Fc-binding protein with improved acid stability, method for producing the protein, and antibody adsorbent using the protein

Abstract

To provide an Fc-binding protein having improved stability against acid compared to natural human FcRn, a method for producing the protein, and an antibody adsorbent including the protein.SOLUTION: An Fc-binding protein at least includes an extracellular region of an amino acid sequence (UniProt No. P55899) of natural human FcRnα chain and a β2 microglobulin region in an amino acid sequence (UniProt No. P61789) of human FcRnβ chain. In amino acid residues of the regions, at least amino acids at specific sites are deleted. There are also provided a method for producing the protein, and an antibody adsorbent including the protein.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to an Fc-binding protein having binding affinity for immunoglobulin G (IgG). More specifically, by deleting amino acid residues at specific positions in the extracellular region of the α-chain of the human neonatal Fc receptor (human FcRn) or the β2-microglobulin region of the human FcRn β-chain, an Fc-binding protein having improved acid stability compared to the native human FcRn, a method for producing the protein, and an antibody adsorbent obtained by immobilizing the protein on an insoluble carrier are provided.

Background Art

[0002] Fc receptors are receptor proteins that bind to the Fc region of immunoglobulin molecules, and bind to immune complexes of antigens and immunoglobulins to conduct signal transduction into cells (Non-Patent Document 1). Each molecule recognizes a single or the same group of immunoglobulin isotypes by a recognition domain belonging to the immunoglobulin superfamily on the recognition domain of the Fc receptor. This determines which accessory cells are activated in the immune response.

[0003] Fc receptors can be further classified into several subtypes, including Fcγ receptors, which are receptors for immunoglobulin G (IgG), Fcα receptors, Fcε receptors, and the like. Each receptor is further classified in detail. In the case of Fcγ receptors, they can be classified into subtypes of FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b) (Non-Patent Documents 1 and 2).

[0004] On the other hand, human neonatal Fc receptor (FcRn) is a major histocompatibility complex (MHC) class I-related molecule, distinct from the human Fcγ receptor which belongs to the immunoglobulin superfamily, and is composed of a heavy chain (α chain) and β2 microglobulin (β chain) (Non-Patent Literature 3). FcRn is involved in the IgG recycling mechanism and has the function of suppressing IgG degradation. Furthermore, FcRn binds to IgG in a pH-dependent manner, binding at pH 6.5 or below (Non-Patent Literature 4).

[0005] The amino acid sequence of the α-chain of human FcRn (SEQ ID NO: 1) is published in public databases such as UniProt (Accession number: P55899). The amino acid sequence of the β-chain (SEQ ID NO: 2) is also published in UniProt (Accession number: P61769). Furthermore, the structural functional domains of human FcRn, the signal peptide sequence for transmembrane transport, and the location of the transmembrane region are also published. Figure 1 shows a schematic diagram of the α-chain of human FcRn, and Figure 2 shows a schematic diagram of the β-chain of human FcRn. The amino acid numbers in Figure 1 correspond to the amino acid numbers listed in SEQ ID NO: 1. Specifically, the first methionine (Met) to the 23rd glycine (Gly) in Sequence ID No. 1 constitutes the signal sequence (S), the 24th alanine (Ala) to the 297th serine (Ser) constitutes the extracellular domain (EC), the 298th valine (Val) to the 321st tryptophan (Trp) constitutes the transmembrane domain (TM), and the 322nd arginine (Arg) to the 365th alanine (Ala) constitutes the intracellular domain (C). Furthermore, the amino acid numbers in Figure 2 correspond to the amino acid numbers listed in Sequence ID No. 2. Specifically, the first methionine (Met) to the 20th alanine (Ala) in Sequence ID No. 2 constitutes the signal sequence (S), and the 21st isoleucine (Ile) to the 119th methionine (Met) constitutes β2-microglobulin (B2M).

[0006] For industrial applications of FcRn, high stability against acids is desirable from the standpoint of use and storage. Patent Document 1 discloses a mutation of natural human FcRn that improves its stability against heat or acid. However, further improvement in acid stability was necessary for industrial applications of human FcRn. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2018-183087 [Non-patent literature]

[0008] [Non-Patent Document 1] Takai T.,Jpn.J.Clin.Immunol.,28,318-326,2005 [Non-Patent Document 2] J.Galon et al.,Eur.J.Immunol.,27,1928-1932,1997 [Non-Patent Document 3] NESimister et al.,Nature,337,184-187,1989 [Non-Patent Document 4] M.Raghavan et al.,Biochemistry,34,14649-14657,1995 [Overview of the project] [Problems that the invention aims to solve]

[0009] The object of the present invention is to provide an Fc-binding protein with improved acid stability compared to natural human FcRn, a method for producing the protein, and an antibody adsorbent using the protein. [Means for solving the problem]

[0010] The inventors of this invention conducted diligent research to solve the above problems and, as a result, identified an amino acid residue involved in improving the acid stability of human FcRn. They found that a mutant lacking this amino acid residue exhibits excellent acid stability, thus completing the present invention.

[0011] In other words, the present invention encompasses the embodiments described in [1] to

[13] below.

[0012] [1] Fc-binding proteins selected from any of (i) to (iii) below: (i) Fc-binding proteins that include at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in SEQ ID NO: 2, wherein the amino acid residues have at least one of the mutations shown in (1) to (5) below; (1) A mutation in which tryptophan at position 82 of sequence number 1 is deleted. (2) A mutation in which tryptophan at position 74 of sequence number 1 is deleted. (3) A mutation in which valine at position 75 of sequence number 1 is deleted. (4) A mutation in which glutamic acid at position 77 of sequence number 1 is deleted. (5) A mutation in which tyrosine at position 83 of sequence number 1 is deleted. (ii) An Fc-binding protein having antibody-binding activity, comprising at least the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence described in Sequence ID No. 2, wherein the amino acid residues have at least one of the mutations described in (1) to (5) above, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions other than those described in (1) to (5) above; (iii) An amino acid sequence having 70% or more homology to an entire amino acid sequence having at least one of the mutations described in (1) to (5) above, in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in Sequence ID No. 1 and from the 21st isoleucine to the 119th methionine of the amino acid sequence described in Sequence ID No. 2, provided that the amino acid sequence contains at least one of the mutations described in (1) to (5) above, and is an Fc-binding protein having antibody-binding activity.

[0013] [2] Fc-binding proteins selected from any of (iv) to (vi) below: (iv) Fc-binding proteins that include at least the amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein at least one of the mutations shown in (1) to (5) below is present in the amino acid residues from the 29th to the 426th; (1) A mutation in which tryptophan at position 87 of sequence number 3 is deleted. (2) A mutation in which tryptophan at position 79 of sequence number 3 is deleted. (3) A mutation in which valine at position 80 of sequence number 3 is deleted. (4) A mutation in which glutamic acid at position 82 of sequence number 3 is deleted. (5) A mutation in which tyrosine at position 88 of sequence number 3 is deleted. (v) An Fc-binding protein having antibody-binding activity, comprising at least the amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th have at least one of the mutations described in (1) to (5) above, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions other than those described in (1) to (5) above; (vi) An amino acid sequence having at least 70% homology with the entire amino acid sequence from alanine at position 29 to methionine at position 426 in the amino acid sequence set forth in SEQ ID NO: 3, provided that it contains an amino acid sequence in which at least one of the mutations shown in (1) to (5) remains, and is an Fc-binding protein having antibody-binding activity.

[0014] [3] The Fc-binding protein according to [1] or [2], having at least the following mutation (1); (1) A mutation in which the tryptophan at position 82 of SEQ ID NO: 1 or position 87 of SEQ ID NO: 3 is deleted.

[0015] [4] The Fc-binding protein according to any one of [1] to [3], further having all of the mutations shown in (6) to (12) below; (6) A mutation in which the cysteine at position 71 of SEQ ID NO: 1 or position 76 of SEQ ID NO: 3 is substituted with arginine (7) A mutation in which the asparagine at position 78 of SEQ ID NO: 1 or position 83 of SEQ ID NO: 3 is substituted with aspartic acid (8) A mutation in which the glycine at position 151 of SEQ ID NO: 1 or position 156 of SEQ ID NO: 3 is substituted with aspartic acid (9) A mutation in which the arginine at position 192 of SEQ ID NO: 1 or position 197 of SEQ ID NO: 3 is substituted with leucine (10) A mutation in which the asparagine at position 196 of SEQ ID NO: 1 or position 201 of SEQ ID NO: 3 is substituted with aspartic acid (11) A mutation in which the glutamine at position 232 of SEQ ID NO: 1 or position 237 of SEQ ID NO: 3 is substituted with leucine (12) A mutation in which the lysine at position 295 of SEQ ID NO: 1 or position 300 of SEQ ID NO: 3 is substituted with glutamic acid.

[0016] [5] The Fc-binding protein according to [4], further having at least one of the mutations shown in (13) to (22) below; (13) A mutation in which the 167th glutamine of SEQ ID NO: 1 or the 172nd glutamine of SEQ ID NO: 3 is replaced by glutamic acid (14) A mutation in which the 26th lysine of SEQ ID NO: 2 or the 333rd lysine of SEQ ID NO: 3 is replaced by isoleucine (15) A mutation in which the 80th tryptophan of SEQ ID NO: 2 or the 387th tryptophan of SEQ ID NO: 3 is replaced by serine (16) A mutation in which the 50th serine of SEQ ID NO: 1 or the 55th serine of SEQ ID NO: 3 is replaced by valine (17) A mutation in which the 68th alanine of SEQ ID NO: 1 or the 73rd alanine of SEQ ID NO: 3 is replaced by valine (18) A mutation in which the 93rd isoleucine of SEQ ID NO: 1 or the 98th isoleucine of SEQ ID NO: 3 is replaced by threonine (19) A mutation in which the 216th phenylalanine of SEQ ID NO: 1 or the 221st phenylalanine of SEQ ID NO: 3 is replaced by serine (20) A mutation in which the 279th histidine of SEQ ID NO: 1 or the 284th histidine of SEQ ID NO: 3 is replaced by arginine (21) A mutation in which the 99th alanine of SEQ ID NO: 2 or the 406th alanine of SEQ ID NO: 3 is replaced by valine (22) A mutation in which the 116th aspartic acid of SEQ ID NO: 2 or the 423rd aspartic acid of SEQ ID NO: 3 is replaced by glutamic acid.

[0017] [6] The Fc-binding protein according to [4], which is selected from any one of the following (vii) to (ix): (vii) An Fc-binding protein comprising at least the amino acid residues from the 29th alanine to the 425th methionine among the amino acid sequences set forth in any of SEQ ID NOs: 22, 26, 30, 34, 37, 39, 41, 43, 47, 51 and 55; (viii) An Fc-binding protein having antibody-binding activity, comprising at least the amino acid residues from the 29th alanine to the 425th methionine in the amino acid sequence described in any of Sequence IDs 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55, wherein the amino acid residues from the 29th to the 425th methionine further have one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions other than the mutations in the aforementioned amino acid sequence; (ix) An Fc-binding protein that contains at least the amino acid residues from the 29th alanine to the 425th methionine of the amino acid sequence described in any of Sequence IDs 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55, provided that it has 70% or more homology to the amino acid sequence from the 29th to the 425th, retains the mutations present in the amino acid sequence, and has antibody-binding activity.

[0018] A polynucleotide encoding an Fc-binding protein as described in any of [7][1] to [6].

[0019] An expression vector containing the polynucleotides described in [8][7].

[0020] A transformant capable of producing Fc-binding proteins, obtained by transforming a host with the recombinant vector described in [9][8].

[0021]

[10] The transformant described in [9], wherein the host is Escherichia coli.

