JP2021073877A - Fc-binding proteins with improved acid stability, production methods thereof and antibody-adsorbing agents using same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an Fc-binding protein having improved acid stability with respect to a natural human FcγRIIb, a method for producing the protein, and an antibody adsorbent using the protein. SOLUTION: The amino acid residue of an extracellular region in the amino acid sequence of a natural human FcγRIIb (UniProt No. P31994) is contained at least, but the amino acid residue of the region contains at least a deletion of an amino acid residue at a specific location. The above-mentioned problems are solved by the Fc-binding protein possessed, the method for producing the protein, and the antibody adsorbent using the protein. [Selection diagram] Fig. 2

Description

The present invention relates to an Fc-binding protein having a binding affinity for immunoglobulin G (IgG). More specifically, the present invention is an Fc-binding protein having improved acid stability as compared with the native human Fcγ receptor IIb by deleting an amino acid residue at a specific position of the human Fcγ receptor IIb. The method and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier.

The Fc receptor is a receptor protein that binds to the Fc region of an immunoglobulin molecule, binds to an immune complex of an antigen and an immunoglobulin, and performs intracellular signal transduction (Non-Patent Document 1). Of particular importance, the Fcγ receptor (FcγR) that binds to immunoglobulin G (IgG) is a sub of FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a) and FcγRIIIb (CD16b). It can be classified into types (Non-Patent Documents 1 and 2). The binding of the IgG immune complex to FcγR occurs by the binding of FcγR to the Fc region of IgG. Each FcγR molecular species has an IgG Fc region recognition domain and recognizes IgGs that belong to a single species or the same subtype. This determines which accessory cells are mobilized in the individual immune response (Non-Patent Documents 1 and 2).

Among FcγRs, FcγRIIb is present on the cell surface of B cells, mast cells, etc., and is the only important receptor involved in inhibitory signal transduction (Non-Patent Document 3). The amino acid sequence of human FcγRIIb (SEQ ID NO: 1) is registered in a public database such as UniProt (Accession Number: P31994). Similarly, the structural functional domain of human FcγRIIb, the signal peptide sequence for penetrating the cell membrane, and the position of the transmembrane region are also published. FIG. 1 shows a schematic structure of human FcγRIIb. The amino acid numbers in FIG. 1 correspond to the amino acid numbers shown in SEQ ID NO: 1. That is, the signal sequence (S) is from the 1st methionine to the 42nd glycine in SEQ ID NO: 1, the intracellular (EC) region is from the 43rd threonine to the 217th proline, and the 240th is from the 218th serine. The region up to alanine is the transmembrane (TM) region and the region from valine 241 to isoleucine 310 is the intracellular (C) region.

In order to apply FcγRIIb industrially, it is preferable that it can be mass-produced and has high stability against heat and acid from the viewpoint of use and storage. By substituting the amino acid residue at a specific position with another amino acid residue among the amino acid residues constituting the EC region of human FcγRIIb, the productivity of human FcγRIIb in transformed Escherichia coli is improved (Patent Documents 1 and 2). Or, the stability of human FcγRIIb to heat and acid is improved (Patent Document 2).

JP-A-2017-178908 JP-A-2018-050616

Takai T.K. , Jpn. J. Clin. Immunol. , 28,318-326,2005 J. Gallon et al. , Euro. J. Immunol. , 27, 1928-1932, 1997 T. Takai, Nat. Rev. Immunol. , 2,580-592,2002

An object of the present invention is to provide an Fc-binding protein having improved acid stability against natural human FcγRIIb, a method for producing the protein, and an antibody adsorbent using the protein.

As a result of diligent studies to solve the above problems, the present inventors have identified amino acid residues involved in improving acid stability in human FcγRIIb, and the mutant lacking the amino acid residues is an acid. It has been found that it has excellent stability with respect to the present invention, and the present invention has been completed.

That is, the present application includes the aspects described in the following [1] to [10].

[1]
Fc-binding protein selected from any of the following (i) to (iii):
(I) Among the amino acid sequences shown in SEQ ID NO: 2, at least the amino acid residues from the 29th threonine to the 201st glutamine are contained, but the amino acid residues from the 29th to the 201st are as follows (1). An Fc-binding protein having at least one deletion of any of the amino acid residues shown in (5) to (5);
(1) Deletion of the 63rd proline of SEQ ID NO: 2 (2) Deletion of the 61st histidine of SEQ ID NO: 2 (3) Deletion of the 62nd serine of SEQ ID NO: 2 (4) 64 of SEQ ID NO: 2 Deletion of the 65th glutamic acid (5) Deletion of the 65th serine of SEQ ID NO: 2 (ii) Of the amino acid sequences shown in SEQ ID NO: 2, at least the amino acid residues from the 29th threonine to the 201st glutamine Including, however, the 29th to 201st amino acid residues have at least one deletion of any of the amino acid residues shown in (1) to (5) above, and further, from (1) to (1) above. In addition to the deletion of amino acid residues shown in 5), it further has one or more of substitution, deletion, insertion and addition of one or several amino acid residues at one or several positions. Fc-binding protein having antibody-binding activity;
(Iii) In the amino acid sequence from the 29th glutamine to the 201st glutamine of the amino acid sequence shown in SEQ ID NO: 2, at least one deletion of any of the amino acid residues shown in (1) to (5) above is made. An Fc-binding protein having an amino acid sequence having 70% or more homology to the entire amino acid sequence having the above, containing an amino acid sequence in which the deletion of at least one amino acid residue remains, and having antibody-binding activity. ..

