JP2024126228A - Fc-binding protein with improved solubility, method for producing said protein, and antibody adsorbent using said protein - Google Patents

Abstract

To provide an Fc-binding protein with improved solubility relative to traditional human FcRn, a method for producing the protein, and an antibody adsorbent including the protein.SOLUTION: The foregoing problem is solved by an Fc-binding protein in which the β2-microglobulin domain of the β chain of the human neonatal Fc receptor (FcRn), the extracellular domain of the α chain of FcRn, and the β2-microglobulin domain of the FcRn β chain are sequentially linked from the N-terminus, a method for producing the protein, and an antibody adsorbent including the protein.SELECTED DRAWING: Figure 3

Description

The present invention relates to an Fc-binding protein that has binding affinity for the Fc region of immunoglobulin (antibody), a method for producing the protein, and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier. More specifically, the present invention relates to an Fc-binding protein that has improved solubility compared to conventional Fc-binding proteins, a method for producing the protein, and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier.

Fc receptors are receptor proteins that bind to the Fc region of immunoglobulin molecules, and transmit signals within the cell by binding to immune complexes between antigens and immunoglobulins (Non-Patent Document 1). Each molecule recognizes a single or same group of immunoglobulin isotypes through a recognition domain on the Fc receptor, which belongs to the immunoglobulin superfamily. This determines which accessory cells are mobilized in the immune response.

Fc receptors can be further classified into several subtypes, including Fcγ receptors, which are receptors for immunoglobulin G (IgG), as well as Fcα receptors and Fcε receptors. Each receptor is further classified into Fcγ receptors, which can be classified into the following subtypes: FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b) (Non-Patent Documents 1 and 2).

On the other hand, human neonatal Fc receptor (FcRn) is a major histocompatibility complex (MHC) class I-related molecule that is different from human Fcγ receptors that belong to the immunoglobulin superfamily, and is composed of a heavy chain (α chain) and β2 microglobulin (β chain) (Non-Patent Document 3). FcRn is involved in the IgG recycling mechanism and has the function of suppressing IgG degradation. In addition, FcRn binds to IgG in a pH-dependent manner, binding at pH 6.5 or lower (Non-Patent Document 4).

The amino acid sequence of the α-chain of human FcRn (SEQ ID NO: 1) has been published in public databases such as UniProt (Accession number: P55899). The amino acid sequence of the β-chain (SEQ ID NO: 2) has also been published in UniProt (Accession number: P61769). Furthermore, the structural functional domains of human FcRn, the signal peptide sequence for penetrating the cell membrane, and the location of the cell membrane-transducing region have also been published. Figure 1 shows a schematic structure of the α-chain of human FcRn, and Figure 2 shows a schematic structure of the β-chain of human FcRn. The amino acid numbers in Figure 1 correspond to the amino acid numbers in SEQ ID NO: 1. That is, in SEQ ID NO:1, the sequence from the 1st methionine (M) to the 23rd glycine (G) is the signal sequence (S), the sequence from the 24th alanine (A) to the 297th serine (S) is the extracellular domain (EC), the sequence from the 298th valine (V) to the 321st tryptophan (W) is the transmembrane domain (TM), and the sequence from the 322nd arginine (R) to the 365th alanine (A) is the intracellular domain (C). The amino acid numbers in FIG. 2 correspond to the amino acid numbers described in SEQ ID NO:2. That is, the sequence from the 1st methionine (M) to the 20th alanine (A) in SEQ ID NO:2 is the signal sequence (S), and the sequence from the 21st isoleucine (I) to the 119th methionine (M) is the β2 microglobulin (B2M).

For industrial application of FcRn, it is preferable that it is highly soluble and highly stable against heat and acid from the viewpoint of use and storage. Patent Document 1 discloses a highly stable FcRn in which the stability against heat or acid is improved by introducing a mutation compared to natural human FcRn. Non-Patent Document 5 discloses an example of expression of FcRn using genetic engineering techniques, in which FcRn is expressed at an expression level of 10 mg/L using animal cells such as HEK293 cells as a host. However, when attempting to express FcRn using E. coli as a host, which can be produced more easily and inexpensively than animal cells, it was difficult to express the FcRn in a large amount in a soluble state.