[0022] A method for producing an Fc-binding protein, comprising the steps of: producing an Fc-binding protein by culturing a transformant described in

[11] [9] or

[10] ; and recovering the Fc-binding protein produced from the obtained culture.

[0023] An antibody adsorbent obtained by immobilizing an Fc-binding protein described in any of

[12] [1] to [6] on an insoluble carrier.

[0024] A method for separating antibodies, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent described in

[13] and

[12] to adsorb the antibody onto the adsorbent; and eluting the antibody adsorbed onto the adsorbent using an eluent.

[0025] The present invention will be described in detail below.

[0026] The Fc-binding protein of the present invention is a protein that binds to the Fc region of an antibody, and contains at least the amino acid residues shown in (I) and (II) below, wherein an amino acid deletion (hereinafter also referred to as "mutation" in this specification) has occurred at a specific position in said amino acid residue. (I) Amino acid residues from alanine at position 24 to serine at position 297, corresponding to the extracellular region (EC region in Figure 1) of the human FcRnα chain consisting of the amino acid sequence described in Sequence ID No. 1 (II) Amino acid residues from isoleucine at position 21 to methionine at position 119, corresponding to the β2 microglobulin region of the human FcRnβ chain (region B2M in Figure 2) consisting of the amino acid sequence described in Sequence ID No. 2 Therefore, the Fc-binding protein of the present invention may include all or part of the signal peptide region (region S in Figures 1 and 2) located at the N-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain or the β2 microglobulin region (region B2M in Figure 2) of the human FcRnβ chain, or it may include all or part of the transmembrane region (region TM in Figure 1) and extracellular region (region C in Figure 1) located at the C-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain.

[0027] In this specification, an Fc-binding protein containing at least the amino acid residues shown in (I) and (II) above means that the amino acid sequence of the protein contains at least the amino acid sequence shown in (I) and the amino acid sequence shown in (II), and the order of the amino acid residues shown in (I) and (II) is irrelevant. That is, the amino acid residue shown in (II) may be on the N-terminal side or the C-terminal side of the amino acid residue shown in (I). Furthermore, the amino acid residues shown in (I) and (II) may be directly linked, or they may be linked via a known linker such as a GS linker (a linker consisting of repeating Gly-Gly-Gly-Ser).

[0028] In this specification, "natural FcRn" is not limited to naturally occurring FcRn, but also includes FcRn containing at least the amino acid residues shown in (I) and (II) above, and in which there are no substitutions, deletions, insertions, or additions of amino acid residues in the amino acid residues shown in (I) and (II). As an example, an Fc-binding protein consisting of the amino acid sequence described in Sequence ID No. 3 is provided, in which the amino acid residues shown in (I) and (II) are linked via a linker sequence. Of Sequence ID No. 3, the first 1 to 26 are the MalE signal peptide sequence (an oligopeptide consisting of amino acid residues 1 to 26 of UniProt No. P0AEX9), the 27th methionine and 28th glycine are linker sequences, the 29th to 302nd are the extracellular region of the FcRnα chain (EC region in Figure 1; region 24 to 297 of Sequence ID No. 1), the 303rd to 327th are GS linker sequences, the 328th to 426th are the β2 microglobulin region of the FcRnβ chain (B2M region in Figure 2; region 21 to 119 of Sequence ID No. 2), the 427th and 428th glycine are linker sequences, and the 429th to 434th are histidine tag sequences.

[0029] Specifically, the mutation at the aforementioned specific position is, in the case of an Fc-binding protein containing at least the amino acid residues shown in (I) and (II) above, an Fc-binding protein consisting of the amino acid sequence described in SEQ ID NO: 3, at least one of the following mutations: ΔTrp79 (this notation indicates the deletion of tryptophan at position 79 in SEQ ID NO: 3, and so on), ΔVal80, ΔGlu82, ΔTrp87, and ΔTyr88. Among these, ΔTrp87 is a mutation that particularly improves stability against acid, so an Fc-binding protein containing at least the ΔTrp87 mutation can be said to be a preferred embodiment of the Fc-binding protein of the present invention. The mutations at the aforementioned specific positions in SEQ ID NO: 1 are, respectively, ΔTrp79 at position 74, ΔVal80 at position 75, ΔGlu82 at position 77, ΔTrp87 at position 82, and ΔTyr88 at position 83. Table 1 shows the correspondence of amino acid residue positions in SEQ ID NOs: 1 and 3.

[0030] [Table 1]

[0031] The Fc-binding protein of the present invention only needs to have at least one mutation at the specific site described above, and as long as it has antibody-binding activity, it may further have one or more of the following: substitution, deletion, insertion, and addition of amino acid residues, in addition to the mutation at the specific site described above. Specific examples of the above embodiments include the Fc-binding proteins having antibody-binding activity shown in (i) to (v) below. When deleting amino acid residues, it is preferable to delete them in such a way that a loop region in the protein's secondary structure is removed, as this improves heat resistance (Manandez-Alias ​​and P. Argos, J.Mol.Biol., 206, 1989).

[0032] (i) Fc-binding proteins that, in addition to the mutations at the specific sites mentioned above, further have mutations in Cys76Arg (this notation indicates that the cysteine ​​at position 76 in SEQ ID NO: 3 (position 71 in SEQ ID NO: 1) is replaced with arginine, and so on), Asn83Asp (position 78 in SEQ ID NO: 1), Gly156Asp (position 151 in SEQ ID NO: 1), Arg197Leu (position 192 in SEQ ID NO: 1), Asn201Asp (position 196 in SEQ ID NO: 1), Glu237Leu (position 232 in SEQ ID NO: 1), and Lys300Glu (position 295 in SEQ ID NO: 1). (ii) Fc-binding proteins having, in addition to the mutation at the specific site mentioned above, one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more sites (provided that at least one of the mutations at the specific site mentioned above remains). (iii) Fc-binding proteins that, in addition to the mutations at the specific sites mentioned above, further have one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more sites (provided that at least one of the mutations at the specific sites mentioned above remains), and further have all of the following mutations: Cys76Arg, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Glu237Leu, and Lys300Glu. (iv) An Fc-binding protein having an amino acid sequence that has 70% or more homology to the entire amino acid sequence of a polypeptide having the aforementioned mutation at a specific site (provided that at least one of the aforementioned mutations at a specific site remains). (v) An Fc-binding protein having an amino acid sequence that has 70% or more homology to the entire amino acid sequence of a polypeptide having the aforementioned mutation at a specific site (provided that at least one of the aforementioned mutations at a specific site remains), provided that the Fc-binding protein has all of the following mutations: Cys76Arg, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Glu237Leu, and Lys300Glu. Of these, the mutations in Cys76Arg, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Glu237Leu, and Lys300Glu described in (i), (iii), and (v) above are mutations that improve thermal stability and productivity by genetically modified organisms (Japanese Patent Publication No. 2018-183087). Therefore, by further adding the seven mutations (Cys76Arg, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Glu237Leu, and Lys300Glu) to the Fc-binding protein of the present invention which has at least one of the aforementioned mutations at specific positions, a protein with improved thermal and acid stability than the Fc-binding protein disclosed in Japanese Patent Publication No. 2018-183087 can be obtained.

[0033] In (ii) and (iii) above, "one or several" means one or more amino acid residues, although this can vary depending on the position and type of amino acid residues in the three-dimensional structure of the protein. For example, it can mean 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3. Furthermore, "one or more of the substitutions, deletions, insertions, and additions" described in (ii) and (iii) above also include naturally occurring mutations (mutants or variants) based on individual differences in the microorganisms from which the gene originates, differences in species, etc. Examples of "substitutions" described in (ii) and (iii) above include Gln172Glu (position 167 in SEQ ID NO: 1), Lys333Ile (position 26 in SEQ ID NO: 2), and Trp387Ser (position 80 in SEQ ID NO: 2). These substitutions improve thermal stability, and by introducing them into the Fc-binding protein of the present invention, a protein with improved stability against heat and acid can be obtained. Furthermore, other examples of the "substitutions" described in (ii) and (iii) above include Ser55Val (number 50 in SEQ ID NO: 1), Ala73Val (number 68 in SEQ ID NO: 1), Ile98Thr (number 93 in SEQ ID NO: 2), Phe221Ser (number 216 in SEQ ID NO: 1), His284Arg (number 279 in SEQ ID NO: 2), Ala406Val (number 99 in SEQ ID NO: 2), and Asp423Glu (number 116 in SEQ ID NO: 2). These substitutions improve acid stability, and by introducing them into the Fc-binding protein of the present invention, a protein with further improved acid stability can be obtained.

[0034] The homology of the amino acid sequences in (iv) and (v) above is sufficient if it is 70% or more, and may have a higher homology (for example, 80% or more, 85% or more, 90% or more, or 95% or more).

[0035] The Fc-binding protein of the present invention may further have conservative substitutions between amino acids whose physical and / or chemical properties are similar. It is known to those skilled in the art that conservative substitutions, not limited to Fc-binding proteins, generally maintain the function of a protein between those with and without substitution. Examples of conservative substitutions include those occurring between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005).

[0036] The Fc-binding protein of the present invention may have an oligopeptide added to its N-terminus or C-terminus, which is useful for separating it from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Furthermore, a cysteine-containing oligopeptide, useful for immobilizing the Fc-binding protein of the present invention onto a solid phase such as a support for chromatography, may be added to the N-terminus or C-terminus of the Fc-binding protein of the present invention. The length of the oligopeptide added to the N-terminus or C-terminus of the Fc-binding protein is not particularly limited as long as it does not impair the IgG binding ability or stability of the Fc-binding protein of the present invention. When adding the oligopeptide to the Fc-binding protein of the present invention, a polynucleotide encoding the oligopeptide may be created and then genetically engineered and added 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 such signal peptides when the host is E. coli include signal peptides that induce protein secretion into the periplasm, such as PelB, DsbA, MalE (the region from amino acid 1 to 26 in the amino acid sequence described in UniProt No. P0AEX9), and TorT (Japanese Patent Publication No. 2011-097898).

[0037] A preferred embodiment of the Fc-binding protein of the present invention is an Fc-binding protein comprising at least a polypeptide consisting of the amino acid sequences shown in (a) to (k) below. These Fc-binding proteins are preferred in that they have improved stability against heat and acid (heat resistance and acid resistance).

[0038] (a) FcRn-m7ΔW79 (amino acid residues from position 29 to 425 in the amino acid sequence described in Sequence ID No. 22) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th are mutated by Cys76Arg, ΔTrp79, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Gln237Leu, and Lys300Glu.

[0039] (b) FcRn-m7ΔV80 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 26) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th methionine are mutated as follows: Cys76Arg, ΔVal80, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Gln237Leu, and Lys300Glu.

[0040] (c)FcRn-m7ΔE82 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 30) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th are mutated as Cys76Arg, ΔGlu82, Asn83Asp, Gly156Asp, Arg197Leu, Asn201Asp, Gln237Leu, and Lys300Glu.

[0041] (d) FcRn-m7ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 34) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th are mutated as Cys76Arg, Asn83Asp, ΔTrp87, Gly156Asp, Arg197Leu, Asn201Asp, Gln237Leu, and Lys300Glu.

[0042] (e) FcRn-m7ΔY88 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 37) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th methionine are mutated by Cys76Arg, Asn83Asp, ΔTyr88, Gly156Asp, Arg197Leu, Asn201Asp, Gln237Leu, and Lys300Glu.

[0043] (f)FcRn-m9ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 39) A polypeptide comprising the amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th are mutated as follows: Cys76Arg, Asn83Asp, ΔTrp87, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, and Lys333Ile.