[2]
The Fc-binding protein according to [1], which has at least a deletion of any of the amino acid residues shown in (1) and (6) to (10) below.
(1) Deletion of the 63rd proline of SEQ ID NO: 2 (6) Deletion of the 61st histidine and 62nd serine of SEQ ID NO: 2 (7) Deletion of the 62nd serine and 63rd proline of SEQ ID NO: 2 Deletion (8) Deletion of 61st histidine, 62nd serine and 63rd proline of SEQ ID NO: 2 (9) Deletion of 63rd proline, 64th glutamic acid and 65th serine of SEQ ID NO: 2 (10) Deletion of 61st histidine, 62nd serine, 63rd proline, 64th glutamic acid and 65th serine of SEQ ID NO: 2 [3]
Fc-binding protein according to [1] or [2], which is selected from the following (iv) to (vi):
(Iv) An Fc-binding protein containing at least an amino acid residue from threonine at position 29 to glutamine at position 201 in the amino acid sequence set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14;
(V) Of the amino acid sequences set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14, the amino acid residues from the 29th threonine to the 201st glutamine are contained at least, but from the 29th position. Among the substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions in addition to the deletion of the amino acid residue of the amino acid sequence in the amino acid residues up to the 201st position. An Fc-binding protein further comprising any one or more and having antibody-binding activity;
(Vi) Of the amino acid sequences set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14, the amino acid residues from the 29th threonine to the 201st glutamine are contained at least, but from the 29th position. An Fc-binding protein having 70% or more homology to the amino acid sequence up to the 201st amino acid sequence, residual deletion of the amino acid residue of the amino acid sequence, and antibody-binding activity.

[4]
A polynucleotide encoding the Fc-binding protein according to any one of [1] to [3].

[5]
An expression vector containing the polynucleotide according to [4].

[6]
A transformant capable of producing an Fc-binding protein obtained by transforming a host with the recombinant vector according to [5].

[7]
The transformant according to [6], wherein the host is Escherichia coli.

[8]
[6] or [7] includes a step of producing an Fc-binding protein by culturing the transformant, and a step of recovering the Fc-binding protein produced from the obtained culture. A method for producing an Fc-binding protein.

[9]
An antibody adsorbent obtained by immobilizing the Fc-binding protein according to any one of [1] to [3] on an insoluble carrier.

[10]
A step of adding a solution containing an antibody to a column packed with the antibody adsorbent according to [9] and adsorbing the antibody to the adsorbent, and a step of eluting the antibody adsorbed on the adsorbent using an eluate. Methods for separating antibodies, including.

Hereinafter, the present invention will be described in detail.

The Fc-binding protein of the present invention is a protein that binds to the Fc region of an antibody, and is the extracellular (EC) region of the native human FcγRIIb consisting of the amino acid sequence set forth in SEQ ID NO: 2 (No. 43 of SEQ ID NO: 1). Contains amino acid residues from at least 29th threonine to 201st glutamine, but at a specific position in the 29th to 201st amino acid residues. A protein that has at least a deletion of amino acid residues. Therefore, the Fc-binding protein of the present invention may contain all or part of the signal peptide (S) region on the N-terminal side of the extracellular region, or transmembrane (TM) on the C-terminal side of the extracellular region. ) Region and all or part of the extracellular (C) region may be included. Of SEQ ID NO: 2, the first methionine to the 26th alanine are MalE signal peptides (oligopeptides consisting of amino acid residues 1 to 26 of UniProt No. P0AEX9), and the 27th methionine and the 27th methionine and The 28th glycine is a linker, the 29th to 201st are extracellular regions of native human FcγRIIb (that is, the 43rd to 215th regions of SEQ ID NO: 1), and the 202nd and 203rd glycines are. It is a linker, and the 204th to 209th are histidine tags.

The deletion of the amino acid residue at the specific position specifically indicates that Del-Pro63 (this notation indicates that the 63rd proline of SEQ ID NO: 2 (77th in SEQ ID NO: 1) is deleted. (Same as above), Del-His61, Del-Ser62, Del-Glu64, Del-Ser65, at least one or more. The Fc-binding protein of the present invention may have at least one deletion of an amino acid residue at the above-mentioned specific position, and as long as it has antibody-binding activity, the above-mentioned amino acid residue at the specific position is missing. In addition to the loss, one or more of the substitutions, deletions, insertions and additions of amino acid residues may be further present. As examples of amino acid residue substitutions to be added in addition to the above-mentioned deletion of amino acid residues at specific positions, JP-A-2017-178908 and JP-A-2018-050616 improve the productivity of human FcγRIIb in transformed Escherichia coli. Isoleucine at position 68 of SEQ ID NO: 2 (82nd in SEQ ID NO: 1) is substituted with valine, which is disclosed as a substitution that improves the stability of human FcγRIIb to heat. (The same applies hereinafter), His80Gln, Ser84Thr, Asn90Thr, Asn91Ser, His125Arg, Lys158Met, Arg162His, Tyr188Asn, Leu190Ser.