JP 2018-183087 A

Takai T. , Jpn. J. Clin. Immunol. , 28, 318-326, 2005 J. Galon et al. , Eur. J. Immunol. , 27, 1928-1932, 1997 N. E. Simister et al. , Nature, 337, 184-187, 1989 M. Raghavan et al. , Biochemistry, 34, 14649-14657, 1995 Y. Feng et al. , Protein ExprPurif. , 79(1), 66-71, 2011

The objective of the present invention is to provide an Fc-binding protein with improved solubility relative to conventional human FcRn, a method for producing said protein, and an antibody adsorbent using said protein.

As a result of intensive research by the inventors to solve the above problems, they discovered that by forming a fusion protein by linking the extracellular domain of the human neonatal Fc receptor (FcRn) α-chain with the β2-microglobulin domain of two molecules of human FcRn β-chain, the solubility of the protein is improved compared to conventional human FcRn when expressed using genetic engineering techniques, and thus completed the present invention.

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

[1] An Fc-binding protein that is linked in the following order from the N-terminus: the β2 microglobulin domain of the human neonatal Fc receptor (FcRn) β chain, the extracellular domain of the FcRn α chain, and the β2 microglobulin domain of the FcRn β chain.

[2] The Fc-binding protein according to [1], wherein the extracellular region of the FcRn α chain is selected from any one of the following (A-1) to (A-3):
(A-1) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1. (A-2) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the amino acid residues have undergone one or more of the following substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions, and which has antibody binding activity. (A-3) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the polypeptide has a homology of 70% or more to an amino acid sequence consisting of the amino acid residues, and which has antibody binding activity.

[3] The Fc binding protein according to [1] or [2], wherein the β2 microglobulin region of the FcRn β chain is selected from any one of the following (B-1) to (B-3):
(B-1) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2; (B-2) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that the amino acid residues have undergone one or more of the following substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions, and which has antibody binding activity; (B-3) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that the polypeptide has a homology of 70% or more to an amino acid sequence consisting of the amino acid residues, and which has antibody binding activity.

[4] A polynucleotide encoding the Fc-binding protein according to any one of [1] to [3]. [5] An expression vector comprising the polynucleotide according to [4].

[6] A transformant capable of producing an Fc-binding protein, obtained by transforming a host with the expression vector described in [5].

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

[8] A method for producing an Fc-binding protein, comprising the steps of expressing an Fc-binding protein by culturing the transformant according to [6] or [7], and recovering the expressed Fc-binding protein from the resulting culture.

[9] An antibody adsorbent comprising an insoluble carrier and an Fc-binding protein according to any one of [1] to [3] immobilized on the carrier.

[10] A method for separating antibodies, comprising the steps of adding a solution containing an antibody to a column packed with the adsorbent described in [9] to adsorb the antibody to the adsorbent, and eluting the antibody adsorbed to the adsorbent with an elution solution.

The Fc-binding protein of the present invention is a fusion protein in which the β2 microglobulin region (B2M region) of the human neonatal Fc receptor (FcRn) β chain, the extracellular region (EC region) of the FcRn α chain, and the B2M region are linked in this order, and is a protein with improved solubility compared to native human FcRn. The Fc-binding protein of the present invention can be produced particularly efficiently using a transformant obtained by transforming Escherichia coli with an expression vector containing a polynucleotide encoding the protein.

The Fc-binding protein of the present invention is also useful as a ligand for an adsorbent for separating antibodies (immunoglobulins).

1 is a schematic diagram of the α-chain of human FcRn, in which the numbers indicate the amino acid sequence numbers set forth in SEQ ID NO: 1. In the diagram, S indicates a signal sequence, EC indicates an extracellular domain, TM indicates a transmembrane domain, and C indicates an intracellular domain. 1 is a schematic diagram of the β-chain of human FcRn, in which the numbers indicate the amino acid sequence numbers set forth in SEQ ID NO: 2. In the diagram, S indicates a signal sequence, and B2M indicates a β2 microglobulin region. 1 is a diagram comparing an Fc-binding protein of the present invention consisting of the amino acid sequence set forth in SEQ ID NO: 3 with an Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 5. The numbers in the diagram indicate the amino acid sequence numbers in each Fc-binding protein. In the diagram, S indicates the MalE signal peptide sequence, B2M indicates the β2 microglobulin region of the human FcRn β chain, L indicates the GS linker sequence, EC indicates the extracellular region of the human FcRn α chain, and 6H indicates the histidine tag sequence.

