolerance was evaluated simultaneously with the mutant obtained by this invention by the method similar to Example 2 (2).
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Abstract
Description
以下、本発明を示す。
(1) 配列番号1~5のいずれかのアミノ酸配列において、第8位のアミノ酸残基がAsp、Glu、His、Ile、Lys、LeuまたはValに置換されているアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(2) 上記(1)に規定されるアミノ酸配列において、上記第8位を除く領域中で1個または数個のアミノ酸がさらに欠失、置換および/または付加されたアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(3) 上記(1)に規定されるアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリン結合性ペプチド(但し、上記(1)に規定されるアミノ酸配列における第8位の置換アミノ酸残基は、(3)においてさらに変異しないものとする)。
上記[6]に記載のアフィニティー分離マトリックスと、該タンパク質を含む液体試料とを接触させる工程と
アフィニティー分離マトリックスに結合した該タンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。
(1) 配列番号1~5のいずれかのアミノ酸配列において、第8位のアミノ酸残基がAsp、Glu、His、Ile、Lys、LeuまたはValに置換されているアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(2) 上記(1)に規定されるアミノ酸配列において、上記第8位を除く領域中で1個または数個のアミノ酸がさらに欠失、置換および/または付加されたアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(3) 上記(1)に規定されるアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリン結合性ペプチド(但し、上記(1)に規定されるアミノ酸配列における第8位の置換アミノ酸残基は、(3)においてさらに変異しないものとする)。
(1) 各種GB1-N08Xの発現プラスミド調製
各種GB1-N08Xのアミノ酸配列から逆翻訳を行い、当該ペプチドをコードするDNA配列を設計した。コードDNAは、3種の一本鎖オリゴDNA(f31/f32/f33)を用いたPCRを行い、その後制限酵素BamHI/EcoRIで消化して、同じ制限酵素で処理した発現ベクターpGEX-6P-1のマルチクローニングサイトに組み込んだ。コードDNA調製時のPCRでは、まず少量のf32(0.2μM、リーディング)とf33(10μM、ラギング)とがオーバーラップPCRで伸張され、次に、f31(10μM、リーディング)と先の反応で伸張したf33(ラギング)とのオーバーラップPCRによって形成される二本鎖DNAが、制限酵素サイトを両端に有したコードDNA配列となる。f31とf32のオーバーラップPCRが起点となる場合もあるが、最終産物は一緒である。各々のGB1-N08Xのf31~f33に該当する各種オリゴDNA配列をこのような反応が進むよう設計し、ユーロフィン社への外注によって合成した。具体的な実験操作については、ポリメラーゼとしてBlendTaq Plus(TOYOBO社)を用い、PCR反応を行い、アガロース電気泳動にかけ、目的のバンドを切り出すことで抽出した二本鎖DNAを、制限酵素BamHIとEcoRI(タカラバイオ社)により切断した。次に、プラスミドベクターpGEX-6P-1(GEヘルスケア・バイオサイエンス社)も、同様にBamHI/EcoRI処理し、次に脱リン酸化酵素CIAP(タカラバイオ社)による脱リン酸化処理を行った後で、Ligation high(TOYOBO社)を用いたライゲーション反応を行った。