In the above (ii) and (v), "1 or several" differs depending on the position of the amino acid substitution in the three-dimensional structure of the Fc-binding protein and the type of amino acid residue, but as an example, 1 to 50, 1 Means any of 30 to 30 pieces, 1 to 20 pieces, and 1 to 10 pieces. Further, in the present specification, any one or more of substitutions, deletions, insertions and additions are naturally occurring mutations based on individual differences, species differences, etc. of microorganisms derived from Fc-binding protein genes. (Mutant or variant) is also included.

The homology of the amino acid sequences in (iii) and (vi) may be 70% or more, and has more homology (for example, 80% or more, 85% or more, 90% or more or 95% or more). May be good.

The Fc-binding proteins of the present invention may further have conservative substitutions in which the 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 the function of the protein between those with and without substitution. Examples of conservative substitutions include substitutions that occur between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, Inc., 9). , 2005).

The Fc-binding protein of the present invention may further have an oligopeptide added to its N-terminal side or C-terminal side, which is useful for separation from the solution in the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid and the like. Further, an oligopeptide containing cysteine, which is useful for immobilizing the Fc-binding protein of the present invention on an insoluble carrier such as a support for chromatography, is provided on the N-terminal side or the C-terminal side of the Fc-binding protein of the present invention. May be further added to. The length of the oligopeptide added to the N-terminal side or the C-terminal side of the Fc-binding protein is not particularly limited as long as the IgG (immunoglobulin G) binding property and stability of the Fc-binding protein of the present invention are not impaired. When adding the oligopeptide to the Fc-binding protein of the present invention, after preparing a polynucleotide encoding the oligopeptide, the N-terminal of the Fc-binding protein is genetically engineered using a method well known to those skilled in the art. It may be added to the side or the C-terminal side, or the chemically synthesized oligopeptide may be added by chemically binding to the N-terminal side or the C-terminal side of the Fc-binding protein of the present invention. Further, 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 periplasms such as PelB, DsbA, MalE (the region from the 1st to the 26th amino acid sequence described in UniProt No. P0AEX9), TorT, and the like secrete proteins. A signal peptide can be exemplified (Japanese Patent Laid-Open No. 2011-097898).

A preferred embodiment of the Fc-binding protein of the present invention is an Fc-binding protein containing at least a polypeptide consisting of the amino acid sequences shown in (a) to (f) below. These Fc-binding proteins are preferable in that they have improved acid stability (acid resistance).

(A) FcγRIIb-Del-Pro63 (amino acid residues from the 29th to the 200th of the amino acid sequence shown in SEQ ID NO: 4)
Of the amino acid sequences shown in SEQ ID NO: 2, the amino acid residues from the 29th threonine to the 201st glutamine, however, in the 29th to 201st amino acid residues, the 63rd proline (Pro63) A polypeptide with a deletion.

(B) FcγRIIb-Del-His61-Ser62 (amino acid residues from position 29 to position 199 of the amino acid sequence shown in SEQ ID NO: 6)
Of the amino acid sequences shown in SEQ ID NO: 2, the amino acid residues from threonine at position 29 to glutamine at position 201, however, in the amino acid residues from position 29 to 201, histidine at position 61 (His61) and A polypeptide having a deletion of the 62nd serine (Ser62).

(C) FcγRIIb-Del-Ser62-Pro63 (amino acid residues from position 29 to position 199 of the amino acid sequence shown in SEQ ID NO: 8)
Of the amino acid sequences shown in SEQ ID NO: 2, it is an amino acid residue from threonine at position 29 to glutamine at position 201, but has a deletion of Ser62 and Pro63 at the amino acid residue from position 29 to 201. Polypeptide.

(D) FcγRIIb-Del-His61-Pro63 (amino acid residues 29 to 198 of the amino acid sequence shown in SEQ ID NO: 10)
Of the amino acid sequences shown in SEQ ID NO: 2, the amino acid residues from the 29th threonine to the 201st glutamine, however, in the 29th to 201st amino acid residues, 3 residues from His61 to Pro63. Polypeptide with a deletion of.

(E) FcγRIIb-Del-Pro63-Ser65 (amino acid residues 29 to 198 of the amino acid sequence shown in SEQ ID NO: 12)
Of the amino acid sequences shown in SEQ ID NO: 2, it is the amino acid residue from the 29th threonine to the 201st glutamine, but in the 29th to 201st amino acid residues, Pro63 to the 65th serine (Ser65). ), A polypeptide having a deletion of 3 residues.

(F) FcγRIIb-Del-His61-Ser65 (amino acid residues 29 to 196 of the amino acid sequence shown in SEQ ID NO: 14)
Of the amino acid sequences shown in SEQ ID NO: 2, the amino acid residues from the 29th threonine to the 201st glutamine, however, in the 29th to 201st amino acid residues, 5 residues from His61 to Ser65. Polypeptide with a deletion of.