The present invention is described in detail below.

The Fc-binding protein of the present invention is a fusion protein whose components are the extracellular domain of the FcRn α chain (hereinafter also referred to as the "EC domain") and the β2 microglobulin domain of the FcRn β chain (hereinafter also referred to as the "B2M domain"), and is a protein in which the B2M domain-EC domain-B2M domain are linked in this order from the N-terminus. The B2M domain-EC domain and the EC domain-B2M domain may be linked directly or via a known linker such as a GS linker (a linker consisting of a repeating oligopeptide consisting of four glycine (G) residues and one serine (S) residue).

A preferred embodiment of the EC region of the present invention is a polypeptide selected from any one of the following (A-1) to (A-3):
(A-1) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1. (A-2) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the amino acid residues have undergone one or more of the following substitutions, deletions, insertions and additions (hereinafter collectively referred to as "modifications") of one or several amino acid residues at one or several positions, and which has antibody-binding activity. (A-3) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the polypeptide has 70% or more homology to an amino acid sequence consisting of the amino acid residues, and which has antibody-binding activity.

In addition, a preferred embodiment of the B2M region in the present invention is a polypeptide selected from any one of the following (B-1) to (B-3):
(B-1) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2; (B-2) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that one or several amino acid residues are modified at one or several positions in the amino acid residues, and having antibody binding activity; (B-3) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that the polypeptide has a homology of 70% or more to the amino acid sequence consisting of the amino acid residues, and having antibody binding activity.

Therefore, in the present invention, the EC region may include all or part of the signal peptide region (S region in Figure 1) located on its N-terminus, and may include all or part of the transmembrane region (TM region in Figure 1) and extracellular region (C region in Figure 1) located on its C-terminus. In addition, in the present invention, the B2M region may include all or part of the signal peptide region (S region in Figure 2) located on its N-terminus.

In the above (A-2) and (B-2), the term "one or several" varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of the amino acid residue. For example,
In the case of the EC region, this means any of 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1;
In the case of the B2M area, this means any of the following: 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.

The modification of "one or several" amino acid residues (one or more of substitution, deletion, insertion, and addition) may occur at positions other than those disclosed in, for example, JP 2018-183087 A, JP 2021-073883 A, JP 2021-136967 A, and JP 2022-076998 A, as long as the modification has immunoglobulin (antibody) binding activity.

The "modification of one or several amino acid residues (one or more of substitution, deletion, insertion, and addition)" in (A-2) and (B-2) may include, in addition to the amino acid modification at the specific position described above, conservative substitutions in which substitution occurs between amino acids with similar physical and/or chemical properties. It is generally known to those skilled in the art that conservative substitutions maintain the function of a protein between a substituted and unsubstituted protein. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). The "modification of one or more of substitution, deletion, insertion, and addition" described in (A-2) and (B-2) also includes naturally occurring mutations (mutants or variants) based on individual differences, species differences, etc. of the microorganism from which the gene is derived.

As an example of (A-2) and (B-2),
Fc-binding proteins disclosed in JP2018-183087A;
Fc binding protein disclosed in JP2021-073883A,
Fc-binding proteins disclosed in JP 2021-136967 A and Fc-binding proteins disclosed in JP 2022-076998 A;
Examples include:

The homology of the amino acid sequences in (A-3) and (B-3) may be 70% or more, and may be higher (e.g., 80% or more, 85% or more, 90% or more, or 95% or more). In this specification, "homology" may mean similarity or identity, and may particularly mean identity. "Homology of amino acid sequences" means homology with respect to the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31 (3), Yodosha). "Similarity" between amino acid sequences means the sum of the ratio of amino acid residues of the same type in those amino acid sequences and the ratio of amino acid residues with similar side chain properties (Experimental Medicine, 31 (3), Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

The Fc-binding protein of the present invention has a total of two molecules of the B2M region, one on the N-terminus side and one on the C-terminus side, and the amino acid sequence of the B2M region on the N-terminus side may be the same as or different from the amino acid sequence of the B2M region on the C-terminus side.

