Modified fc-binding protein and method for producing same

Abstract

The present invention addresses the problem of providing a polypeptide that exhibits binding ability to a substance having an Fc region such as an immunoglobulin and has excellent productivity and a method for producing the polypeptide. The problem is solved by an Fc-binding protein in which an amino acid at a specific position among the amino acids constituting the Fc-binding protein is substituted with another amino acid.

Description

Modified Fc-binding proteins and methods for producing same

[0001] The present disclosure relates to an Fc-binding protein that has binding affinity to the Fc region of immunoglobulins (e.g., IgG), etc. More specifically, the present disclosure relates to a human neonatal Fc receptor (human FcRn) with improved productivity and a method for producing the receptor.

[0002] Fc receptors are receptor proteins that bind to the Fc region of immunoglobulin molecules and transmit signals intracellularly by binding to immune complexes of antigens and immunoglobulins (Non-Patent Document 1). Each molecule recognizes a single or the same group of immunoglobulin isotypes through a recognition domain on the Fc receptor, which belongs to the immunoglobulin superfamily. This determines which accessory cells are mobilized in the immune response.

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

[0004] On the other hand, human FcRn is a major histocompatibility complex (MHC) class I-related molecule and is composed of a heavy chain (α chain) and a β2 microglobulin (β chain) (Non-Patent Document 3). The amino acid sequence of the α chain of human FcRn (SEQ ID NO: 1) has been published in public databases such as UniProt (Accession number: P55899). The amino acid sequence of the β chain (SEQ ID NO: 2) has been published in UniProt (Accession number: P61769). Figure 1 shows a schematic structure of the α chain of human FcRn, and Figure 2 shows a schematic structure of the β chain. The amino acid numbers in Figure 1 correspond to those in SEQ ID NO: 1. That is, in SEQ ID NO: 1, the sequence from methionine (M) at position 1 to glycine (G) at position 23 is the signal sequence (S), the sequence from alanine (A) at position 24 to serine (S) at position 297 is the extracellular domain (EC), the sequence from valine (V) at position 298 to tryptophan (W) at position 321 is the transmembrane domain (TM), and the sequence from arginine (R) at position 322 to alanine (A) at position 365 is the intracellular domain (C). The amino acid numbers in FIG. 2 correspond to the amino acid numbers set forth in SEQ ID NO: 2. That is, the sequence from methionine (M) at position 1 to alanine (A) at position 20 in SEQ ID NO: 2 is the signal sequence (S), and the sequence from isoleucine (I) at position 21 to methionine (M) at position 119 is the β2 microglobulin (B2M).

[0005] The binding of human FcRn to IgG (Fc region) is pH-dependent, binding at pH 6.0 to 6.5 and dissociating at pH 7.4 or higher. This pH dependence is involved in the recycling and transport mechanisms of IgG by human FcRn in the body, and IgG and Fc fusion proteins that bind and dissociate from human FcRn in a pH-dependent manner are known to have long in vivo lifetimes (Non-Patent Document 4). Taking advantage of this characteristic of human FcRn, a method is known for evaluating the in vivo lifetime of human IgG using an affinity column with recombinant human FcRn as a ligand (Non-Patent Document 5).

[0006] As mentioned above, recombinant human FcRn has the property of functioning as a ligand for affinity columns. Recombinant human FcRn with improved functions and methods for producing the same have been disclosed (Patent Documents 1 to 4), but further improvements in productivity have been desired.

[0007] JP 2018-183087 A JP 2021-073883 A JP 2021-136967 A JP 2022-076998 A

[0008] Takai T. , Jpn. J. Clin. Immunol. , 28, 318-326, 2005J. Galon et al. , Eur. J. Immunol. , 27, 1928-1932, 1997N. E. Simister et al., Nature, 337, 184-187, 1989M. Raghavan et al., Biochemistry, 34, 14649-14657, 1995F. Cymer et al., Bioanalysis, 9, 1305-1317, 2017

[0009] An object of one aspect of the present disclosure is to provide a polypeptide that exhibits binding to a substance having an Fc region, such as immunoglobulin, and that is highly productive, and a method for producing the same.An object of another aspect of the present disclosure is to provide a polypeptide that exhibits binding to a substance having an Fc region, such as immunoglobulin, and that has excellent alkali resistance, and a method for producing the same.

[0010] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that the productivity of an Fc-binding protein can be improved by substituting an amino acid residue at a specific position among the amino acids constituting the Fc-binding protein with another specific amino acid residue.