Of the Fc-binding proteins set forth in SEQ ID NOs: 4, 6, 8, 10, 12 and 14, the first methionine to the 26th alanine are MalE signal peptide sequences, and the 27th methionine and the 28th methionine. Glycine is the linker sequence, and glutamine at positions 200 (SEQ ID NO: 4), 199 (SEQ ID NOs: 6 and 8), 198 (SEQ ID NOs: 10 and 12) or 196 (SEQ ID NO: 14) from the 29th threonine. Polypeptides capable of binding to the Fc region of the antibody (FcγRIIb-Del-Pro63 (SEQ ID NO: 4), FcγRIIb-Del-His61-Ser62 (SEQ ID NO: 6), FcγRIIb-Del-Ser62-Pro63 (SEQ ID NO: 8), It is an amino acid sequence of FcγRIIb-Del-His61-Pro63 (SEQ ID NO: 10), FcγRIIb-Del-Pro63-Ser65 (SEQ ID NO: 12) or FcγRIIb-Del-His61-Ser65 (SEQ ID NO: 14), and is the C of the polypeptide. A linker sequence consisting of 2 residues of glycine and a tag sequence consisting of 6 residues of histidine are added to the terminal side.

As an example of a method for producing a polynucleotide encoding the Fc-binding protein of the present invention (hereinafter, also simply referred to as the polynucleotide of the present invention).
(1) A method of converting the amino acid sequence of the Fc-binding protein of the present invention into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence, or
(2) A polynucleotide containing the whole or partial sequence of an Fc-binding protein is prepared directly artificially or from the cDNA of the Fc-binding protein by a DNA amplification method such as PCR, and the prepared polynucleotide is suitable. A method of connecting in various ways can be exemplified.

In the method (1) above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to convert in consideration of the frequency of use of codons in the host to be transformed. As an example, when the host is Escherichia coli, arginine (Arg) is AGA / AGG / CGG / CGA, isoleucine (Ile) is ATA, leucine (Leu) is CTA, and glycine (Gly) is GGA. However, in proline (Pro), since each CCC is used infrequently (because it is a so-called rare codon), it may be converted so as to avoid those codons. Analysis of codon usage frequency is also possible by using a public database (eg, Codon Usage Database on the Kazusa DNA Research Institute website).

When introducing a mutation into the polynucleotide of the present invention, the error prone PCR method can be used. The reaction conditions in the error-prone PCR method are not particularly limited as long as the desired mutation can be introduced into the polynucleotide encoding human FcγRIIb (or Fc-binding protein). Polynucleotides are subjected to PCR by making the concentration of dATP / dTTP / dCTP / dGTP non-uniform _{and adding MnCl 2} to the PCR reaction solution at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM). Mutations can be introduced into. As a mutagen introduction method other than the error prone PCR method, a polynucleotide containing the entire or partial sequence of human FcγRIIb is mutated into a polynucleotide by contacting / acting with a mutagen drug or irradiating with ultraviolet rays. Can be mentioned as a method of producing by introducing. As the drug used as a mutagen in the method, a mutagen commonly used by those skilled in the art such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, and hydrazine may be used. ..

When transforming a host with the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but an expression vector (for example, a bacterial plasmid, cosmid or cosmid, which is usually used for transformation of prokaryotic cells or eukaryotic cells, or the like. It is more preferable to use a polynucleotide in which the polynucleotide of the present invention is inserted at an appropriate position (plasmid, etc.). The expression vector is not particularly limited as long as it exists stably in the transforming host and can be replicated. When using Escherichia coli as a host, the pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, and pCDF plasmid vector are used. , PBBR plasmid vector can be exemplified.
Further, the appropriate position means a position that does not destroy the replication function of the expression vector, a desired antibiotic marker, and a region related to transmissibility. When inserting the polynucleotide of the present invention into the expression vector, it is preferable to insert it in a state of being linked to a functional polynucleotide such as a promoter required for expression. Examples of the promoter include, when the host is Escherichia coli, a trp promoter, a tac promoter, a trc promoter, a lac promoter, a T7 promoter, a recA promoter, a lpp promoter, a λ phage promoter, a λPR promoter, and the like.

In order to transform a host using an expression vector containing the polynucleotide of the present invention prepared by the above method (hereinafter referred to as the expression vector of the present invention), a method usually used by those skilled in the art may be used. For example, when a microorganism belonging to the genus Escherichia (Escherichia coli JM109 strain, Escherichia coli BL21 (DE3) strain, Escherichia coli W3110 strain, etc.) is selected as a host, known documents (for example, Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992) are selected. ) May be used for transformation. The transformant obtained by transforming by the above-mentioned method is a transformant capable of expressing the Fc-binding protein of the present invention by screening by an appropriate method (hereinafter referred to as the transformant of the present invention). ) Can be obtained. The host expressing the Fc-binding protein of the present invention is not particularly limited, and examples thereof include animal cells (CHO (Chinese Hamster Ovaly) cells, HEK cells, Hela cells, COS cells, etc.), yeast (Schizosaccharomyces cerevisiae, etc.). Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces octosporus, Schizosaccharomyces octopus, Schizosaccharomyces pof, etc. It is preferable to use animal cells or Escherichia coli as a host in terms of productivity, and it is more preferable to use Escherichia coli as a host.