The Fc-binding protein of the present invention may further have an oligopeptide added to its N-terminus or C-terminus that is useful for separating the Fc-binding protein of the present invention from a solution in the presence of contaminants. Examples of the oligopeptide include polyhistidine, polylysine, polyarginine, polyglutamic acid, polyaspartic acid, and the like. Furthermore, an oligopeptide containing cysteine that is useful for immobilizing the Fc-binding protein of the present invention on a solid phase such as a support for chromatography may further be added to the N-terminus or C-terminus of the Fc-binding protein of the present invention. There is no particular limit to the length of the oligopeptide added to the N-terminus or C-terminus of the Fc-binding protein, as long as it does not impair the antibody binding ability or stability of the Fc-binding protein of the present invention. When the oligopeptide is added to the Fc-binding protein of the present invention, a polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to add it to the N-terminus or C-terminus of the Fc-binding protein using a method well known to those skilled in the art, or a chemically synthesized oligopeptide may be chemically bonded to the N-terminus or C-terminus of the Fc-binding protein of the present invention. Furthermore, a signal peptide may be added to the N-terminus of the Fc-binding protein of the present invention to promote efficient expression in the host. Examples of the signal peptide when the host is Escherichia coli include signal peptides that secrete proteins into the periplasm, such as PelB, DsbA, MalE (the region from the 1st to the 26th amino acid sequence described in UniProt No. P0AEX9), and TorT (JP Patent Publication No. 2011-097898).

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),
<A> 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;
<B> A method in which a polynucleotide containing the entire or partial sequence of an Fc-binding protein is prepared artificially directly or from the cDNA of the Fc-binding protein by using a DNA amplification method such as PCR, and the prepared polynucleotides are then ligated by an appropriate method.

In the method of <A>, when converting an amino acid sequence to a nucleotide sequence, it is preferable to convert taking into consideration the frequency of codon usage in the host to be transformed. As an example, when the host is Escherichia coli, AGA/AGG/CGG/CGA for arginine (R), ATA for isoleucine (I), CTA for leucine (L), GGA for glycine (G), and CCC for proline (P) are used infrequently (so-called rare codons), so conversion should be made to avoid these codons. Analysis of codon usage can also be done by using a public database (for example, the Codon Usage Database on the website of the Kazusa DNA Research Institute).

When transforming a host using the polynucleotide of the present invention, the polynucleotide of the present invention itself may be used, but it is more preferable to use an expression vector (e.g., a bacteriophage, cosmid, or plasmid, which is usually used for transforming prokaryotic or eukaryotic cells) into which the polynucleotide of the present invention has been inserted at an appropriate position. The expression vector is not particularly limited as long as it can stably exist and replicate in the host to be transformed. When Escherichia coli is used as the host, examples of the expression vector include pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector, and pBBR plasmid vector. The appropriate position means a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, and the 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 where it is linked to a functional polynucleotide such as a promoter required for expression. Examples of such promoters, when the host is Escherichia coli, include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and further the λPL promoter and λPR promoter of λ phage.

To transform a host using the expression vector (hereinafter referred to as the expression vector of the present invention) into which the polynucleotide of the present invention is inserted, which is prepared by the above-mentioned method, a method commonly used by those skilled in the art may be used. For example, when a microorganism belonging to the genus Escherichia (E. coli JM109 strain, E. coli BL21 (DE3) strain, E. coli W3110 strain, etc.) is selected as a host, the host may be transformed by a method described in a known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). The transformant obtained by transformation by the above-mentioned method can be screened by an appropriate method to obtain a transformant capable of expressing the Fc-binding protein of the present invention (hereinafter referred to as the transformant of the present invention). There are no particular limitations on the host in which the Fc-binding protein of the present invention is expressed, and examples include animal cells (CHO (Chinese Hamster Ovary) cells, HEK cells, HeLa cells, COS cells, etc.), yeast (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, etc.), insect cells (Sf9, Sf21, etc.), Escherichia coli (JM109 strain, BL21 (DE3) strain, W3110 strain, etc.), and Bacillus subtilis. In terms of productivity, it is preferable to use animal cells or E. coli as a host, and it is even more preferable to use E. coli as a host.