[0044] (g)FcRn-m10ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 41) A polypeptide comprising amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th are mutated as follows: Cys76Arg, Asn83Asp, ΔTrp87, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, Lys333Ile, and Trp387Ser.

[0045] (h)FcRn-m11ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 43) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th methionine are mutated as follows: Cys76Arg, Asn83Asp, ΔTrp87, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, Lys333Ile, Trp387Ser, and Asp423Glu.

[0046] (i) FcRn-m12ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 47) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th amino acid residues are mutated as follows: Cys76Arg, Asn83Asp, ΔTrp87, Ile98Thr, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, Lys333Ile, Trp387Ser, and Asp423Glu.

[0047] (j)FcRn-m13ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 51) A polypeptide comprising amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th amino acid residues are mutated as follows: Cys76Arg, Asn83Asp, ΔTrp87, Ile98Thr, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, Lys333Ile, Trp387Ser, Ala406Val, and Asp423Glu.

[0048] (k)FcRn-m14ΔW87 (amino acid residues from position 29 to 425 in the amino acid sequence described in SEQ ID NO: 55) The polypeptide is the amino acid residue from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th amino acid residues are mutated as follows: Ser55Val, Cys76Arg, Asn83Asp, ΔTrp87, Ile98Thr, Gly156Asp, Gln172Glu, Arg197Leu, Asn201Asp, Gln237Leu, Lys300Glu, Lys333Ile, Trp387Ser, Ala406Val, and Asp423Glu.

[0049] Of the Fc-binding proteins described in SEQ ID NOs. 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55, the first methionine to the 26th alanine is the MalE signal peptide, the 27th methionine and the 28th glycine are the linker sequence, the 29th alanine to the 301st serine is the amino acid sequence of the FcRnα chain (the extracellular region (EC) of SEQ ID NO. 1), the 302nd to the 326th is the GS linker sequence, the 327th isoleucine to the 425th methionine is the amino acid sequence of the FcRnβ chain (the β2 microglobulin region (B2M) of SEQ ID NO. 2), the 426th and 427th glycines are the linker sequence, and the 428th to the 433rd histidine are the tag sequence.

[0050] 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 for converting the amino acid sequence of the Fc-binding protein of the present invention into a nucleotide sequence, and artificially synthesizing a polynucleotide containing said nucleotide sequence, (B) An example of a method is to prepare polynucleotides containing the whole or partial sequence of an Fc-binding protein directly or artificially, or from the cDNA of an Fc-binding protein using a DNA amplification method such as PCR, and then ligate the prepared polynucleotides in an appropriate manner.

[0051] In the method described in (A) above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to consider the frequency of codon use in the host being transformed. For example, if the host is Escherichia coli, the codons AGA / AGG / CGG / CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) are used infrequently (they are so-called rare codons), so the conversion should avoid these codons. Codon usage frequency can also be analyzed using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website).

[0052] When introducing mutations 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 they are conditions that can introduce the desired mutation into the polynucleotide encoding human FcRn (or Fc-binding protein). For example, by making the concentrations of the four types of deoxynucleotides (dATP / dTTP / dCTP / dGTP) that are substrates heterogeneous and adding MnCl2 at a concentration of 0.01 to 10 mM (preferably 0.1 to 1 mM) to the PCR reaction mixture and performing PCR, mutations can be introduced into the polynucleotide. In addition to the error-prone PCR method, methods for introducing mutations into polynucleotides other than the error-prone PCR method include contacting and acting on a polynucleotide containing the whole or partial sequence of human FcRn with a mutagenic agent or irradiating it with ultraviolet light. As the agent used as the mutagenic agent in this method, any mutagenic agent commonly used by those skilled in the art may be used, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, etc.

[0053] 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 (for example, a bacteriophage, cosmid, or plasmid commonly used for the transformation of 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 within the host to be transformed. When E. coli is used as the host, examples include the pET plasmid vector, pUC plasmid vector, pTrc plasmid vector, pCDF plasmid vector, and pBBR plasmid vector. The appropriate position refers to a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region related to transduction. 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 necessary for expression. Examples of such promoters when the host is E. coli include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and also the λPL promoter and λPR promoter of λ phage.

[0054] To transform a host using an expression vector containing the polynucleotide of the present invention (hereinafter referred to as the "expression vector of the present invention"), which was prepared by the method described above, the procedure can be carried out by methods commonly used by those skilled in the art. For example, when selecting a microorganism belonging to the genus Escherichia (such as Escherichia coli strain JM109, Escherichia coli strain BL21(DE3), or Escherichia coli strain W3110) as the host, transformation can be carried out by methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). By screening the transformants obtained by the method described above using an appropriate method, transformants capable of expressing the Fc-binding protein of the present invention (hereinafter referred to as the "transformants of the present invention") can be obtained. There are no particular restrictions on the host used to express the Fc-binding protein of the present invention. 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. Using animal cells or Escherichia coli as the host is preferable in terms of productivity, and using Escherichia coli as the host is even more preferable.

[0055] To prepare the expression vector of the present invention from the transformant of the present invention, it may be prepared from the culture obtained by culturing the transformant of the present invention using an alkaline extraction method or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen). 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 this specification, the culture includes not only the cultured cells of the transformant of the present invention themselves, but also the culture medium used for cultivation. The transformant used in the protein production method of the present invention should be cultured in a medium suitable for culturing the target host, and in the case of E. coli, LB (Luria-Bertani) medium supplemented with the necessary nutrients is an example of a preferred medium. In order to selectively grow the transformant of the present invention depending on whether or not the vector of the present invention is introduced, it is preferable to add a drug corresponding to the drug resistance gene contained in the vector to the culture medium. For example, if the vector contains a kanamycin resistance gene, kanamycin should be added to the culture medium. In addition to carbon, nitrogen, and inorganic salt sources, the culture medium may also contain appropriate nutrients, and optionally, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. Furthermore, reagents that promote protein secretion from the transformants to the culture medium, such as glycine, may be added. Specifically, when the host is E. coli, it is preferable to add glycine to the culture medium at a concentration of 2% (w / v) or less. The culture temperature when the host is E. coli is generally 10°C to 40°C, preferably 20°C to 37°C, more preferably around 25°C, but should be selected according to the characteristics of the protein to be expressed. The pH of the culture medium when the host is E. coli is pH 6.8 to pH 7.4, preferably around pH 7.0. If the vector of the present invention contains an inducible promoter, it is preferable to induce it under conditions that allow for good expression of the Fc-binding protein of the present invention. An example of an inducible agent is IPTG (isopropyl-β-D-thiogalactopyranoside).When the host is E. coli, the turbidity of the culture medium (absorbance at 600 nm) is measured, and when it reaches approximately 0.5 to 1.0, an appropriate amount of IPTG is added, followed by continued cultivation to induce the expression of Fc-binding proteins. The concentration of IPTG can be appropriately selected from the range of 0.005 to 1.0 mM, but the range of 0.01 to 0.5 mM is preferred. Various conditions for IPTG induction can be carried out using conditions that are well known in the art.

[0056] To recover the Fc-binding protein of the present invention from a culture obtained by culturing the transformant of the present invention, the Fc-binding protein of the present invention can be recovered by separating and purifying it from the culture using a method suitable for the expression mode of the Fc-binding protein of the present invention in the transformant of the present invention. For example, if the protein is expressed in the culture supernatant, the bacterial cells can be separated by centrifugation, and the Fc-binding protein of the present invention can be purified from the resulting culture supernatant. If the protein is expressed intracellularly (including in the periplasm), the bacterial cells can be collected by centrifugation, then the cells can be disrupted by adding an enzyme treatment agent or surfactant to extract the Fc-binding protein of the present invention, and then it can be purified. To purify the Fc-binding protein of the present invention, any method known in the art can be used, and one example is separation and purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and by combining these chromatography methods for purification, the Fc-binding protein of the present invention can be prepared in high 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 can be measured using the Enzyme-Linked ImmunoSorbent Assay (ELISA) method or surface plasmon resonance method. The IgG used for measuring the binding activity is preferably human IgG, and any of human IgG1, human IgG2, human IgG3, or human IgG4 may be used.

[0057] 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 include carriers made from polysaccharides such as agarose, alginate (alginate 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 preferred carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellfine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited, and it may be granular or non-granular, porous or non-porous.

[0058] To immobilize the Fc-binding protein of the present invention onto an insoluble carrier, an active group such as an N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, or haloacetamide (iodoacetamide, bromoacetamide, etc.) is attached to the insoluble carrier, and the human Fc-binding protein is immobilized by covalent bonding between the insoluble carrier and the active group via this active group. The carrier to which the active group is attached may be a commercially available carrier as is, or it may be prepared by introducing the active group to the surface of the carrier under appropriate reaction conditions. Examples of commercially available carriers with added active groups 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 Cytiva Corporation), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0059] On the other hand, as a method for introducing active groups to the surface of a support, one example is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the surface of the support. Examples of such compounds that introduce epoxy groups to hydroxyl groups or amino groups on the surface of the support include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of compounds that introduce epoxy groups to the surface of the support using the above compound and then introduce carboxyl groups to the surface of the support include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0060] Compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and 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-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include pionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-6-aminohexanoic acid, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0061] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the carrier 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, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide. Another example is a method in which hydroxyl or amino groups present on the carrier surface are reacted with an ω-alkenyl alkane glycidyl ether, and then the ω-alkenyl moiety is halogenated and activated with a halogenating agent. Examples of ω-alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether, while examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0062] Another method for introducing active groups to a support surface involves introducing active groups to carboxyl groups present on the support surface using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0063] Examples of buffers used when immobilizing the Fc-binding protein of the present invention onto 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 for immobilization can be appropriately set within a temperature 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.

[0064] To purify antibodies using the adsorbent of the present invention, which is obtained by immobilizing the Fc-binding protein of the present invention on an insoluble carrier, for example, a buffer containing the antibody can be added to a column packed with the adsorbent of the present invention using a liquid delivery means such as a pump, thereby specifically adsorbing the antibody to the adsorbent of the present invention, and then the antibody can be eluted by adding an appropriate eluent to the column. Antibodies that can be purified with the adsorbent of the present invention are those that contain at least the Fc region of an antibody that has affinity for the Fc-binding protein. Examples include chimeric antibodies, humanized antibodies, human antibodies, and their amino acid substitutions, which are commonly used as antibodies in antibody drugs. Furthermore, even artificially modified 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 the buffer containing the antibody to the column, as this allows for higher purity purification of the antibody. Examples of buffer solutions include phosphate buffers and other buffer solutions containing inorganic salts, and the pH of the buffer solution is between 3.0 and 10.0, preferably between 5.0 and 8.0.

[0065] To elute antibodies adsorbed onto the adsorbent of the present invention, the interaction between the antibody and the ligand (the Fc-binding protein of the present invention) can be weakened. Specifically, examples include pH changes using a buffer, counterpeptides, temperature changes, and salt concentration changes. A specific example of an elution solution for elutering antibodies adsorbed onto the adsorbent of the present invention is a buffer solution that is more acidic than the solution used to adsorb the antibody onto the adsorbent of the present invention. Examples of buffer solutions include citrate buffer, glycine hydrochloride buffer, and acetate buffer, all of which have buffering capacity on the acidic side. The pH of the buffer should be set within a range that does not impair the function of the antibody, preferably pH 4.0 to 8.0, and more preferably pH 5.0 to 6.0. [Effects of the Invention]

[0066] The Fc-binding protein of the present invention is a protein in which mutations have been introduced at specific positions in the extracellular region and / or the β2 microglobulin region of the β chain of the native human FcRn α chain. The Fc-binding protein of the present invention has improved acid stability compared to the native human FcRn. When producing Fc-binding proteins industrially, it is efficient to use transformants obtained by transforming a host with an expression vector containing the nucleotide sequence encoding the Fc-binding protein. However, it is preferable that inactivation and denaturation of the Fc-binding protein are suppressed during the culture of the transformant and during the extraction and purification of the Fc-binding protein expressed from the transformant. Since the Fc-binding protein of the present invention has improved acid resistance and a reduced risk of inactivation and denaturation, it can be said to be a protein suitable for the industrial production described above.