上記(1)で得られた、各種GB1-N08X遺伝子またはGB1#-N08X遺伝子を導入した各形質転換細胞を、アンピシリン含有2×YT培地にて、37℃で終夜培養した。これらの培養液を、100倍量程度のアンピシリン含有2×YT培地に接種し、37℃で約1時間、その後25℃で約1時間培養した後で、終濃度0.1mMになるようイソプロピル1-チオ-β-D-ガラクシド(IPTG)を添加し、さらに25℃にて約18時間培養した。
(1) Fabフラグメント/Fcフラグメントの調製
ヒト化モノクローナルIgG製剤をパパインによってFabフラグメントとFcフラグメントに断片化し、FcフラグメントとFabフラグメントを分離精製した(以下、各フラグメントを単にそれぞれ「Fc」および「Fab」と略す)。具体的には、抗ヒトTNFαモノクローナルIgG製剤(一般名「インフリキシマブ」,製品名「レミケード」,田辺三菱製薬)を、パパイン消化用緩衝液(0.1M AcOH-AcONa,2mM EDTA,1mMシステイン,pH5.5)に溶解し、Papain Agarose from papaya latexパパイン固定化アガロース(SIGMA社)を添加し、ローテーターで混和させながら、37℃で約8時間インキュベートした。パパイン固定化アガロースから分離した反応溶液(FabとFcが混在)から、KanCapA(カネカ)を利用したアフィニティークロマトグラフィーにより、素通り画分でFabを回収することでFcと分離精製した。カラムに吸着したFcは、0.05M 酢酸/酢酸ナトリウム緩衝液(pH3.5)で溶出し、即座に0.1M Tris-塩酸緩衝液(pH8.0)で中和した。Fabを含む溶液およびFcを含む溶液を、Superdex 75 10/300 GLカラム(GEヘルスケア社)を用いたゲルろ過クロマトグラフィーにて精製(平衡化および分離には標準緩衝液を使用)し、FabおよびFcの溶液を得た。なお、実施例1と同様に、クロマトグラフィーによるペプチド精製は、AKTAprime plusシステムを利用して実施した。
超純水を用いて透析した各種GB1-N08Xを濃度調整し、20μM水溶液を得た。当該水溶液0.2mLに半量の45mM水酸化ナトリウム水溶液0.1mLを添加し、25℃で2時間インキュベ―トした後、50mM酢酸(pH3.0)0.1mLで中和した。中和されていることをpH試験紙にて確認し、標準緩衝液で5倍希釈した。
表面プラズモン共鳴を利用したバイオセンサーBiacore 3000(GEヘルスケア・バイオサイエンス社)を用いて、実施例1(2)で取得した各種GB1-N08X変異体のFcに対する結合レスポンスを観測した。本実施例では、実施例2(1)で取得したFcをセンサーチップに固定化し、各種GB1-N08X変異体をチップ上に流して、両者の相互作用を検出した。FcのセンサーチップCM5への固定化は、N-ヒドロキシスクシンイミド(NHS)およびN-エチル-N’-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)を用いたアミンカップリング法にて行い、ブロッキングにはエタノールアミンを用いた。センサーチップや固定化用試薬は、全てGEヘルスケアバイオサイエンス社製のものを用いた。Fc溶液は、固定化用緩衝液(10mM CH3COOH-CH3COONa,pH4.5)を用いて10倍程度に希釈し、Biacore 3000付属のプロトコルに従い、センサーチップへ固定化した。また、チップ上の別のフローセルに対して、EDC/NHSにより活性化した後にエタノールアミンのみを固定化する処理を行うことで、ネガティブ・コントロールとなるリファレンスセルも用意した。固定化されたFcの量は5000RU程であった。なお、固定化量を5000RU以上と高くし、流速を遅くすることで、検出感度およびアナライト濃度への依存度が向上する。すなわち、マストランスポート・リミテッドがかかっている環境下では、結合レスポンスの親和性への依存度が低下し、相対的に濃度への依存度が上がる。