[0011] That is, the present disclosure includes aspects described in the following [1] to

[11] . [1] An Fc-binding protein selected from any of the following to <c>: An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the following amino acid substitutions (1) to (7) has occurred in the amino acid residues: (1) cysteine ​​at position 71 in SEQ ID NO: 1 is substituted with arginine; (2) asparagine at position 78 in SEQ ID NO: 1 is substituted with aspartic acid; (3) arginine at position 192 in SEQ ID NO: 1 is substituted with leucine; (4) asparagine at position 196 in SEQ ID NO: 1 is substituted with aspartic acid; (5) glutamine at position 232 in SEQ ID NO: 1 is substituted with leucine; (6) lysine at position 295 in SEQ ID NO: 1 is substituted with glutamic acid; (7) cysteine ​​at position 274 in SEQ ID NO: 1 is substituted with another amino acid; An Fc-binding protein comprising the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence set forth 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 set forth in SEQ ID NO: 2, with the proviso that the amino acid substitutions shown in (1) to (7) above have occurred in the amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitutions shown in (1) to (7), and which has antibody-binding activity; <c> An Fc-binding protein comprising the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, with the amino acid sequence in which the amino acid substitutions shown in (1) to (7) above have occurred, and which has antibody-binding activity.[2] The Fc-binding protein according to [1], selected from any of the following <d> to <f>: <d> An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid residues have undergone the amino acid substitutions shown in (1) to (7) above and one or more of the amino acid substitutions shown in (8) to (10) below; (8) A substitution of valine at position 80 in SEQ ID NO: 1 with aspartic acid; (9) A substitution of lysine at position 96 in SEQ ID NO: 1 with glutamic acid; and (10) A substitution of asparagine at position 172 in SEQ ID NO: 1 with aspartic acid. <e> An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid residues have at least one of the amino acid substitutions set forth in (1) to (7) above and (8) to (10) above, and further have at least one of substitution, deletion, insertion, and addition of one or several amino acid residues at one or several positions other than the amino acid substitutions set forth in (1) to (10), and has antibody-binding activity; <f> An Fc-binding protein comprising an amino acid sequence in which the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2 has amino acid substitutions shown in (1) to (7) and at least one of amino acid substitutions shown in (8) to (10), and which has 70% or more identity to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (10) remain, and which has antibody-binding activity.[3] An Fc-binding protein according to [1] or [2], selected from any of the following <g> to : <g> an Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the amino acid substitutions set forth in (1) to (10) has occurred in said amino acid residues; <h> an Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the amino acid substitutions set forth in (1) to (10) has occurred in said amino acid residues, and further comprising any one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions set forth in (1) to (10), and having antibody-binding activity; An Fc-binding protein comprising an amino acid sequence that has 70% or more identity to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (10) have occurred in the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, wherein the amino acid substitutions shown in (1) to (10) remain, and that has antibody-binding activity. [4] An Fc-binding protein according to any of [1] to [3], wherein the other amino acid in (7) is any of serine, glutamic acid, and histidine. [5] A polynucleotide encoding the Fc-binding protein according to any of [1] to [4]. [6] An expression vector comprising the polynucleotide according to [5]. [7] A transformant capable of producing an Fc-binding protein, obtained by transforming a host with the expression vector according to [6]. [8] The transformant according to [7], wherein the host is Escherichia coli.[9] A method for producing an Fc-binding protein, comprising the steps of producing the Fc-binding protein by culturing the transformant according to [7] or [8] and recovering the Fc-binding protein produced from the resulting culture.

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

[11] A method for separating a substance having an Fc region, comprising the steps of adding a solution containing a substance having an Fc region to a column packed with the adsorbent according to

[10] to adsorb the substance having the Fc region to the adsorbent, and eluting the substance having the Fc region adsorbed to the adsorbent with an eluent.

[0012] 1 is a schematic diagram of the α-chain of human FcRn. The numbers in the diagram indicate the numbers of the amino acid sequence set forth in SEQ ID NO: 1. In the diagram, S indicates the signal sequence, EC indicates the extracellular domain, TM indicates the transmembrane domain, and C indicates the intracellular domain. It is a schematic diagram of the β-chain of human FcRn. The numbers in the diagram indicate the numbers of the amino acid sequence set forth in SEQ ID NO: 2. In the diagram, S indicates the signal sequence, and B2M indicates β2-microglobulin. It shows the results of comparing the expression levels (brightness) of Fc-binding proteins in which the 274th cysteine ​​(Cα274) of SEQ ID NO: 1 has been substituted with other amino acids. Note that brightness is expressed as a relative value, with the value for the unsubstituted Fc-binding protein (i.e., Cα274 intact) being taken as 1. 9 shows the results of comparing the purification yields of FcRnm7b-2 (abbreviated as m7b in the figure, corresponding to the portion from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 92) and FcRnm10 (abbreviated as m10 in the figure, corresponding to the portion from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 93). The purification yields are expressed as relative values, with the purification yield for m7b defined as 1. This figure shows the results of measuring the binding activity of FcRnm10 (corresponding to the portion from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 93) to IgG. This figure shows the results of separating a monoclonal antibody (Zenyaku Kogyo Co., Ltd., Rituxan) using a column packed with gel immobilized with FcRnm10 (corresponding to the portion from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 93). This figure shows the results of separating various antibodies using a column packed with gel immobilized with FcRnm10 (corresponding to the sequence from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 93). The <No. > in the figure corresponds to the antibody listed in Table 6. This figure shows the results of evaluating the alkaline resistance of Fc-binding proteins. WT represents FcRnWT, m7a represents FcRnm7a, m7b represents FcRnm7b, and m10 represents FcRnm10.