[0067] Furthermore, the Fc-binding protein of the present invention is also useful as a ligand for an adsorbent used to separate antibodies (immunoglobulins). [Brief explanation of the drawing]

[0068] [Figure 1]This is a schematic diagram of the α-chain of human FcRn. The numbers in the diagram indicate the amino acid sequence numbers listed in Sequence ID No. 1. In the diagram, S represents the signal sequence, EC represents the extracellular region, TM represents the transmembrane region, and C represents the intracellular region. [Figure 2] This is a schematic diagram of the β-chain of human FcRn. The numbers in the diagram indicate the amino acid sequence numbers listed in Sequence ID No. 2. In the diagram, S represents the signal sequence, and B2M represents β2-microglobulin. [Figure 3] This figure shows the results of evaluating the heat resistance of an Fc-binding protein (Control) with a specific mutation introduced at a single site compared to an Fc-binding protein (Control) consisting of the amino acid sequence described in Sequence ID No. 4. [Figure 4] This figure shows the results of evaluating the acid resistance of an Fc-binding protein in which a specific amino acid residue was deleted compared to an Fc-binding protein (Control) consisting of the amino acid sequence described in Sequence ID No. 4. [Figure 5] This figure shows the results of evaluating the acid resistance of an Fc-binding protein (FcRn-m10ΔW87) with the amino acid sequence described in SEQ ID NO: 41, in which a mutation was introduced at a specific site. [Examples]

[0069] The following examples illustrate the present invention in more detail, but the present invention is not limited to these examples. Reference examples do not constitute the present invention.

[0070] Reference example 1 Random mutagenesis was performed on the polynucleotide portion (SEQ ID NO: 5) encoding the Fc-binding protein of the expression vector pET-FcRn_m7, which expresses an Fc-binding protein consisting of the amino acid sequence described in SEQ ID NO: 4, prepared by the method described in Japanese Patent Publication No. 2018-183087, using error-prone PCR. The Fc-binding protein consisting of the sequence described in SEQ ID NO: 4 is a polypeptide obtained by introducing the following seven amino acid substitutions (mutations) into the Fc-binding protein consisting of the sequence described in SEQ ID NO: 3, which includes the extracellular region of the natural human FcRnα chain consisting of the sequence described in SEQ ID NO: 1 and the β2 microglobulin region of the β chain consisting of the sequence described in SEQ ID NO: 2. A mutation in which the 76th cysteine ​​molecule in sequence number 3 (the 71st in sequence number 1) is replaced with arginine. A mutation in which asparagine at position 83 (position 78 in SEQ ID NO: 3) is replaced with aspartic acid. A mutation in which the glycine at position 156 in SEQ ID NO: 3 (position 151 in SEQ ID NO: 1) is replaced with aspartic acid. A mutation in which the arginine at position 197 (position 192 in sequence number 1) is replaced with leucine. A mutation in which asparagine at position 201 in SEQ ID NO: 3 (position 196 in SEQ ID NO: 1) is replaced with aspartic acid. A mutation in which the 237th glutamic acid molecule in sequence number 3 (the 232nd in sequence number 1) is replaced with leucine. A mutation in which lysine at position 300 (position 295 in sequence number 1) is replaced with glutamic acid. (1) Error-prone PCR was performed using the aforementioned pET-FcRn_m7 as the template DNA. Error-prone PCR was performed by preparing a reaction solution with the composition shown in Table 2 using the primers described in SEQ ID NO: 6 (forward) and SEQ ID NO: 7 (reverse), then heat-treating the reaction solution at 95°C for 2 minutes, performing 35 cycles of a reaction consisting of a first step at 95°C for 30 seconds, a second step at 60°C for 30 seconds, and a third step at 72°C for 90 seconds, and finally heat-treating at 72°C for 7 minutes.

[0071] [Table 2]

[0072] (2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with the same restriction enzymes.

[0073] (3) After the ligation reaction was completed, the reaction solution was introduced into Escherichia coli BL21(DE3) strain by heat shock and cultured in LB (Luria-Bertani) plate medium containing 50 μg / mL kanamycin (at 37°C for 18 hours). The colonies formed on the plate were then used as a random mutant library.

[0074] Reference example 2 (1) The random mutant library (transformed organisms) prepared in Reference Example 1 was inoculated into 200 μL of 2YT liquid medium containing 50 μg / mL kanamycin (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride), and incubated overnight with shaking at 30°C using a 96-well deep-well plate.

[0075] (2) After culturing, 5 μL of culture medium was subpoenaed into 500 μL of 2YT liquid medium containing 0.05 mM IPTG (isopropyl-β-D-thiogalactopyranoside), 0.3% (w / v) glycine, and 50 μg / mL kanamycin, and the cultures were further incubated overnight at 20°C in a 96-well deep-well plate with shaking.

[0076] (3) After culturing, the culture supernatant containing each Fc-binding protein obtained by centrifugation was heat-treated at 45°C for 10 minutes.

[0077] The antibody binding activity of the Fc-binding protein after the heat treatment described in (4)(3) and the antibody binding activity of the Fc-binding protein without the heat treatment described in (3) were measured using the ELISA method shown below. The residual activity was calculated by dividing the antibody binding activity of the Fc-binding protein after heat treatment by the antibody binding activity of the Fc-binding protein without heat treatment. (4-1) A human antibody gamma globulin preparation (manufactured by the Institute of Chemotherapy and Serum Therapy) was immobilized at 1 μg / well in the wells of a 96-well microplate (at 4°C for 18 hours). After immobilization, the plates were blocked with 20 mM phosphate buffer (pH 6.0) containing 2% (w / v) skim milk (manufactured by BD) and 150 mM sodium chloride. (4-2) After washing with a washing buffer (20 mM Tris hydrochloride buffer (pH 6.0) containing 0.05% [w / v] Tween 20 (trade name) and 150 mM NaCl), a solution containing an Fc-binding protein to evaluate antibody binding activity was added, and the Fc-binding protein was reacted with immobilized gamma globulin (at 30°C for 1 hour). (4-3) After the reaction was complete, the samples were washed with the washing buffer and 100 μL / well of Anti-6His antibody (Bethyl Laboratories), diluted to 100 ng / mL, was added. (4-4) The mixture was reacted at 30°C for 1 hour, washed with the aforementioned washing buffer, and then 50 μL / well of TMB Peroxidase Substrate (KPL) was added. The color development was stopped by adding 50 μL / well of 1 M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (Tecan).

[0078] Approximately 700 transformant strains were evaluated using the method described in (5)(4), and from these, transformants expressing an Fc-binding protein with improved thermal stability compared to the Fc-binding protein consisting of the amino acid sequence described in SEQ ID NO: 4 were selected. The selected transformants were cultured, and expression vectors were prepared using the QIAprep Spin Miniprep kit (Qiagen).

[0079] (6) The sequence of the polynucleotide region encoding the Fc-binding protein inserted into the obtained expression vector was subjected to a cycle sequencing reaction using the Big Dye Terminator Cycle Sequencing FS read Reaction kit (PE Applied Biosystems, Inc.) based on the chain terminator method, and the base sequence was analyzed using the ABI Prism 3700 DNA analyzer (PE Applied Biosystems, Inc.) to identify amino acid mutation sites. During this analysis, either an oligonucleotide consisting of the sequence described in SEQ ID NO: 6 or SEQ ID NO: 7 was used as a sequencing primer.

[0080] Figure 3 summarizes the mutation sites and residual activity [%] after heat treatment of the Fc-binding protein expressed by the transformants selected in (5) compared to the Fc-binding protein (Control) consisting of the amino acid sequence described in SEQ ID NO: 4. It can be seen that the Fc-binding proteins with the following mutations are improved in thermal stability compared to the Control: Ser55Thr (this notation indicates that the 55th proline in SEQ ID NO: 3 (50th in SEQ ID NO: 1) is substituted with threonine, the same applies below), Thr94Val, Gln172Glu, Lys174Asn, Lys333Ile, Trp387Ser, or ΔTrp81 (this notation indicates that the 81st tryptophan in SEQ ID NO: 3 (76th in SEQ ID NO: 1) is deleted). Therefore, it can be seen that having at least one of the above seven mutations in the extracellular region of the human FcRnα chain and the β2 microglobulin region of the β chain improves thermal stability (heat resistance). In particular, ΔTrp81 exhibits the highest residual activity, indicating that Fc-binding proteins with mutations in which at least ΔTrp81 is deleted in the extracellular domain of the human FcRnα chain show particularly improved thermal stability (heat resistance).

[0081] The amino acid sequence of the Fc-binding protein with improved thermal stability obtained in this reference example, specifically the Fc-binding protein with a mutation lacking ΔTrp81, is shown as Sequence ID No. 8, and the sequence of the polynucleotide encoding the said protein is shown as Sequence ID No. 9. In Sequence ID No. 8, the first methionine (Met) to the 26th alanine (Ala) constitute the MalE signal peptide, the 27th methionine (Met) and the 28th glycine (Gly) constitute the linker sequence, the 29th alanine (Ala) to the 301st serine (Ser) constitute the extracellular region of FcRn (corresponding to the region from the 24th to the 297th in Sequence ID No. 1), the 302nd glycine (Gly) to the 326th serine (Ser) constitute the GS linker, the 327th isoleucine (Ile) to the 425th methionine (Met) constitute the β2 microglobulin region (the region from the 21st to the 119th in Sequence ID No. 2), the 426th and 427th glycine (Gly) constitute the linker sequence, and the 428th to 433rd histidine (His) constitute the tag sequence. Furthermore, among the Fc-binding proteins consisting of the amino acid sequence described in Sequence ID No. 8, the polypeptide consisting of amino acid residues from the 29th alanine to the 425th methionine will be named FcRn-m7ΔW81.

[0082] Reference example 3 To further improve the thermal stability of Fc-binding proteins, we introduced amino acid substitutions (mutations) that were identified in Reference Example 2 as being involved in improving the thermal stability of Fc-binding proteins into FcRn-m7ΔW81. Mutation introduction (accumulation of amino acid substitutions) was mainly performed using PCR, and the Fc-binding proteins shown in (a) and (b) below were produced. (a) A polypeptide (named FcRn-m9ΔW81) obtained by introducing further amino acid substitutions of Gln172Glu and Lys333Ile into FcRn-m7ΔW81. (b) A polypeptide obtained by introducing further amino acid substitutions of Gln172Glu, Lys333Ile, and Trp387Ser into FcRn-m7ΔW81 (named FcRn-m10ΔW81). The following describes in detail the methods for producing each Fc-binding protein.

[0083] (a)FcRn-m9ΔW81 From the mutations involved in improving thermal stability, as revealed in Reference Example 2, Gln172Glu and Lys333Ile were selected, and FcRn-m9ΔW81 was created by accumulating these mutations in FcRn-m7ΔW81. Specifically, FcRn-m9ΔW81 was created by introducing a mutation that produces Lys333Ile into the polynucleotide encoding FcRn-m7ΔW81 obtained in Reference Example 2 (named FcRn-m8ΔW81), and then introducing a mutation that produces Gln172Glu.