In order to prepare an expression vector of the present invention from the transformant of the present invention, an alkali extraction method or a commercially available QIAprep Spin Miniprep kit (manufactured by Kiagen) or the like is commercially available from the culture obtained by culturing the transformant of the present invention. It may be prepared using the extraction kit of. 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 but also the 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, and when the host is Escherichia coli, an LB (Luria-Bertani) medium supplemented with necessary nutrient sources is preferable. It is given as an example of a medium. In order to selectively grow the transformant of the present invention depending on the presence or absence of the introduction of the 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 the transformant. For example, if the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to carbon, nitrogen and inorganic salt sources, suitable nutrient sources may be added to the medium, optionally selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol. May contain one or more reducing agents. Further, a reagent such as glycine that promotes protein secretion from the transformant into the culture medium may be added. Specifically, when the host is Escherichia coli, it is preferable to add glycine to the medium at a concentration of 2% (w / v) or less. When the host is Escherichia coli, the culture temperature is generally 10 ° C. to 40 ° C., preferably 20 ° C. to 37 ° C., more preferably 25 ° C., but it 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 pH 7.0. When the vector of the present invention contains an inducible promoter, it is preferable to induce the vector under conditions that allow good expression of the Fc-binding protein of the present invention. As the inducer, IPTG (isopropanol-β-D-thiogalactopylanoside) can be exemplified. When the host is Escherichia coli, the turbidity of the culture solution (absorbance at 600 nm) is measured, and when it becomes about 0.5 to 1.0, an appropriate amount of IPTG is added, and then the culture is continued. Can induce protein expression. The concentration of IPTG added 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 IPTG induction may be performed under conditions well known in the art.

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. Then, 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 may be separated by a centrifugation operation, and the Fc-binding protein of the present invention may be purified from the obtained culture supernatant. When expressed in cells (including periplasm), the cells are collected by centrifugation and then crushed by adding an enzyme treatment agent, a surfactant, or the like to disrupt the Fc of the present invention. After extracting the binding protein, it may be purified. In order to purify the Fc-binding protein of the present invention, a method known in the art may be used, and one example is separation / purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and the Fc-binding protein of the present invention can be obtained by a purification operation combining these chromatographies. Can be adjusted to purity. 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 by using the Enzyme-Linked ImmunoSorbent Assay (hereinafter referred to as ELISA) method, surface plasmon resonance method, or the like. You just have to measure. As the IgG used for measuring the binding activity, human IgG is preferable, and any of human IgG1, human IgG2, human IgG3, and human IgG4 may be used.

By binding the Fc-binding protein of the present invention to an insoluble carrier, the antibody adsorbent of the present invention can be produced. The insoluble carrier is not particularly limited, and carriers made from polysaccharides such as agarose, alginate (alginate), carrageenan, chitin, cellulose, dextrin, dextrin, and starch, polyvinyl alcohol, polymethacrate, and poly (2-). Examples thereof include carriers made from synthetic polymers such as hydroxyethyl methacrylate) and polyurethane, and carriers made from ceramics such as silica. Of these, a carrier made from a polysaccharide or a carrier made from a synthetic polymer is preferable as an insoluble carrier. Examples of the preferred carrier include a hydroxyl group-introduced polymethacrylate gel such as Toyopearl (manufactured by Tosoh Corporation), an agarose gel such as Sepharose (manufactured by GE Healthcare), and a cellulose gel such as Cellfine (manufactured by JNC). .. The shape of the insoluble carrier is not particularly limited, and examples thereof include a particle shape, a mesh shape, a flat film shape, a flat plate shape, a fibrous shape, and the like, and these may be either porous or non-porous. Further, the shape (monolith type column) may be integrated with the column container filled with the antibody adsorbent of the present invention.

To immobilize an Fc-binding protein on an insoluble carrier, N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, trecil group, formyl group haloacetamide are used on the insoluble carrier. It may be immobilized by imparting an active group such as, etc., and covalently binding the human Fc-binding protein and the insoluble carrier via the active group. As the carrier to which the active group is added, a commercially available carrier may be used as it is, or the active group may be introduced into the surface of the carrier under appropriate reaction conditions to prepare the carrier. Commercially available carriers to which an active group has been added include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (all manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sephaf Examples thereof include 6B (both manufactured by GE Healthcare) and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