[0013] DETAILED DESCRIPTION OF THE DISCLOSURE In the following detailed description of the present disclosure, the singular forms "a," "an," and "the" are intended to include plural referents unless the context clearly dictates otherwise.

[0014] The Fc-binding protein of the present disclosure is a protein that has binding activity to the Fc region and contains at least the amino acid residues shown in (I) and (II) below, with the proviso that amino acid substitutions have occurred at specific positions in the amino acid residues: (I) amino acid residues from alanine at position 24 to serine at position 297, which correspond to the extracellular region of the human FcRnα chain (the EC region in Figure 1 ) consisting of the amino acid sequence set forth in SEQ ID NO: 1; (II) amino acid residues from isoleucine at position 21 to methionine at position 119, which correspond to the β2 microglobulin region of the human FcRnβ chain (the B2M region in Figure 2 ) consisting of the amino acid sequence set forth in SEQ ID NO: 2. Thus, the Fc-binding protein of the present disclosure may contain all or a part of the signal peptide region (the S region in Figures 1 and 2 ) located on the N-terminal side of the extracellular region of the human FcRnα chain (the EC region in Figure 1 ) or the β2 microglobulin region of the human FcRnβ chain (the B2M region in Figure 2 ). The Fc-binding protein may also comprise all or part of the transmembrane domain (the TM domain in Figure 1 ) and extracellular domain (the C domain in Figure 1 ) located on the C-terminal side of the extracellular domain (the EC domain in Figure 1 ) of the human FcRn α chain.

[0015] As used herein, the term "Fc region" refers to the Fc region of an immunoglobulin. "Immunoglobulin" may be interpreted as "antibody." In other words, the Fc region of an immunoglobulin is also referred to as the Fc region of an antibody. Proteins capable of binding to an Fc region may be capable of binding not only to antibodies but also to any substance that has an Fc region. A substance that has an Fc region is, in other words, a substance that has the Fc region of an antibody and may be a substance consisting of the Fc region of an antibody and any substance, for example, a protein having any amino acid sequence or any small molecule compound. Examples of substances that have an Fc region include antibodies, Fc fusion proteins, and complexes of an Fc region and a drug. The Fc-binding protein of the present disclosure, which is a protein capable of binding to an Fc region, may be a protein that can bind to any substance, including substances that have an Fc region.

[0016] As used herein, an Fc-binding protein containing at least the amino acid residues shown in (I) and (II) refers to a protein whose amino acid sequence 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) does not matter. That is, the amino acid residue shown in (II) may be located on the N-terminal or 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 may be linked via a known linker such as a GS linker (e.g., a linker consisting of four glycine (G) residues and one serine (S) residue repeat). The length of the linker may be any length and is not particularly limited. Specifically, the length of the linker may be, for example, 5 to 50 amino acid residues.

[0017] The amino acid substitutions at specific positions specifically refer to the following (1) to (7). It has been found that the productivity of Fc-binding proteins is improved by having the amino acid substitutions shown below. (1) Cysteine ​​at position 71 of SEQ ID NO: 1 is substituted with arginine. (2) Asparagine at position 78 of SEQ ID NO: 1 is substituted with aspartic acid. (3) Arginine at position 192 of SEQ ID NO: 1 is substituted with leucine. (4) Asparagine at position 196 of SEQ ID NO: 1 is substituted with aspartic acid. (5) Glutamine at position 232 of SEQ ID NO: 1 is substituted with leucine. (6) Lysine at position 295 of SEQ ID NO: 1 is substituted with glutamic acid. (7) Cysteine ​​at position 274 of SEQ ID NO: 1 is substituted with another amino acid.