[0084] (a-1) Using the polynucleotide encoding an Fc-binding protein containing FcRn-m7ΔW81 obtained in Reference Example 2 (SEQ ID NO: 9) as the template DNA, PCR was performed using oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 10 (reverse) as PCR primers. PCR was performed by preparing a reaction solution with the composition shown in Table 3, heat-treating the reaction solution at 98°C for 5 minutes, and performing 30 cycles of a reaction consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, and finally heat-treating at 72°C for 5 minutes. The amplified PCR product was subjected to agarose gel electrophoresis, and the gel was purified using the QIAquick Gel Extraction Kit (Qiagen). The purified PCR product was named m8ΔW81-F.

[0085] [Table 3]

[0086] (a-2) PCR and purification of the PCR product were performed in the same manner as in (a-1), except that the same polynucleotide as in (a-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 11 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m8ΔW81-R.

[0087] (a-3) The two PCR products (m8ΔW81-F and m8ΔW81-R) obtained in (a-1) and (a-2) were mixed to prepare a reaction solution with the composition shown in Table 4. After heat treatment of the reaction solution at 98°C for 5 minutes, PCR was performed by carrying out 5 cycles of a reaction consisting of a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 1 minute at 72°C, thereby obtaining the PCR product m8ΔW81-FR, which is a ligation of m8ΔW81-F and m8ΔW81-R.

[0088] [Table 4]

[0089] The PCR product m8ΔW81-FR obtained in (a-4)(a-3) was used as the template DNA, and PCR was performed using oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 7 (reverse) as PCR primers. For PCR, a reaction solution with the composition shown in Table 5 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes. The reaction consisted of a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 1 minute at 72°C, and this reaction was repeated for 30 cycles. This produced the polynucleotide encoding FcRn-m8ΔW81.

[0090] [Table 5]

[0091] After purifying the polynucleotides obtained in (a-5) and (a-4), they were digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0092] (a-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m8ΔW81, which contains a polynucleotide encoding a polypeptide (FcRn-m8ΔW81) in which a Lys333Ile mutation was further introduced into FcRn-m7ΔW81.

[0093] (a-7)(a-6) Of the pET-m8ΔW81 obtained in (a-7)(a-6), the polynucleotide encoding the Fc-binding protein was used as the template DNA, and the oligonucleotide consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 12 (reverse) was used as the PCR primer. PCR and purification of the PCR product were performed in the same manner as in (a-1). The purified PCR product was named m9ΔW81-F.

[0094] PCR and purification of the PCR product were performed in the same manner as in (a-1), except that the same polynucleotide as in (a-8) and (a-7) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 13 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m9ΔW81-R.

[0095] (a-9) PCR was performed in the same manner as in (a-3), except that a mixture of the two PCR products (m9ΔW81-F and m9ΔW81-R) obtained in (a-7) and (a-8) was used as the PCR product, to obtain the PCR product m9ΔW81-FR, which is a ligated form of m9ΔW81-F and m9ΔW81-R.

[0096] PCR was performed in the same manner as in (a-4), except that the PCR product m9ΔW81-FR obtained in (a-9) was used as the template DNA. This produced polynucleotides encoding FcRn-m9ΔW81.

[0097] The polynucleotides obtained in (a-11) and (a-10) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0098] (a-12) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m9ΔW81, which contains polynucleotides encoding FcRn-m9ΔW81, a polypeptide in which further mutations of Gln172Glu and Lys333Ile were introduced into FcRn-m7ΔW81.

[0099] (a-13) The nucleotide sequence of pET-m9ΔW81 was analyzed using the same method as in Reference Example 2(6).

[0100] Sequence ID 14 shows the amino acid sequence of FcRn-m9ΔW81 with the signal sequence and polyhistidine tag attached, and Sequence ID 15 shows the sequence of the polynucleotide encoding FcRn-m9ΔW81. In Sequence ID 14, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m9ΔW81 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0101] (b)FcRn-m10ΔW81 From the mutations involved in improving thermal stability that were identified in Example 2, Gln172Glu, Lys333Ile, and Trp387Ser were selected, and FcRn-m10ΔW81 was created by accumulating them in FcRn-m7ΔW81. Specifically, FcRn-m10ΔW81 was created by introducing a mutation that produces Trp387Ser into the polynucleotide encoding FcRn-m9ΔW81 obtained in (a).

[0102] (b-1) Using the polynucleotide encoding an Fc-binding protein containing FcRn-m9ΔW81 obtained in (a) (SEQ ID NO: 15) as the template DNA, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 16 (reverse) as PCR primers, PCR and purification of the PCR product were performed in the same manner as in (a-1). The purified PCR product was named m10ΔW81-F.

[0103] (b-2) PCR and purification of the PCR product were performed in the same manner as in (a-1), except that the same polynucleotide as in (b-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 17 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m10ΔW81-R.

[0104] (b-3) PCR was performed in the same manner as in (a-3), except that a mixture of the two PCR products (m10ΔW81-F and m10ΔW81-R) obtained in (b-1) and (b-2) was used as the PCR product, and the PCR product m10ΔW81-FR was obtained by ligating m10ΔW81-F and m10ΔW81-R.

[0105] (b-4) PCR was performed in the same manner as in (a-4), except that the PCR product m10ΔW81-FR obtained in (b-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m10ΔW81.

[0106] The polynucleotides obtained in (b-5) and (b-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0107] (b-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m10ΔW81, which contains a polynucleotide encoding FcRn-m10ΔW81, a polypeptide in which a Trp387Ser mutation was further introduced into FcRn-m9ΔW81.

[0108] (b-7) The nucleotide sequence of pET-m10ΔW81 was analyzed using the same method as in Reference Example 2(6).

[0109] Sequence ID 18 shows the amino acid sequence of FcRn-m10ΔW81 with the signal sequence and polyhistidine tag attached, and Sequence ID 19 shows the sequence of the polynucleotide encoding FcRn-m10ΔW81. In Sequence ID 18, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m10ΔW81 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0110] Example 1: Introduction of amino acid deletion To clarify the contribution of amino acid deletion to acid stability, we attempted to create mutants by deleting one amino acid residue around Trp81 (this notation represents tryptophan at position 81 in SEQ ID NO: 3 (position 76 in SEQ ID NO: 1), the same applies below), which showed improved thermal stability due to deletion in Reference Example 2, and to evaluate their acid stability. Amino acid residue deletion was mainly performed using PCR, and five types of polypeptides shown in (a) to (e) below were produced. (a) A polypeptide in which Trp79 is deleted from FcRn-m7 (sequence number 4) (named FcRn-m7ΔW79) (b) A polypeptide in which Val80 is deleted from FcRn-m7 (named FcRn-m7ΔV80) (c) A polypeptide in which Glu82 is deleted from FcRn-m7 (named FcRn-m7ΔE82) (d) A polypeptide in which Trp87 is deleted from FcRn-m7 (named FcRn-m7ΔW87) (e) A polypeptide in which Tyr88 is deleted from FcRn-m7 (named FcRn-m7ΔY88) The following describes in detail the methods for producing each Fc-binding protein.

[0111] (a)FcRn-m7ΔW79 Trp79 was selected from the surrounding amino acids of Trp81, and FcRn-m7ΔW79 was created by deleting this amino acid from FcRn-m7. Specifically, FcRn-m7ΔW79 was created by introducing a mutation that deleted Trp79 into the polynucleotide encoding FcRn-m7 (SEQ ID NO: 4).

[0112] (a-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding FcRn-m7 (SEQ ID NO: 5) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 20 (reverse) were used as PCR primers. The purified PCR product was named m7ΔW79-F.

[0113] (a-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (a-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 21 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m7ΔW79-S.

[0114] (a-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m7ΔW79-F and m7ΔW79-S) obtained in (a-1) and (a-2) was used as the PCR product, to obtain the PCR product m7ΔW79-FL, which is a ligated form of m7ΔW79-F and m7ΔW79-S.

[0115] (a-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m7ΔW79-FL obtained in (a-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m7ΔW79.

[0116] After purifying the polynucleotides obtained in (a-5) and (a-4), they were digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0117] (a-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m7ΔW79, which contains a polynucleotide encoding FcRn-m7ΔW79, a polypeptide in which Trp79 is deleted from FcRn-m7.

[0118] (a-7) The nucleotide sequence of pET-m7ΔW79 was analyzed using the same method as in Reference Example 3(6).

[0119] Sequence ID 22 shows the amino acid sequence of FcRn-m7ΔW79 with the signal sequence and polyhistidine tag attached, and Sequence ID 23 shows the sequence of the polynucleotide encoding FcRn-m7ΔW79. In Sequence ID 22, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m7ΔW79 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0120] (b)FcRn-m7ΔV80 Val80 was selected from the surrounding amino acids of Trp81, and FcRn-m7ΔV80 was created by deleting this amino acid from FcRn-m7. Specifically, FcRn-m7ΔV80 was created by introducing a mutation that deletes Val80 into the polynucleotide encoding FcRn-m7 (SEQ ID NO: 4).

[0121] (b-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding FcRn-m7 (SEQ ID NO: 5) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 24 (reverse) were used as PCR primers. The purified PCR product was named m7ΔV80-F.

[0122] (b-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (b-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 25 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m7ΔV80-S.

[0123] (b-3) Except for using a mixture of the two PCR products (m7ΔV80-F and m7ΔV80-S) obtained in (b-1) and (b-2) as the PCR product, PCR was performed in the same manner as in Reference Example 3(a-3) to obtain the PCR product m7ΔV80-FL, which is a ligated form of m7ΔV80-F and m7ΔV80-S.

[0124] (b-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m7ΔV80-FL obtained in (b-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m7ΔV80.

[0125] The polynucleotides obtained in (b-5) and (b-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0126] (b-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m7ΔV80, which contains a polynucleotide encoding FcRn-m7ΔV80, a polypeptide in which Val80 is deleted from FcRn-m7.

[0127] (b-7) The nucleotide sequence of pET-m7ΔV80 was analyzed using the same method as in Reference Example 3(6).

[0128] Sequence ID 26 shows the amino acid sequence of FcRn-m7ΔV80 with the signal sequence and polyhistidine tag attached, and Sequence ID 27 shows the sequence of the polynucleotide encoding FcRn-m7ΔV80. In Sequence ID 26, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m7ΔV80 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0129] (c)FcRn-m7ΔE82 Glu82 was selected from the surrounding amino acids of Trp81, and FcRn-m7ΔE82 was created by deleting this amino acid from FcRn-m7. Specifically, FcRn-m7ΔE82 was created by introducing a mutation that deletes Glu82 into the polynucleotide encoding FcRn-m7 (SEQ ID NO: 4).

[0130] (c-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding FcRn-m7 (SEQ ID NO: 5) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 28 (reverse) were used as PCR primers. The purified PCR product was named m7ΔE82-F.

[0131] (c-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (c-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 29 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m7ΔE82-S.

[0132] (c-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m7ΔE82-F and m7ΔE82-S) obtained in (c-1) and (c-2) was used as the PCR product, to obtain the PCR product m7ΔE82-FL, which is a ligated form of m7ΔE82-F and m7ΔE82-S.

[0133] (c-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m7ΔE82-FL obtained in (c-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m7ΔE82.

[0134] The polynucleotides obtained in (c-5)(c-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0135] (c-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m7ΔE82, which contains a polynucleotide encoding FcRn-m7ΔE82, a polypeptide in which Glu82 is deleted from FcRn-m7.

[0136] The nucleotide sequence of (c-7)pET-m7DelE82 was analyzed using the same method as in Reference Example 3(6).

[0137] Sequence ID 30 shows the amino acid sequence of FcRn-m7ΔE82 with the signal sequence and polyhistidine tag attached, and Sequence ID 31 shows the sequence of the polynucleotide encoding FcRn-m7DelE82. In Sequence ID 30, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m7ΔE82 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0138] (d)FcRn-m7ΔW87 Trp87 was selected from the surrounding amino acids of Trp81, and FcRn-m7ΔW87 was created by deleting this amino acid from FcRn-m7. Specifically, FcRn-m7ΔW87 was created by introducing a mutation that deleted Trp87 into the polynucleotide encoding FcRn-m7 (SEQ ID NO: 4).