To prepare the expression vector of the present invention from the transformant of the present invention, the vector may be prepared from the culture obtained by culturing the transformant of the present invention by an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (Qiagen). The Fc-binding protein of the present invention can be produced by expressing the Fc-binding protein of the present invention by culturing the transformant of the present invention and recovering the expressed Fc-binding protein of the present invention from the resulting culture. In this specification, the culture includes not only the cells of the transformant of the present invention that have been cultured, 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. When the host is Escherichia coli, LB (Luria-Bertani) medium supplemented with necessary nutrient sources is an example of a preferred medium. In order to selectively grow the transformant of the present invention depending on the presence or absence of 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, when the vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to carbon, nitrogen and inorganic salt sources, the medium may contain an appropriate nutrient source, and may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate and dithiothreitol, if desired. Furthermore, a reagent such as glycine that promotes protein secretion from the transformant into the culture medium may be added. Specifically, when the host is E. coli, it is preferable to add glycine to the medium at 2% (w/v) or less. When the host is E. coli, the culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, more preferably around 25°C, but may be selected depending on the characteristics of the protein to be expressed. When the host is E. coli, the pH of the medium is pH 6.8 to 7.4, preferably around pH 7.0. When the vector of the present invention contains an inducible promoter, it is preferable to induce it under conditions that allow the Fc-binding protein of the present invention to be well expressed. An example of an inducer is IPTG (IsoPropyl-β-D-ThioGalactopyranoside). When the host is Escherichia coli, the turbidity of the culture solution (absorbance at 600 nm) is measured, and when it reaches about 0.5 to 1.0, an appropriate amount of IPTG is added and the culture is continued, thereby inducing expression of the Fc-binding protein. The concentration of IPTG added may be appropriately selected from the range of 0.005 mM to 1.0 mM, but is preferably in the range of 0.01 mM to 0.5 mM. Various conditions for IPTG induction may be performed under conditions well known in the art.

To recover the Fc-binding protein of the present invention expressed by the transformant from the culture obtained by culturing the transformant, the Fc-binding protein of the present invention may be recovered by separating/purifying the protein from the culture using a method suitable for the expression form of the Fc-binding protein of the present invention in the transformant. For example, when the protein is expressed in the culture supernatant, the cells may be separated by centrifugation, and the Fc-binding protein of the present invention may be purified from the resulting culture supernatant. When the protein is expressed intracellularly (including the periplasm), the cells may be collected by centrifugation, and then the cells may be disrupted by adding an enzyme treatment agent or a surfactant, and the Fc-binding protein of the present invention may be extracted and purified. 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, and the like, and the Fc-binding protein of the present invention may be prepared to a high purity by performing a purification operation using a combination of these chromatographies. The binding activity of the obtained Fc-binding protein of the present invention to an antibody can be measured, for example, by measuring the binding activity to immunoglobulin G (IgG) using an enzyme-linked immunosorbent assay (hereinafter referred to as ELISA) method or a surface plasmon resonance method. The IgG used to measure the binding activity is preferably human IgG, and any of human IgG1, human IgG2, human IgG3, and human IgG4 may be used.

The antibody adsorbent of the present invention (hereinafter, also simply referred to as "the adsorbent of the present invention") can be produced by binding (immobilizing) the Fc-binding protein of the present invention to an insoluble carrier. The insoluble carrier is not particularly limited, and examples thereof include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch, carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane, and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with hydroxyl groups introduced such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC Corporation). There are no particular limitations on the shape of the insoluble carrier, and it may be granular or non-granular, porous or non-porous.

To immobilize the Fc-binding protein of the present invention on an insoluble carrier, an active group such as an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetamide (iodoacetamide, bromoacetamide, etc.) may be imparted to the insoluble carrier, and the human Fc-binding protein may be covalently bonded to the insoluble carrier via the active group, thereby immobilizing the protein. A commercially available carrier having an active group imparted thereto may be used as is, or the carrier may be prepared by introducing the active group onto the carrier surface under appropriate reaction conditions. Examples of commercially available carriers to which active groups have been added include TOYOPEARL AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by GE Healthcare), and SulfoLink Coupling Resin (manufactured by Thermo Scientific).