On the other hand, as a method for introducing an active group onto the surface of a carrier, a method of reacting one of compounds having two or more active sites with a hydroxyl group, an epoxy group, a carboxyl group, an amino group or the like existing on the surface of the carrier is exemplified. it can. Among the examples of the compound, epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether can be exemplified as a compound for introducing an epoxy group into a hydroxyl group or an amino group on the surface of the carrier. Examples of the compound for introducing an epoxy group on the carrier surface with the above compound and then introducing 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 for introducing a maleimide group into the hydroxyl group, epoxy group, carboxyl group, and amino group existing on the surface of the carrier include N- (ε-maleimide caproic acid) hydrazide, N- (ε-maleimide propionic acid) hydrazide, and 4- ( 4-N-maleimidephenyl) acetate hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1- (4-aminophenyl) maleimide, 1- (3-aminophenyl) maleimide, 4- (Maleimide) phenylisocyanate, 2-maleimideacetic acid, 3-maleimidepropionic acid, 4-maleimidebutyric acid, 6-maleimidehexanoic acid, N- (α-maleimideacetoxy) succinimide ester, (m-maleimidebenzoyl) N- Hydroxysuccinimide ester, succinimidyl-4- (maleimidemethyl) cyclohexane-1-carbonyl-6-aminohexanoic acid, succinimidyl-4- (maleimidemethyl) cyclohexane-1-carboxylic acid, (p-maleimidebenzoyl) N-hydroxysuccinimide ester , (M-Maleimidebenzoyl) N-hydroxysuccinimide ester can be exemplified.

Compounds that introduce haloacetyl groups into hydroxyl groups and amino groups existing on the surface of the carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetate chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetate anhydride, bromoacetic acid anhydride, and the like. Iodoacetic anhydride, 2- (iodoacetamide) acetic acid-N-hydroxysuccinimide ester, 3- (bromoacetamide) propionic acid-N-hydroxysuccinimide ester, 4- (iodoacetyl) aminobenzoic acid-N-hydroxysuccinimide ester It can be exemplified. A method of reacting the hydroxyl group or amino group existing on the surface of the carrier with the ω-alkenyl alkanoglycidyl ether and then halogenating and activating the ω-alkenyl moiety with a halogenating agent can also be exemplified. Examples of the ω-alkenyl alkaneglycidyl ether include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide. it can.

As another example of the method of introducing an active group on the surface of a carrier, there is a method of introducing an active group into a carboxyl group existing on the surface of a carrier by 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.

Examples of the buffer solution used for immobilizing the Fc-binding protein of the present invention on the insoluble carrier include an acetate buffer solution, a phosphate buffer solution, a MES (2-Morphorinoethanesulfonic acid) buffer solution, and HEPES (2- [4- (2-) Hydroxyethyl) -1-piperazinyl] ethanesulphonic acid) buffer solution, Tris buffer solution, borate buffer solution can be exemplified. The reaction temperature at the time of immobilization may be appropriately set from the temperature range of 5 ° C. to 50 ° C. in consideration of the reactivity of the active group and the stability of the Fc-binding protein of the present invention, preferably. It is in the range of 10 ° C to 35 ° C.

To purify an antibody using the antibody adsorbent of the present invention obtained by immobilizing the Fc-binding protein of the present invention on an insoluble carrier, for example, a buffer containing the antibody in a column packed with the antibody adsorbent of the present invention. Is added using a liquid feeding means such as a pump to specifically adsorb the antibody to the antibody adsorbent of the present invention, and then an appropriate eluate may be added to the column to elute the antibody. The antibody that can be purified by the antibody adsorbent of the present invention may be an antibody that contains at least the Fc region of an antibody that has an affinity for an Fc-binding protein. Examples thereof include chimeric antibodies, humanized antibodies, human antibodies and their amino acid substitutions, which are generally used as antibodies used in antibody drugs. In addition, it is an artificially structurally modified antibody such as a bispecific antibody (bispecific antibody), a fusion antibody between the Fc region of an antibody and another protein, and a complex (ADC) between the Fc region of an antibody and a drug. Can also be purified by the antibody adsorbent of the present invention. It is also preferable to equilibrate the column with an appropriate buffer before adding the buffer containing the antibody to the column, because the antibody can be purified to a higher purity. Examples of the buffer solution include a buffer solution containing an inorganic salt as a component, such as a phosphate buffer solution, and the pH of the buffer solution is pH 3.0 to 10.0, preferably pH 5.0 to 8.0.

In order to elute the antibody adsorbed on the antibody adsorbent of the present invention, the interaction between the antibody and the ligand (Fc-binding protein of the present invention) may be weakened. Examples thereof include changes in peptide, temperature, and salt concentration. Specific examples of the eluate for eluting the antibody adsorbed on the adsorbent of the present invention include a buffer solution on the more acidic side than the solution used for adsorbing the antibody on the antibody adsorbent of the present invention. Examples of the type of buffer include a citric acid buffer, a glycine-hydrochloric acid buffer, and an acetate buffer having a buffering ability on the acidic side. The pH of the buffer solution 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, and even more preferably pH 3.3 to 4. It is 0.

The Fc-binding protein of the present invention is a protein lacking an amino acid residue at a specific position in the extracellular region of native human FcγRIIb. The Fc-binding protein of the present invention has improved acid stability as compared to native human FcγRIIb. Therefore, the Fc-binding protein of the present invention is useful as a ligand for an adsorbent for separating an antibody (immunoglobulin).

It is a schematic diagram of human FcγRIIb. The numbers in the figure indicate the positions of the amino acid sequences shown in SEQ ID NO: 1. In the figure, S indicates a signal sequence, EC indicates an extracellular region, TM indicates a transmembrane region, and C indicates an intracellular region. It is a figure which shows the result of having evaluated the acid resistance of the Fc-binding protein which deleted 5 amino acid residues with respect to the Fc-binding protein (Control) which consists of the amino acid sequence shown in SEQ ID NO: 2.