[0139] (d-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding FcRn-m7 (SEQ ID NO: 5) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 32 (reverse) were used as PCR primers. The purified PCR product was named m7ΔW87-F.

[0140] (d-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (d-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 33 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m7ΔW87-S.

[0141] (d-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m7ΔW87-F and m7ΔW87-S) obtained in (d-1) and (d-2) was used as the PCR product, to obtain the PCR product m7ΔW87-FL, which is a ligated product of m7ΔW87-F and m7ΔW87-S.

[0142] (d-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m7ΔW87-FL obtained in (d-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m7ΔW87.

[0143] After purifying the polynucleotides obtained in (d-5) and (d-4), they were digested with restriction enzymes NcoI and HindIII and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0144] (d-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m7ΔW87, which contains a polynucleotide encoding FcRn-m7ΔW87, a polypeptide in which Trp87 is deleted from FcRn-m7.

[0145] The nucleotide sequence of (d-7)pET-m7ΔW87 was analyzed using the same method as in Reference Example 3(6).

[0146] Sequence ID 34 shows the amino acid sequence of FcRn-m7ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 35 shows the sequence of the polynucleotide encoding FcRn-m7ΔW87. In Sequence ID 34, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m7ΔW87 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0147] (e)FcRn-m7ΔY88 Tyr88 was selected from the surrounding amino acids of Trp81, and FcRn-m7ΔY88 was created by deleting this amino acid from FcRn-m7. Specifically, FcRn-m7ΔY88 was created by introducing a mutation that deleted Tyr88 into the polynucleotide encoding FcRn-m7 (SEQ ID NO: 4).

[0148] (e-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding FcRn-m7 (SEQ ID NO: 5) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 32 (reverse) were used as PCR primers. The purified PCR product was named m7ΔY88-F.

[0149] (e-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (e-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 36 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m7ΔY88-S.

[0150] (e-3) Except for using a mixture of the two PCR products (m7ΔY88-F and m7ΔY88-S) obtained in (e-1) and (e-2) as the PCR product, PCR was performed in the same manner as in Reference Example 3(a-3) to obtain the PCR product m7ΔY88-FL, which is a ligated form of m7ΔY88-F and m7ΔY88-S.

[0151] (e-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m7ΔY88-FL obtained in (e-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m7ΔY88.

[0152] After purifying the polynucleotides obtained in (e-5)(e-4), they were digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0153] (e-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m7ΔY88, which contains a polynucleotide encoding FcRn-m7ΔY88, a polypeptide in which Tyr88 is deleted from FcRn-m7.

[0154] The nucleotide sequence of (e-7)pET-m7ΔY88 was analyzed using the same method as in Reference Example 3(6).

[0155] Sequence ID 37 shows the amino acid sequence of FcRn-m7ΔY88 with the signal sequence and polyhistidine tag attached, and Sequence ID 38 shows the sequence of the polynucleotide encoding FcRn-m7ΔY88. In Sequence ID 37, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m7ΔY88 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0156] Example 2: Evaluation of the acid stability of Fc-binding proteins (Part 1) (1) Transformants expressing FcRn-m7, FcRn-m7ΔW81 obtained in Reference Example 2, and FcRn-m7ΔW79, FcRn-m7ΔV80, FcRn-m7ΔE82, FcRn-m7ΔW87, and FcRn-m7ΔY88 prepared in Example 1 were each inoculated into 3 mL of 2YT liquid medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) containing 50 μg / mL kanamycin, and pre-cultured by aerobic shaking at 37°C overnight.

[0157] (2) 600 μL of the preculture solution was inoculated into 20 mL of 2YT liquid medium supplemented with 50 μg / mL of kanamycin, and cultured aerobically with shaking at 37°C.

[0158] (3) 1.5 hours after the start of culture, the culture temperature was changed to 20°C and the culture was shaken for 30 minutes. Then, IPTG was added to a final concentration of 0.01 mM, and the culture was continued aerobically at 20°C overnight with shaking.

[0159] (4) After the culture was completed, the cells were collected by centrifugation and a protein extract was prepared using the BugBuster Protein Extraction Kit (Merck Millipore).

[0160] The antibody-binding activity of FcRn-m7, FcRn-m7ΔW81, FcRn-m7ΔW79, FcRn-m7ΔV80, FcRn-m7ΔE82, FcRn-m7ΔW87, and FcRn-m7ΔY88 in the protein extract prepared in (5)(4) was measured by the ELISA method described in Reference Example 2(4). At this time, a calibration curve was prepared using purified and quantified FcRn-m7, and the protein concentration was measured.

[0161] (6) After diluting each Fc-binding protein with pure water to a concentration of 10 μg / mL, 20 μL of the diluted solution was mixed with 80 μL of 0.1 M glycine hydrochloride buffer (pH 3.0), and the mixture was left to stand at 25°C for 24 hours.

[0162] The antibody binding activity of the Fc-binding protein after acid treatment (7)(6) and the antibody binding activity of the Fc-binding protein without acid treatment (6) were measured using the ELISA method described in Reference Example 2(4), and the residual activity was calculated by dividing the antibody binding activity of the Fc-binding protein after acid treatment by the antibody binding activity of the Fc-binding protein without acid treatment.

[0163] The results are shown in Figure 4. The Fc-binding proteins produced in Example 1, FcRn-m7ΔW79 (SEQ ID NO: 22), FcRn-m7ΔV80 (SEQ ID NO: 26), FcRn-m7ΔE82 (SEQ ID NO: 30), FcRn-m7ΔW87 (SEQ ID NO: 34), and FcRn-m7ΔY88 (SEQ ID NO: 37), all showed higher residual activity compared to the control FcRn-m7 (SEQ ID NO: 4) and FcRn-m7ΔW81 (SEQ ID NO: 8) obtained in Reference Example 2. This confirms that they exhibit improved acid stability compared to FcRn-m7 and FcRn-m7ΔW81. In particular, the Fc-binding protein with the ΔTrp87 mutation (FcRn-m7ΔW87) showed the highest residual activity, indicating that Fc-binding proteins with at least the ΔTrp87 mutation exhibit particularly improved acid stability.

[0164] Example 3: Introduction of further amino acid substitutions From the amino acid residue deletions (mutations) involved in improving the acid stability of Fc-binding proteins identified in Example 2, ΔTrp87 was selected, and acid stability was improved by introducing this deletion into the Fc-binding proteins FcRn-m9 and FcRn-m10. The amino acid deletion was mainly performed using PCR, and two types of polypeptides shown in (a) and (b) below were produced.

[0165] FcRn-m9 is an Fc-binding protein obtained by further introducing the amino acid substitutions Gln172Glu and Lys333Ile into FcRn-m7 (SEQ ID NO: 4), and FcRn-m10 is an Fc-binding protein obtained by further introducing the amino acid substitution Trp387Ser into FcRn-m9. (a) A polypeptide in which Trp87 is deleted from FcRn-m9 (named FcRn-m9ΔW87) (b) A polypeptide in which Trp87 is deleted from FcRn-m10 (named FcRn-m10ΔW87) The following describes in detail the methods for producing each Fc-binding protein.

[0166] (a)FcRn-m9ΔW87 FcRn-m9ΔW87 was created by deleting Trp87 from FcRn-m9. Specifically, FcRn-m9ΔW87 was created by introducing mutations that resulted in the insertion of the deleted Trp81 and the deletion of Trp87 into the polynucleotide encoding FcRn-m9ΔW81 obtained in Reference Example 3(a).

[0167] (a-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding an Fc-binding protein containing FcRn-m9ΔW81 obtained in Reference Example 3(a) (SEQ ID NO: 15) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 32 (reverse) were used as PCR primers. The purified PCR product was named m9ΔW87-F.

[0168] (a-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (a-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 33 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m9ΔW87-S.

[0169] (a-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m9ΔW87-F and m9ΔW87-S) obtained in (a-1) and (a-2) was used as the PCR product, to obtain the PCR product m9ΔW87-FL, which is a ligated form of m9ΔW87-F and m9ΔW87-S.

[0170] (a-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m9ΔW87-FL obtained in (a-3) was used as the template DNA. This produced the polynucleotide encoding FcRn-m9ΔW87.

[0171] After purifying the polynucleotides obtained in (a-5) and (a-4), they were digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0172] (a-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m9ΔW87, which contains a polynucleotide encoding FcRn-m9ΔW87, a polypeptide in which Trp87 is deleted from FcRn-m9.

[0173] (a-7) The nucleotide sequence of pET-m9ΔW87 was analyzed using the same method as in Reference Example 3(6).

[0174] Sequence ID 39 shows the amino acid sequence of FcRn-m9ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 40 shows the sequence of the polynucleotide encoding FcRn-m9ΔW87. In Sequence ID 39, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m9ΔW87 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0175] (b)FcRn-m10ΔW87 FcRn-m10ΔW87 was created by deleting Trp87 from FcRn-m10. Specifically, FcRn-m10ΔW87 was created by introducing mutations that resulted in the insertion of the deleted Trp81 and the deletion of Trp87 into the polynucleotide encoding FcRn-m10ΔW81 obtained in Reference Example 3(b).

[0176] (b-1) Using the polynucleotide encoding an Fc-binding protein containing FcRn-m10ΔW81 obtained in Reference Example 3(b) (SEQ ID NO: 19) as the template DNA, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 32 (reverse) as PCR primers, PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1). The purified PCR product was named m10ΔW87-F.

[0177] (b-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (b-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 33 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m10ΔW87-S.

[0178] (b-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m10ΔW87-F and m10ΔW87-S) obtained in (b-1) and (b-2) was used as the PCR product, to obtain the PCR product m10ΔW87-FL, which is a ligated product of m10ΔW87-F and m10ΔW87-S.

[0179] (b-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m10ΔW87-FL obtained in (b-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m10ΔW87.

[0180] The polynucleotides obtained in (b-5) and (b-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0181] (b-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m10ΔW87, which contains a polynucleotide encoding FcRn-m10ΔW87, a polypeptide in which Trp87 is deleted from FcRn-m10.

[0182] (b-7) The nucleotide sequence of pET-m10ΔW87 was analyzed using the same method as in Reference Example 3(6).

[0183] Sequence ID 41 shows the amino acid sequence of FcRn-m10ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 42 shows the sequence of the polynucleotide encoding FcRn-m10ΔW87. In Sequence ID 41, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m10ΔW87 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0184] Example 4: Evaluation of the acid stability of Fc-binding proteins (Part 2) Fc-binding protein extracts were prepared from transformants expressing FcRn-m9ΔW81 and FcRn-m10ΔW81 prepared in Reference Example 3, and FcRn-m9ΔW87 and FcRn-m10ΔW87 prepared in Example 3, using the same method as in Examples 2(1) to (4), and the acid stability of the proteins was evaluated using the same method as in Examples 2(5) to (7).

[0185] The results are shown in Table 6. It can be seen that acid stability is improved by changing the amino acid residue deletion in FcRn-m9 and FcRn-m10 from ΔTrp81 to ΔTrp87.

[0186] [Table 6]

[0187] Example 5: Evaluation of the thermal stability of Fc-binding proteins (1) An Fc-binding protein extract was prepared from transformants expressing FcRn-m10ΔW81 prepared in Reference Example 3 and FcRn-m10ΔW87 prepared in Example 3, using the same method as in Examples 2(1) to (4).