On the other hand, an example of a method for introducing active groups onto the carrier surface is a method in which one of the compounds having two or more active sites is reacted with a hydroxy group, epoxy group, carboxy group, amino group, or the like present on the carrier surface. Examples of such compounds that introduce epoxy groups into hydroxy groups or amino groups on the carrier surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of compounds that introduce carboxy groups onto the carrier surface after introducing epoxy groups onto the carrier surface using the above compounds include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

Compounds that introduce maleimide groups to hydroxyl, epoxy, carboxyl, or amino groups present on the support surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-6-aminohexanoic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the support surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester. Another example of a method is to react hydroxyl or amino groups present on the support surface with an ω-alkenylalkane glycidyl ether, and then activate the ω-alkenyl moiety by halogenating it with a halogenating agent. Examples of ω-alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, and examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

Another example of a method for introducing active groups onto the carrier surface is to introduce active groups onto carboxy groups present on the carrier surface using a condensation agent and an additive. Condensation agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenzotriazole.

Buffer solutions used when immobilizing the Fc-binding protein of the present invention on an insoluble carrier include acetate buffer, phosphate buffer, MES (2-MorpholinoEthaneSulfonic acid) buffer, HEPES (2-[4-(2-HydroxyEthyl)-1-Piperazinyl]EthaneSulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature during immobilization may be appropriately set within the range of 5°C to 50°C, taking into consideration the reactivity of the active group and the stability of the Fc-binding protein of the present invention, and is preferably in the range of 10°C to 35°C.

To purify (separate) an antibody using the adsorbent of the present invention obtained by immobilizing the Fc-binding protein of the present invention on an insoluble carrier, for example, a buffer containing the antibody is added to a column packed with the adsorbent of the present invention using a liquid delivery means such as a pump, so that the antibody is specifically adsorbed to the adsorbent of the present invention, and then an appropriate eluent is added to the column to elute the antibody. The antibody that can be purified with the adsorbent of the present invention may be any antibody that contains at least the Fc region of an antibody that has affinity with the Fc-binding protein. Examples include chimeric antibodies, humanized antibodies, human antibodies, and amino acid substitutions thereof, which are commonly used as antibodies for antibody drugs. In addition, even artificially structured antibodies such as bispecific antibodies, fusion antibodies of the Fc region of an antibody with other proteins, and complexes (ADCs) of the Fc region of an antibody with a drug can be purified with the adsorbent of the present invention. In addition, it is preferable to equilibrate the column with an appropriate buffer before adding a buffer containing the antibody to the column, because this allows the antibody to be purified to a higher purity. Examples of buffer solutions include buffer solutions containing inorganic salts, such as phosphate buffer solutions, and the pH of the buffer solution is from pH 3.0 to pH 10.0, preferably from pH 5.0 to pH 8.0.

To elute the antibody adsorbed to the adsorbent of the present invention, the interaction between the antibody and the ligand (the Fc-binding protein of the present invention) may be weakened, and specific examples include a pH change by a buffer solution, a counter peptide, a temperature change, and a salt concentration change. Specific examples of elution solutions for eluting the antibody adsorbed to the adsorbent of the present invention include buffer solutions that are more acidic than the solution used to adsorb the antibody to the adsorbent of the present invention. Examples of types of buffer solutions include citrate buffer, glycine hydrochloride buffer, and acetate buffer, which have buffering capacity on the acidic side. The pH of the buffer solution may be set within a range that does not impair the functions of the antibody, and is preferably pH 4.0 or more and pH 8.0 or less, more preferably pH 5.0 or more and pH 6.0 or less.

The following examples are provided to further explain the present invention, but the present invention is not limited to these examples.