Hereinafter, examples will be shown in order to explain the present invention in more detail, but the present invention is not limited to these examples.

Example 1 Preparation of amino acid residue deletion Amino acid located in the loop region of FcγRIIb based on the knowledge of the crystal structure data (PDB ID: 2FCB) of FcγRIIb registered in a known database for improving acid stability. The residue was deleted. Specifically, in the amino acid sequence shown in SEQ ID NO: 2, an Fc bond in which the amino acid residue located in the loop region (the region from the 73rd aspartic acid to the 81st threonine of SEQ ID NO: 2) is deleted. The polypeptides shown in (a) to (f) below, which are sex proteins, were prepared.
(A) Of the Fc-binding proteins shown in SEQ ID NO: 2, the Fc-binding protein lacking proline at position 63 (SEQ ID NO: 4, named FcγRIIb-Del-Pro63).
(B) Among the Fc-binding proteins shown in SEQ ID NO: 2, the Fc-binding protein lacking the 61st histidine and the 62nd serine (SEQ ID NO: 6, named FcγRIIb-Del-His61-Ser62).
(C) Of the Fc-binding proteins shown in SEQ ID NO: 2, the Fc-binding protein lacking serine at position 62 and proline at position 63 (SEQ ID NO: 8, named FcγRIIb-Del-Ser62-Pro63).
(D) Among the Fc-binding proteins shown in SEQ ID NO: 2, the Fc-binding protein lacking the 61st histidine, the 62nd serine and the 63rd proline (SEQ ID NO: 10, FcγRIIb-Del-His61-). Named Pro63)
(E) Of the Fc-binding proteins shown in SEQ ID NO: 2, Fc-binding proteins lacking proline at position 63, glutamic acid at position 64, and serine at position 65 (SEQ ID NO: 12, FcγRIIb-Del-Pro63-). Named Ser65)
(F) Of the Fc-binding proteins shown in SEQ ID NO: 2, the Fc-binding protein lacking the 61st histidine, the 62nd serine, the 63rd proline, the 64th glutamic acid, and the 65th serine ( SEQ ID NO: 14, named FcγRIIb-Del-His61-Ser65)
(1) A polynucleotide encoding the Fc-binding protein of (a) to (f) ((a) FcγRIIb-Del-Pro63: SEQ ID NO: 5, (b) FcγRIIb-Del-His61-Ser62: SEQ ID NO: 7, (C) FcγRIIb-Del-Ser62-Pro63: SEQ ID NO: 9, (d) FcγRIIb-Del-His61-Pro63: SEQ ID NO: 11, (e) FcγRIIb-Del-Pro63-Ser65: SEQ ID NO: 13, (f) FcγRIIb- DelHis61-Ser65: SEQ ID NO: 15) were prepared by artificial gene synthesis (requested by Fasmac).

(2) The polynucleotide prepared in (1) was ligated to an expression vector pETMalE (Japanese Patent Laid-Open No. 2011-206046) digested with restriction enzymes NcoI and HindIII, and Escherichia coli BL21 (DE3) strain was transformed using this. did.

(3) The obtained transformants were inoculated into 3 mL of 2YT liquid medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 50 μg / mL kanamycin, respectively, at 37 ° C. In the evening, preculture was performed by aerobically shaking culture.

(4) 600 μL of the preculture solution of (3) was inoculated into 20 mL of 2YT liquid medium supplemented with 50 μg / mL kanamycin, and aerobic shaking culture was performed at 37 ° C.

(5) 1.5 hours after the start of culturing, the culturing temperature was changed to 20 ° C. and the cultivated with shaking for 30 minutes. Then, IPTG (isopropanol-β-D-thiogalactopylanoside) was added so as to have a final concentration of 0.05 mM, followed by aerobic shaking culture at 20 ° C. overnight to express Fc-binding protein.

Example 2 Acid resistance evaluation of amino acid residue deletion product (1) After culturing Example 1 (5), the culture solution was centrifuged, and 20 μL of the culture supernatant containing each Fc-binding protein obtained and 0. 80 μL of 1 M glycine hydrochloride buffer (pH 3.0) was mixed, and the mixture was allowed to stand at 25 ° C. for 24 hours.

(2) The antibody-binding activity of the Fc-binding protein when the acid treatment of (1) is performed and the antibody-binding activity of the Fc-binding protein when the acid treatment of (1) is not performed are shown below. Residual activity is calculated by dividing the antibody-binding activity of the Fc-binding protein when acid-treated by the antibody-binding activity of the Fc-binding protein when not acid-treated, as measured by the ELISA method. did.
(2-1) A gamma globulin preparation (manufactured by the Institute of Chemistry and Serum Therapy), which is a human antibody, was immobilized at 1 μg / well in a well of a 96-well microplate (18 hours at 4 ° C.), and after the immobilization was completed, 2 Blocking was performed with 20 mM Tris-hydrochloric acid buffer (pH 7.4) containing% (w / v) SKIM MILK (manufactured by BD) and 150 mM sodium chloride.
(2-2) After washing with a washing buffer (20 mM Tris-HCl buffer (pH 7.4) containing 0.05% [w / v] Protein 20 (trade name) and 150 mM NaCl), antibody binding is performed. A solution containing the Fc-binding protein whose activity was evaluated was added, and the Fc-binding protein was reacted with immobilized gamma globulin (1 hour at 30 ° C.).
(2-3) After completion of the reaction, the reaction was washed with the washing buffer, and Anti-6His antibody (manufactured by Bethyl Laboratories) diluted to 100 ng / mL was added at 100 μL / well.
(2-4) After reacting at 30 ° C. for 1 hour and washing with the washing buffer, 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 the absorbance at 450 nm was measured with a microplate reader (manufactured by Tecan).