[0188] (2) The antibody binding activity of FcRn-m10ΔW81 and FcRn-m10ΔW87 in the protein extract prepared in (1) was measured by the ELISA method described in Reference Example 2(4). At this time, a calibration curve was prepared using the purified and quantified Fc-binding protein (FcRn-m7) consisting of the amino acid sequence described in SEQ ID NO: 4, and the protein concentration was measured.

[0189] (3) After diluting each Fc-binding protein with pure water to a concentration of 10 μg / mL, 100 μL of the diluted solution was heat-treated at 45°C for 10 minutes.

[0190] (4) The residual activity was calculated by dividing the antibody binding activity when the heat treatment described in (3) was performed by the antibody binding activity when the heat treatment described in (3) was not performed, and the thermal stability was evaluated.

[0191] The results are shown in Table 7. It can be seen that thermal stability is also improved by changing the amino acid residue deletion in FcRn-m10 from ΔTrp81 to ΔTrp87.

[0192] [Table 7]

[0193] Example 6: Mutation introduction into Fc-binding proteins and library preparation In Example 3(b), a random mutagenesis was performed on the polynucleotide portion (SEQ ID NO: 42) encoding the Fc-binding protein FcRn-m10ΔW87 (SEQ ID NO: 41) of the expression vector pET-m10ΔW87, which expresses the Fc-binding protein FcRn-m10ΔW87.

[0194] (1) Error-prone PCR was performed in the same manner as in Reference Example 1(1), except that the aforementioned pET-m10ΔW87 was used as the template DNA.

[0195] (2) The PCR product obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with the same restriction enzymes.

[0196] (3) After the ligation reaction was completed, the reaction solution was introduced into Escherichia coli BL21(DE3) strain by heat shock method, and cultured in LB plate medium containing 50 μg / mL kanamycin (at 37°C for 18 hours). The colonies formed on the plate were then used as a random mutant library.

[0197] Example 7: Screening of acid-resistant Fc-binding proteins (1) The random mutant library (transformers) prepared in Example 6 was cultured using the methods described in Reference Examples 2(1) and (2). The culture medium was centrifuged, and 20 μL of the culture supernatant containing each Fc-binding protein was mixed with 80 μL of 0.1 M glycine hydrochloride buffer (pH 3.0), and the mixture was acid-treated by standing at 25°C for 24 hours.

[0198] (2) The antibody binding activity of the Fc-binding protein when the acid treatment described in (1) was performed, and the antibody binding activity of the Fc-binding protein when the acid treatment described in (1) was not performed, were measured using the ELISA method shown below. The residual activity was calculated by dividing the antibody binding activity of the Fc-binding protein when the acid treatment was performed by the antibody binding activity of the Fc-binding protein when the acid treatment was not performed. (2-1) A human antibody gamma globulin preparation (manufactured by the Institute of Chemotherapy and Serum Therapy) was immobilized at 1 μg / well in the wells of a 96-well microplate (at 4°C for 18 hours). After immobilization, the plates were blocked with 20 mM Tris hydrochloride buffer (pH 7.4) containing 2% (w / v) skim milk (manufactured by BD) and 150 mM sodium chloride. (2-2) After washing with a washing buffer (20 mM Tris hydrochloride buffer (pH 7.4) containing 0.05% [w / v] Tween 20 (trade name) and 150 mM NaCl), a solution containing an Fc-binding protein to evaluate antibody binding activity was added, and the Fc-binding protein was reacted with the immobilized gamma globulin (at 30°C for 1 hour). (2-3) After the reaction was complete, the samples were washed with the washing buffer and 100 μL / well of Anti-6His antibody (Bethyl Laboratories), diluted to 100 ng / mL, was added. (2-4) The mixture was reacted at 30°C for 1 hour, washed with the aforementioned washing buffer, and then 50 μL / well of TMB Peroxidase Substrate (KPL) was added. The color development was stopped by adding 50 μL / well of 1 M phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (Tecan).

[0199] (3)Approximately 800 transformant strains were evaluated using the method described in (2), and from these, transformants expressing an Fc-binding protein with improved acid stability compared to FcRn-m10ΔW87 (SEQ ID NO: 41) were selected. The selected transformants were cultured, and expression vectors were prepared using the QIAprep Spin Miniprep kit (Qiagen).

[0200] (4) Sequence analysis of the polynucleotide region encoding the Fc-binding protein inserted into the obtained expression vector was performed using the same method as in Reference Example 2(6).

[0201] Figure 5 summarizes the mutation sites and residual activity [%] after acid treatment of the Fc-binding protein expressed by the transformants selected in (3) relative to FcRn-m10ΔW87 (SEQ ID NO: 41). It can be seen that the Fc-binding protein into which mutations at Ser55Val, Ala73Val, Ile98Thr, Phe221Ser, His284Arg, Ala406Val, or Asp423Glu have been introduced has improved acid stability compared to FcRn-m10ΔW87. Therefore, it can be seen that acid stability (acid resistance) is improved by having at least one of the above seven mutation sites in the extracellular domain of the human FcRn α chain and the β2 microglobulin domain of the β chain. Among them, Asp423Glu exhibits the highest residual activity, indicating that Fc-binding proteins with at least an amino acid substitution (mutation) of Asp423Glu in the extracellular domain of the human FcRnα chain show particularly improved acid stability (acid resistance).

[0202] In this embodiment, among the Fc-binding proteins with improved acid stability obtained, the amino acid sequence of the Fc-binding protein obtained by further introducing the amino acid substitution of Asp423Glu to FcRn-m10ΔW87 is shown as Sequence ID No. 43, and the sequence of the polynucleotide encoding the said protein is shown as Sequence ID No. 44. In Sequence ID No. 43, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m10ΔW87 (corresponding to the region from the 29th to the 426th in Sequence ID No. 4), the 426th and 427th glycine (Gly) are linker sequences, and the 428th to 433rd histidine (His) are the tag sequence. Furthermore, among the Fc-binding proteins consisting of the amino acid sequence described in Sequence ID No. 43, the polypeptide comprising amino acid residues from the 29th alanine to the 425th methionine will be named FcRn-m11ΔW87.

[0203] Example 8: Preparation of an acid-resistant Fc-binding protein From the amino acid substitutions (mutations) involved in improving the acid stability of Fc-binding proteins, as identified in Example 7, Ser55Val, Ile98Thr, and Ala406Val were selected, and these substitutions were introduced into FcRn-m11ΔW87 to further improve acid stability. Mutation introduction (accumulation of amino acid substitutions) was mainly performed using PCR, and three types of polypeptides shown in (a) to (c) below were produced. (a) A polypeptide obtained by introducing an additional amino acid substitution at Ile98Thr to FcRn-m11ΔW87 (sequence number 43) (named FcRn-m12ΔW87). (b) A polypeptide obtained by introducing further amino acid substitutions of Ile98Thr and Ala406Val to FcRn-m11W87 (named FcRn-m13ΔW87) (c) A polypeptide obtained by introducing further amino acid substitutions of Ser55Val, Ile98Thr, and Ala406Val to FcRn-m11ΔW87 (named FcRn-m14ΔW87). The following describes in detail the methods for producing each Fc-binding protein.

[0204] (a)FcRn-m12ΔW87 From the mutations involved in improving thermal stability identified in Example 7, Ile98Thr was selected, and FcRn-m12ΔW87 was created by accumulating this mutation in FcRn-m11ΔW87. Specifically, FcRn-m12ΔW87 was created by introducing a mutation that produces Ile98Thr into the polynucleotide encoding FcRn-m11ΔW87 obtained in Example 7.

[0205] (a-1) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding an Fc-binding protein containing FcRn-m11ΔW87 obtained in Example 7 (SEQ ID NO: 44) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 45 (reverse) were used as PCR primers. The purified PCR product was named m12ΔW87-F.

[0206] (a-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (a-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 46 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m12ΔW87-R.

[0207] (a-3) Except for using a mixture of the two PCR products (m12ΔW87-F and m12ΔW87-R) obtained in (a-1) and (a-2) as the PCR product, PCR was performed in the same manner as in Reference Example 3(a-3) to obtain the PCR product m12ΔW87-FR, which is a ligated product of m12ΔW87-F and m12ΔW87-R.

[0208] (a-4) PCR was performed in the same manner as in reference example (a-4), except that the PCR product m12-FR obtained in (a-3) was used as the template DNA. This produced the polynucleotide encoding FcRn-m12ΔW87.

[0209] After purifying the polynucleotides obtained in (a-5) and (a-4), they were digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0210] (a-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m12ΔW87, which contains a polynucleotide encoding FcRn-m12ΔW87, a polypeptide in which the Ile98Thr mutation was further introduced into FcRn-m11ΔW87.

[0211] (a-7) The nucleotide sequence of pET-m12ΔW87 was analyzed using the same method as in Reference Example 2(6).

[0212] Sequence ID 47 shows the amino acid sequence of FcRn-m12ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 48 shows the sequence of the polynucleotide encoding FcRn-m12ΔW87. In Sequence ID 47, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m12ΔW87 (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0213] (b)FcRn-m13ΔW87 From the mutations involved in improving thermal stability that were identified in Example 6, Ile98Thr and Ala406Val were selected, and FcRn-m13ΔW87 was created by accumulating these mutations in FcRn-m11ΔW87. Specifically, FcRn-m13ΔW87 was created by introducing a mutation that produces Ala406Val into the polynucleotide encoding FcRn-m12ΔW87 obtained in (a).

[0214] (b-1) Using the polynucleotide encoding an Fc-binding protein containing FcRn-m12ΔW87 obtained in (a) (SEQ ID NO: 48) as the template DNA, and using oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 49 (reverse) as PCR primers, PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1). The purified PCR product was named m13ΔW87-F.

[0215] (b-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (b-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 50 (forward) and SEQ ID NO: 7 (reverse) were used as PCR primers. The purified PCR product was named m13ΔW87-R.

[0216] (b-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m13ΔW87-F and m13ΔW87-R) obtained in (b-1) and (b-2) was used as the PCR product, to obtain the PCR product m13ΔW87-FR, which is a ligated product of m13ΔW87-F and m13ΔW87-R.

[0217] (b-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m13ΔW87-FR obtained in (b-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m13ΔW87.

[0218] The polynucleotides obtained in (b-5) and (b-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0219] (b-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m13ΔW87, which contains a polynucleotide encoding FcRn-m13ΔW87, a polypeptide in which an Ala406Val mutation was further introduced into FcRn-m12ΔW87.

[0220] (b-7) The nucleotide sequence of pET-m13ΔW87 was analyzed using the same method as in Reference Example 2(6).

[0221] Sequence ID 51 shows the amino acid sequence of FcRn-m13ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 52 shows the sequence of the polynucleotide encoding FcRn-m13ΔW87. In Sequence ID 51, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m8Δ1A (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0222] (c)FcRn-m14ΔW87 From the mutations involved in improving thermal stability that were identified in Example 6, Ser55Val, Ile98Thr, and Ala406Val were selected, and FcRn-m14ΔW87 was created by accumulating these mutations in FcRn-m11ΔW87. Specifically, FcRn-m14ΔW87 was created by introducing a mutation that produces Ser55Val into the polynucleotide encoding FcRn-m13ΔW87 obtained in (b).

[0223] PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the polynucleotide encoding an Fc-binding protein containing FcRn-m13ΔW87 obtained in (c-1)(b) (SEQ ID NO: 52) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 6 (forward) and SEQ ID NO: 53 (reverse) were used as PCR primers. The purified PCR product was named m14ΔW87-F.

[0224] (c-2) PCR and purification of the PCR product were performed in the same manner as in Reference Example 3(a-1), except that the same polynucleotide as in (c-1) was used as the template DNA, and oligonucleotides consisting of the sequences described in SEQ ID NO: 54 (forward) and SEQ ID NO: 21 (reverse) were used as PCR primers. The purified PCR product was named m14ΔW87-R.