Example 1: Preparation of an expression vector for the Fc-binding protein of the present invention Using the method described below, a vector capable of expressing the Fc-binding protein of the present invention (SEQ ID NO: 3) was prepared, which contains a polynucleotide (SEQ ID NO: 4) encoding the Fc-binding protein of the present invention, in which the β2 microglobulin region (B2M region) of the human neonatal Fc receptor (FcRn) β chain, the extracellular region (EC region) of the FcRn α chain, and the B2M region are linked in this order from the N-terminus via a linker. Note that, among the Fc-binding proteins consisting of the amino acid sequence described in SEQ ID NO: 3, the amino acid sequence of the B2M region (amino acid residues 29 to 127 and 447 to 545 of SEQ ID NO: 3) is identical to the amino acid sequence of the native B2M region (amino acid residues 21 to 117 of SEQ ID NO: 2), and the EC region (amino acid residues 148 to 421 of SEQ ID NO: 3) is a polypeptide in which the following seven amino acid substitutions have been introduced into the native EC region (amino acid residues 24 to 297 of SEQ ID NO: 1);
Cysteine (C) at position 195 of SEQ ID NO:3 (position 71 in SEQ ID NO:1) is substituted with arginine (R). Asparagine (N) at position 202 of SEQ ID NO:3 (position 78 in SEQ ID NO:1) is substituted with aspartic acid (D). Glycine (G) at position 275 of SEQ ID NO:3 (position 151 in SEQ ID NO:1) is substituted with aspartic acid (D). Arginine (R) at position 316 of SEQ ID NO:3 (position 192 in SEQ ID NO:1) is substituted with leucine (L). Asparagine (N) at position 320 of SEQ ID NO:3 (position 196 in SEQ ID NO:1) is substituted with aspartic acid (D). Glutamine (Q) at position 356 of SEQ ID NO:3 (position 232 in SEQ ID NO:1) is substituted with leucine (L). Lysine (K) at position 419 of SEQ ID NO:3 (position 295 in SEQ ID NO:1) is substituted with glutamic acid (E).

(1) A polynucleotide was artificially synthesized by adding an NcoI recognition sequence (CCATGG) to the 5' end of the nucleotide sequence shown in SEQ ID NO:4 and a HindIII recognition sequence (AAGCTT) to the 3' end of the nucleotide sequence.

(2) The polynucleotide synthesized in (1) was digested with the restriction enzymes NcoI and HindIII, and ligated to the expression vector pETMalE (JP Patent Publication No. 2011-206046) that had been previously digested with NcoI and HindIII, and the ligation product was used to transform E. coli BL21 (DE3).

(3) The obtained transformant was cultured in LB (Luria-Bertani) medium containing 50 μg/mL kanamycin, and then a vector capable of expressing the Fc-binding protein described in SEQ ID NO:3 was extracted using a QIAprep Spin Miniprep kit (Qiagen) (named pETMalE-FcRn(B2M-EC(m7)-B2M)).

(4) Of the expression vector pETMalE-FcRn (B2M-EC(m7)-B2M) prepared in (3), the polynucleotide encoding FcRn and its surrounding region were subjected to cycle sequencing reaction using the Big Dye Terminator Cycle Sequencing FS read Reaction kit (Thermo Fisher Scientific) based on the chain terminator method, and the nucleotide sequence was analyzed using a fully automated DNA sequencer ABI Prism 3700 DNA analyzer (Thermo Fisher Scientific). In this analysis, the oligonucleotides shown in SEQ ID NO: 6 (5'-TAATACGACTCACTATAGGG-3'), SEQ ID NO: 7 (5'-ATGCTAGTTATTGCTCAGCGG-3'), or SEQ ID NO: 8 (5'-AGAGGATTCGGCAGGGGATTC-3') were used as sequencing primers.

The amino acid sequence of the polypeptide expressed by the expression vector pETMalE-FcRn (B2M-EC(m7)-B2M) is shown in SEQ ID NO: 3, and the sequence of the polynucleotide encoding said polypeptide is shown in SEQ ID NO: 4. In SEQ ID NO: 3, the region from the 1st methionine (M) to the 26th alanine (A) is the MalE signal peptide (the region from the 1st to the 26th of UniProt No. P0AEX9), the 27th methionine (M) and the 28th glycine (G) are linkers, the region from the 29th isoleucine (I) to the 127th methionine (M) is the B2M region, the region from the 128th glycine (G) to the 147th serine (S) is the GS linker (a linker consisting of repeated oligopeptides consisting of 4 glycine (G) residues and 1 serine (S) residue), and the region from the 148th The region from the alanine (A) at position 421 to the serine (S) at position 421 is the EC region with the seven substitutions introduced as described above, the region from the glycine (G) at position 422 to the serine (S) at position 446 is the GS linker, the region from the isoleucine (I) at position 447 to the methionine (M) at position 545 is the B2M region, the glycines (G) at positions 546 and 547 are the linker, and the histidine (H) at positions 548 to 553 is the histidine tag.

Example 2 Preparation of Fc-binding protein of the present invention (1) Escherichia coli (transformant) BL21(DE3) strain transformed with the expression vector pETMalE-FcRn(B2M-EC(m7)-B2M) prepared in Example 1 was 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, and cultured overnight at 37° C. with shaking.