The results are shown in FIG. The amino acid residue deletion products prepared in Example 1 (SEQ ID NOs: 4, 6, 8, 10, 12 and 14) all have higher residual activity than the native FcγRIIb (SEQ ID NO: 2) and are resistant to acids. It was confirmed that the stability was improved.

Claims (10)

Fc-binding protein selected from any of the following (i) to (iii):
(I) Among the amino acid sequences shown in SEQ ID NO: 2, at least the amino acid residues from the 29th threonine to the 201st glutamine are contained, but the amino acid residues from the 29th to the 201st are as follows (1). An Fc-binding protein having at least one deletion of any of the amino acid residues shown in (5) to (5);
(1) Deletion of the 63rd proline of SEQ ID NO: 2 (2) Deletion of the 61st histidine of SEQ ID NO: 2 (3) Deletion of the 62nd serine of SEQ ID NO: 2 (4) 64 of SEQ ID NO: 2 Deletion of the 65th glutamic acid (5) Deletion of the 65th serine of SEQ ID NO: 2 (ii) Of the amino acid sequences shown in SEQ ID NO: 2, at least the amino acid residues from the 29th threonine to the 201st glutamine Including, however, the 29th to 201st amino acid residues have at least one deletion of any of the amino acid residues shown in (1) to (5) above, and further, from (1) to (1) above. In addition to the deletion of amino acid residues shown in 5), it further has one or more of substitution, deletion, insertion and addition of one or several amino acid residues at one or several positions. Fc-binding protein having antibody-binding activity;
(Iii) In the amino acid sequence from the 29th glutamine to the 201st glutamine of the amino acid sequence shown in SEQ ID NO: 2, at least one deletion of any of the amino acid residues shown in (1) to (5) above is made. An Fc-binding protein having an amino acid sequence having 70% or more homology to the entire amino acid sequence having the above, containing an amino acid sequence in which the deletion of at least one amino acid residue remains, and having antibody-binding activity. .. The Fc-binding protein according to claim 1, which has at least a deletion of any of the amino acid residues shown in (1) and (6) to (10) below.
(1) Deletion of the 63rd proline of SEQ ID NO: 2 (6) Deletion of the 61st histidine and 62nd serine of SEQ ID NO: 2 (7) Deletion of the 62nd serine and 63rd proline of SEQ ID NO: 2 Deletion (8) Deletion of 61st histidine, 62nd serine and 63rd proline of SEQ ID NO: 2 (9) Deletion of 63rd proline, 64th glutamic acid and 65th serine of SEQ ID NO: 2 (10) Deletion of 61st histidine, 62nd serine, 63rd proline, 64th glutamic acid and 65th serine of SEQ ID NO: 2 The Fc-binding protein according to claim 1 or 2, selected from (iv) to (vi) below:
(Iv) An Fc-binding protein containing at least an amino acid residue from threonine at position 29 to glutamine at position 201 in the amino acid sequence set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14;
(V) Of the amino acid sequences set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14, the amino acid residues from the 29th threonine to the 201st glutamine are contained at least, but from the 29th position. Among the substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions in addition to the deletion of the amino acid residue of the amino acid sequence in the amino acid residues up to the 201st position. An Fc-binding protein further comprising any one or more and having antibody-binding activity;
(Vi) Of the amino acid sequences set forth in any of SEQ ID NOs: 4, 6, 8, 10, 12 and 14, the amino acid residues from the 29th threonine to the 201st glutamine are contained at least, but from the 29th position. An Fc-binding protein having 70% or more homology to the amino acid sequence up to the 201st amino acid sequence, residual deletion of the amino acid residue of the amino acid sequence, and antibody-binding activity. A polynucleotide encoding the Fc-binding protein according to any one of claims 1 to 3. An expression vector containing the polynucleotide according to claim 4. A transformant capable of producing an Fc-binding protein obtained by transforming a host with the recombinant vector according to claim 5. The transformant according to claim 6, wherein the host is Escherichia coli. Fc binding comprising a step of producing an Fc-binding protein by culturing the transformant according to claim 6 or 7 and a step of recovering the Fc-binding protein produced from the obtained culture. Method for producing sex protein. An antibody adsorbent obtained by immobilizing the Fc-binding protein according to any one of claims 1 to 3 on an insoluble carrier. A step of adding a solution containing an antibody to a column packed with the antibody adsorbent according to claim 9 to adsorb the antibody to the adsorbent, and a step of eluting the antibody adsorbed on the adsorbent using an eluate. Methods for separating antibodies, including.

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