アルカリ処理後の各種GB1-N08X変異体も、ランニング緩衝液で10倍希釈して濃度を200nMに調整し、同様に流速10μL/minで2分間センサーチップに添加し、添加1分後のFc結合レスポンスを求めた。先に求めたアルカリ処理前の200nMの結合レスポンス値と図1のプロットで求めた傾きから、アルカリ処理後の結合レスポンスに対応するアナライト濃度を算出した。そして、その濃度を、結合活性を維持した変異体濃度とし、処理前(100%)に対する濃度比率を結合残存活性として算出し、その値をグラフとして図2(1)に示した。
実施例2(2)~(4)と同様の手法にて、GB1#-N08KのFabに対する結合残存活性を評価すると、GB1#-N08Aが68%(N=4)であったのに対し、GB1#-N08Kは80%(N=4)であった。以上の結果より、ベースとなるSpGの配列が異なる場合にも、同様の効果が期待できることが確認できた。
本実施例の比較対照として用いたGB1-N08A、GB1-N08G、および、GB1#-N08Aについては、表2に示す配列番号のオリゴDNAf31~f33を用いて、実施例1(1)と同様の方法で発現プラスミドを調製した。そして、実施例1(2)と同様の方法でタンパク質サンプルを調製し、実施例2(2)と同様の方法で、本発明で得られた変異体と同時にアルカリ耐性を評価した。
Claims (10)
- 下記(1)~(3)のいずれかの改変型免疫グロブリン結合性ペプチド。
(1) 配列番号1~5のいずれかのアミノ酸配列において、第8位のアミノ酸残基がAsp、Glu、His、Ile、Lys、LeuまたはValに置換されているアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(2) 上記(1)に規定されるアミノ酸配列において、上記第8位を除く領域中で1個または数個のアミノ酸がさらに欠失、置換および/または付加されたアミノ酸配列を有する免疫グロブリン結合性ペプチド;
(3) 上記(1)に規定されるアミノ酸配列に対して80%以上の配列同一性を有するアミノ酸配列を有する免疫グロブリン結合性ペプチド(但し、上記(1)に規定されるアミノ酸配列における第8位の置換アミノ酸残基は、(3)においてさらに変異しないものとする)。 - 上記(1)に規定されるアミノ酸配列において、第8位がIle、LeuまたはLysに置換されている請求項1に記載の改変型免疫グロブリン結合性ペプチド。
- 上記(2)に規定されるアミノ酸配列において、上記欠失、置換および/または付加されるアミノ酸の部位が、第6位、第7位、第13位、第15位、第18位、第19位、第21位、第24位、第28位、第29位、第30位、第31位、第33位、第35位、第39位、第40位、第42位および第47位から必須的になる群より選択される1以上の部位である請求項1~2のいずれかに記載の改変型免疫グロブリン結合性ペプチド。
- 上記(2)に規定されるアミノ酸配列において、上記欠失、置換および/または付加されるアミノ酸の部位がN末端および/またはC末端である請求項1~3のいずれかに記載の改変型免疫グロブリン結合性ペプチド。
- 請求項1~4のいずれかに記載の改変型免疫グロブリン結合性ペプチドを2個以上連結した複数ドメインを有することを特徴とする改変型免疫グロブリン結合性ペプチド多量体。
- 請求項1~4のいずれかに記載の改変型免疫グロブリン結合性ペプチドまたは請求項5に記載の改変型免疫グロブリン結合性ペプチド多量体と水不溶性担体を含み、該改変型免疫グロブリン結合性ペプチドまたは該改変型免疫グロブリン結合性ペプチドがリガンドとして水不溶性担体に固定化されていることを特徴とするアフィニティー分離マトリックス。
- 免疫グロブリンのFc領域および/またはFab領域を含むタンパク質を製造する方法であって、
請求項6に記載のアフィニティー分離マトリックスと、該タンパク質を含む液体試料とを接触させる工程と
アフィニティー分離マトリックスに結合した該タンパク質を、アフィニティー分離マトリックスから分離する工程を含むことを特徴とする方法。 - 請求項1~4のいずれかに記載の改変型免疫グロブリン結合性ペプチド、または請求項5に記載の改変型免疫グロブリン結合性ペプチド多量体をコードすることを特徴とするDNA。
- 請求項8に記載のDNAを含むことを特徴とするベクター。
- 請求項9に記載のベクターにより形質転換されたものであることを特徴とする形質転換体。
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