(2) After cultivation, 3 mL of the culture was transferred to 100 mL of 2YT liquid medium and cultured with shaking at 37°C for 2 hours. Protein expression was then induced with 0.05 mM IPTG (IsoPropyl-β-D-ThioGalactopyranoside), and the mixture was further cultured with shaking overnight at 20°C.

(3) After cultivation, the medium was removed by centrifugation and the E. coli cells were collected.

(4) A disruption solution (50 mM Tris-HCl buffer (pH 8.0) containing 150 mM sodium chloride) was added to the resulting E. coli in an amount five times the volume of the cells, and the E. coli was disrupted using an ultrasonic disrupter (Tomy Seiko Co., Ltd.). The supernatant was separated by centrifugation, and a protein extract of the soluble fraction was prepared.

(5) The extract from (4) was applied to a Ni-Sepharose (Cytiva) column, washed with a washing buffer (20 mM Tris-HCl buffer (pH 8.0) containing 150 mM sodium chloride and 5 mM imidazole), and then eluted with an elution buffer (20 mM Tris-HCl buffer (pH 8.0) containing 150 mM sodium chloride and 250 mM imidazole).

(6) After confirming by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) that the Fc-binding protein contained in the soluble fraction was present in the elution fraction of (5), the fraction was concentrated and the imidazole was removed using a desalting column. The amount of Fc-binding protein contained in the soluble fraction was quantified by the Bradford method to be 8.2 mg.

Comparative Example 1
A transformant capable of expressing the Fc-binding protein FcRn_m7 (SEQ ID NO: 5) prepared by the method described in JP 2018-183087 A was used as the transformant, and the Fc-binding protein was expressed by the method described in Example 2, and the Fc-binding protein contained in the soluble fraction was purified and quantified. As a result, the amount of Fc-binding protein contained in the soluble fraction was 0.12 mg.

Figure 3 shows a conceptual diagram showing the difference in amino acid sequence between the Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, evaluated in Example 2, and the Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 5, evaluated in Comparative Example 1. It can be seen that the solubility of the Fc-binding protein in the form of a fusion protein (SEQ ID NO: 3) in which the B2M region, EC region, and BM2 region are linked in this order from the N-terminus is improved compared to the form of a fusion protein (SEQ ID NO: 5) in which the B2M region and EC region are linked in this order from the N-terminus.

Claims (10)

An Fc-binding protein that is linked in the following order from the N-terminus: the β2 microglobulin domain of the human neonatal Fc receptor (FcRn) β chain, the extracellular domain of the FcRn α chain, and the β2 microglobulin domain of the FcRn β chain. The Fc binding protein according to claim 1, wherein the extracellular region of the FcRn α chain is selected from any one of the following (A-1) to (A-3):
(A-1) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1. (A-2) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the amino acid residues have undergone one or more of the following substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions, and which has antibody binding activity. (A-3) A polypeptide comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO:1, provided that the polypeptide has a homology of 70% or more to an amino acid sequence consisting of the amino acid residues, and which has antibody binding activity. The Fc binding protein according to claim 1, wherein the β2 microglobulin region of the FcRn β chain is selected from any one of the following (B-1) to (B-3):
(B-1) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2; (B-2) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that the amino acid residues have undergone one or more of the following substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions, and which has antibody binding activity; (B-3) A polypeptide comprising at least the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, provided that the polypeptide has a homology of 70% or more to an amino acid sequence consisting of the amino acid residues, and which has antibody binding activity. A polynucleotide encoding an Fc-binding protein according to any one of claims 1 to 3. An expression vector comprising the polynucleotide of claim 4. A transformant capable of producing an Fc-binding protein, obtained by transforming a host with the expression vector according to claim 5. The transformant according to claim 6, wherein the host is Escherichia coli. A method for producing an Fc-binding protein, comprising the steps of expressing an Fc-binding protein by culturing the transformant according to claim 6, and recovering the expressed Fc-binding protein from the resulting culture. An antibody adsorbent comprising an insoluble carrier and an Fc-binding protein according to any one of claims 1 to 3 immobilized on the carrier. A method for separating an antibody, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent according to claim 9 to adsorb the antibody to the adsorbent; and eluting the antibody adsorbed to the adsorbent with an elution solution.