[0225] (c-3) PCR was performed in the same manner as in Reference Example 3(a-3), except that a mixture of the two PCR products (m14ΔW87-F and m14ΔW87-R) obtained in (c-1) and (c-2) was used as the PCR product, to obtain the PCR product m14ΔW87-FR, which is a ligated form of m14ΔW87-F and m14ΔW87-R.

[0226] (c-4)PCR was performed in the same manner as in Reference Example 3(a-4), except that the PCR product m14ΔW87-FR obtained in (c-3) was used as the template DNA. This produced polynucleotides encoding FcRn-m14ΔW87.

[0227] The polynucleotides obtained in (c-5)(c-4) were purified, digested with restriction enzymes NcoI and HindIII, and ligated into the expression vector pETMalE (Japanese Patent Publication No. 2011-206046), which had been previously digested with restriction enzymes NcoI and HindIII. This was then used to transform Escherichia coli BL21(DE3) strain.

[0228] (c-6) The obtained transformants were cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the recovered bacterial cells (transformants) to obtain plasmid pET-m14ΔW87, which contains a polynucleotide encoding FcRn-m14ΔW87, a polypeptide in which a Ser55Val mutation was further introduced into FcRn-m13ΔW87.

[0229] The nucleotide sequence of (c-7)pET-m14ΔW87 was analyzed using the same method as in Reference Example 2(6).

[0230] Sequence ID 55 shows the amino acid sequence of FcRn-m14ΔW87 with the signal sequence and polyhistidine tag attached, and Sequence ID 56 shows the sequence of the polynucleotide encoding FcRn-m14ΔW87. In Sequence ID 55, the first methionine (Met) to the 26th alanine (Ala) is the MalE signal peptide, the 27th methionine (Met) to the 28th glycine (Gly) is the linker sequence, the 29th alanine (Ala) to the 425th methionine (Met) is the amino acid sequence of FcRn-m8Δ1A (corresponding to the region from the 29th to the 426th in Sequence ID 4), the 426th and 427th glycine (Gly) are the linker sequence, and the 428th to 433rd histidine (His) are the tag sequence.

[0231] Example 9: Evaluation of the acid stability of Fc-binding proteins (Part 3) Fc-binding protein extracts were prepared from transformants expressing FcRn-m10ΔW87 prepared in Example 3(b), FcRn-m11ΔW87 prepared in Example 7, and FcRn-m12ΔW87, FcRn-m13ΔW87, and FcRn-m14ΔW87 prepared in Example 8, using the same method as in Examples 2(1) to (4), and the acid stability of the proteins was evaluated using the same method as in Examples 2(5) to (7).

[0232] The results are shown in Table 8. The Fc-binding proteins prepared in Example 8 (FcRn-m12ΔW87, FcRn-m13ΔW87, and FcRn-m14ΔW87) all showed improved acid stability compared to FcRn-m10ΔW87 (Example 3(b)) and FcRn-m11ΔW87 (Example 7). From these results, it can be seen that acid stability is improved if there is at least one mutation (amino acid substitution) in Ser55Val, Ile98Thr, or Ala406Val.

[0233] [Table 8]

Claims

1. An Fc-binding protein selected from any of the following (i) through (iii): (i) Fc-binding proteins comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein the amino acid residues consist only of any of the mutations shown in (1) to (5) below and all of the mutations shown in (6) to (12) below; (1) A mutation in which the 82nd tryptophan molecule in sequence number 1 is deleted. (2) A mutation in which the 74th tryptophan molecule in sequence number 1 is deleted. (3) A mutation in which the valine at position 75 of sequence number 1 is deleted. (4) A mutation in which the 77th glutamic acid molecule of sequence number 1 is deleted. (5) A mutation in which the tyrosine at position 83 of sequence number 1 is deleted. (6) A mutation in which the 71st cysteine ​​in SEQ ID NO: 1 or the 76th cysteine ​​in SEQ ID NO: 3 is replaced with arginine. (7) A mutation in which the asparagine at position 78 of SEQ ID NO: 1 or position 83 of SEQ ID NO: 3 is replaced with aspartic acid. (8) A mutation in which the glycine at position 151 of SEQ ID NO: 1 or position 156 of SEQ ID NO: 3 is replaced with aspartic acid. (9) A mutation in which the arginine at position 192 of SEQ ID NO: 1 or position 197 of SEQ ID NO: 3 is replaced with leucine. (10) A mutation in which the 196th asparagine molecule in SEQ ID NO: 1 or the 201st asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (11) A mutation in which glutamine at position 232 of SEQ ID NO: 1 or glutamine at position 237 of SEQ ID NO: 3 is replaced with leucine. (12) A mutation in which lysine at position 295 of SEQ ID NO: 1 or position 300 of SEQ ID NO: 3 is replaced with glutamic acid. (ii) An Fc-binding protein comprising at least the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein the amino acid residues consist only of any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12), and further comprising one or more substitutions, deletions, insertions, and additions of 1 to 30 amino acid residues in addition to the mutations shown in (1) to (12), and having the ability to bind to the Fc region of an antibody; (iii) An amino acid sequence having 90% or more identity with the entire amino acid sequence of an Fc-binding protein consisting only of any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12), in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in Sequence ID No. 1 and from the 21st isoleucine to the 119th methionine of the amino acid sequence described in Sequence ID No. 2, provided that the amino acid sequence contains any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12), and is an Fc-binding protein that binds to the Fc region of an antibody.

2. An Fc-binding protein selected from any of the following (iv) to (vi): (iv) An Fc-binding protein comprising at least the amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th consist only of any of the mutations shown in (1) to (5) below and all of the mutations shown in (6) to (12) below; (1) A mutation in which tryptophan at position 87 of sequence number 3 is deleted. (2) A mutation in which the 79th tryptophan in SEQ ID NO: 3 is deleted. (3) A mutation in which the valine at position 80 of sequence number 3 is deleted. (4) A mutation in which the 82nd glutamic acid molecule of sequence number 3 is deleted. (5) A mutation in which the tyrosine at position 88 of sequence number 3 is deleted. (6) A mutation in which the 71st cysteine ​​in SEQ ID NO: 1 or the 76th cysteine ​​in SEQ ID NO: 3 is replaced with arginine. (7) A mutation in which the asparagine at position 78 of SEQ ID NO: 1 or position 83 of SEQ ID NO: 3 is replaced with aspartic acid. (8) A mutation in which the glycine at position 151 of SEQ ID NO: 1 or position 156 of SEQ ID NO: 3 is replaced with aspartic acid. (9) A mutation in which the arginine at position 192 of SEQ ID NO: 1 or position 197 of SEQ ID NO: 3 is replaced with leucine. (10) A mutation in which the 196th asparagine molecule in SEQ ID NO: 1 or the 201st asparagine molecule in SEQ ID NO: 3 is replaced with aspartic acid. (11) A mutation in which glutamine at position 232 of SEQ ID NO: 1 or glutamine at position 237 of SEQ ID NO: 3 is replaced with leucine. (12) A mutation in which lysine at position 295 of SEQ ID NO: 1 or position 300 of SEQ ID NO: 3 is replaced with glutamic acid. (v) An Fc-binding protein comprising at least the amino acid residues from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, wherein the amino acid residues from the 29th to the 426th consist only of any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12), and further comprising one or more substitutions, deletions, insertions, and additions of 1 to 30 amino acid residues in addition to the mutations shown in (1) to (12), and having binding ability to the Fc region of an antibody; (vi) An amino acid sequence having 90% or more identity with the entire amino acid sequence of an Fc-binding protein consisting only of any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12) in the amino acid sequence from the 29th alanine to the 426th methionine of the amino acid sequence described in Sequence ID No. 3, provided that the amino acid sequence includes any of the mutations shown in (1) to (5) and all of the mutations shown in (6) to (12) that remain, and is an Fc-binding protein that binds to the Fc region of an antibody.

3. Furthermore, the Fc-binding protein according to claim 1 or 2 has at least one of the mutations shown in (13) to (22) below; (13) A mutation in which glutamine at position 167 of SEQ ID NO: 1 or glutamic acid at position 172 of SEQ ID NO: 3 is replaced with glutamic acid. (14) A mutation in which the 26th lysine in SEQ ID NO: 2 or the 333rd lysine in SEQ ID NO: 3 is replaced with isoleucine. (15) A mutation in which the 80th tryptophan in SEQ ID NO: 2 or the 387th tryptophan in SEQ ID NO: 3 is replaced with serine. (16) A mutation in which the 50th serine in SEQ ID NO: 1 or the 55th serine in SEQ ID NO: 3 is replaced with valine. (17) A mutation in which alanine at position 68 of SEQ ID NO: 1 or position 73 of SEQ ID NO: 3 is replaced with valine. (18) A mutation in which isoleucine at position 93 of SEQ ID NO: 1 or position 98 of SEQ ID NO: 3 is replaced by threonine. (19) A mutation in which phenylalanine at position 216 of SEQ ID NO: 1 or position 221 of SEQ ID NO: 3 is replaced with serine. (20) A mutation in which the histidine at position 279 of SEQ ID NO: 1 or position 284 of SEQ ID NO: 3 is replaced with arginine. (21) A mutation in which alanine at position 99 of SEQ ID NO: 2 or position 406 of SEQ ID NO: 3 is replaced with valine. (22) A mutation in which aspartic acid at position 116 of SEQ ID NO: 2 or position 423 of SEQ ID NO: 3 is replaced with glutamic acid.

4. An Fc-binding protein according to claim 1 or 2, selected from any of (vii) to (ix) below: (vii) Fc-binding protein comprising at least the amino acid residues from the 29th alanine to the 425th methionine of the amino acid sequence described in any of SEQ ID NOs: 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55; (viiii) An Fc-binding protein having binding ability to the Fc region of an antibody, comprising at least the amino acid residues from the 29th alanine to the 425th methionine in the amino acid sequence described in any of Sequence IDs 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55, wherein the amino acid residues from the 29th to the 425th methionine further have at least one of the substitutions, deletions, insertions, and additions of 1 to 30 amino acid residues in addition to the at least one mutation described in (1) to (5) above and all the mutations described in (6) to (12) above; (ix) An Fc-binding protein that includes at least the amino acid residues from the 29th alanine to the 425th methionine in the amino acid sequence described in any of Sequence IDs 22, 26, 30, 34, 37, 39, 41, 43, 47, 51, and 55, provided that it has 90% or more identity with the amino acid sequence from the 29th to the 425th, and that at least one of the mutations described in (1) to (5) and all of the mutations described in (6) to (12) of the amino acid sequence remain, and that is capable of binding to the Fc region of an antibody.

5. A polynucleotide encoding an Fc-binding protein according to any one of claims 1 to 4.

6. An expression vector comprising the polynucleotide described in claim 5.

7. A transformant capable of producing Fc-binding proteins, obtained by transforming a host with the recombinant vector described in claim 6.

8. The transformant according to claim 7, wherein the host is Escherichia coli.

9. A method for producing an Fc-binding protein, comprising the steps of: producing an Fc-binding protein by culturing a transformant according to claim 7 or 8; and recovering the Fc-binding protein produced from the obtained culture.

10. An antibody adsorbent obtained by immobilizing an Fc-binding protein according to any one of claims 1 to 4 on an insoluble carrier.

11. A method for separating antibodies, comprising the steps of: adding a solution containing an antibody to a column packed with the adsorbent described in claim 10 to adsorb the antibody onto the adsorbent; and eluting the antibody adsorbed onto the adsorbent using an eluent.

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