[0018] In one embodiment, the Fc-binding protein may be any of the following proteins: An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the following amino acid substitutions (1) to (7) has occurred in the amino acid residues: (1) cysteine ​​at position 71 in SEQ ID NO: 1 is substituted with arginine; (2) asparagine at position 78 in SEQ ID NO: 1 is substituted with aspartic acid; (3) arginine at position 192 in SEQ ID NO: 1 is substituted with leucine; (4) asparagine at position 196 in SEQ ID NO: 1 is substituted with aspartic acid; (5) glutamine at position 232 in SEQ ID NO: 1 is substituted with leucine; (6) lysine at position 295 in SEQ ID NO: 1 is substituted with glutamic acid; and (7) cysteine ​​at position 274 in SEQ ID NO: 1 is substituted with another amino acid. An Fc-binding protein comprising the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence set forth 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 set forth in SEQ ID NO: 2, with the proviso that the amino acid substitutions shown in (1) to (7) above have occurred in the amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions in addition to the amino acid substitutions shown in (1) to (7), and having antibody-binding activity. <c> An Fc-binding protein comprising the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, with the amino acid sequence in which the amino acid substitutions shown in (1) to (7) have occurred, and in which the amino acid substitutions shown in (1) to (7) remain, and having antibody-binding activity.<d> An Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid residues have undergone the amino acid substitutions shown in (1) to (7) above and at least one of the amino acid substitutions shown in (8) to (10) below: (8) substitution of valine at position 80 in SEQ ID NO: 1 with aspartic acid; (9) substitution of lysine at position 96 in SEQ ID NO: 1 with glutamic acid; and (10) substitution of asparagine at position 172 in SEQ ID NO: 1 with aspartic acid.

[0019] <e> An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid residues have at least one of the amino acid substitutions shown in (1) to (7) and (8) to (10), and further has one or more of the substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (10), and has antibody-binding activity. <f> An Fc-binding protein comprising an amino acid sequence that has 70% or more identity to the entire amino acid sequence comprising the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, in which the amino acid substitutions set forth in (1) to (7) and at least one of the amino acid substitutions set forth in (8) to (10) have occurred, wherein the amino acid substitutions set forth in (1) to (10) remain, and which has antibody-binding activity. <g> An Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, in which at least one of the amino acid substitutions set forth in (1) to (10) has occurred in said amino acid residues. <h> An Fc-binding protein comprising the amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that the amino acid substitutions shown in (1) to (10) above have occurred in these amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (10), and having antibody-binding activity. An Fc-binding protein comprising an amino acid sequence in which the amino acid substitutions (1) to (10) have occurred in the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, and which has 70% or more identity to the entire amino acid sequence in which the amino acid substitutions (1) to (10) have occurred, and which has antibody-binding activity.

[0020] The Fc-binding protein of the present disclosure is sufficient as long as it has at least the amino acid substitutions at the specific positions described above, and may further have one or more of substitutions, deletions, insertions, and additions of amino acid residues (hereinafter collectively referred to as "modifications") in addition to the amino acid substitutions at the specific positions described above, as long as it has antibody-binding activity.

[0021] In the above , <e>, and <h>, "one or several" varies depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but means, for example, any of 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, and 1 to 2. The position of the amino acid residue modification may be any position in the amino acid residue portion from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1, or the amino acid residue portion from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2, except for those explicitly excluded in the above , <e>, and <h>. Furthermore, the modification of "one or several" amino acid residues may occur at positions other than those disclosed in JP 2018-183087 A, JP 2021-073883 A, JP 2021-136967 A, and JP 2022-076998 A, as long as the antibody binding activity is maintained. Furthermore, "any one or more of substitution, deletion, insertion, and addition" may also include naturally occurring mutations (mutants or variants) based on individual differences, species differences, etc. of the microorganism from which the gene is derived.

[0022] The identity of the amino acid sequences in <c>, <f>, and may be 70% or more, and may be higher, for example, 80% or more, 85% or more, 90% or more, or 95% or more.

[0023] As used herein, the "identity" of amino acid sequences refers to the percentage obtained by aligning two amino acid sequences to be compared so that as many amino acid residues as possible are identical, and dividing the number of identical amino acid residues by the total number of amino acid residues. During the alignment, gaps may be inserted as needed into one or both of the two sequences to be compared. The method for aligning such sequences is not particularly limited, and can be performed using well-known sequence comparison programs such as BLAST, FASTA, and CLUSTALW. When gaps are inserted, the total number of amino acid residues is calculated by counting each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences to be compared, the sequence identity (%) is calculated by dividing the number of identical amino acid residues by the total number of amino acid residues in the longer sequence.

[0024] As used herein, "antibody binding activity" refers to the activity of binding to an antibody as a target molecule. The "antibody binding activity" in , <e>, <h>, <c>, <f>, and may also refer to the activity of binding to the Fc region, i.e., Fc binding activity.

[0025] The Fc-binding proteins of the present disclosure may further have conservative substitutions between amino acids that have similar physical and / or chemical properties. Conservative substitutions are generally known to those skilled in the art, not just for Fc-binding proteins, because protein function is maintained between those with and without the substitution. Examples of conservative substitutions include substitutions between glycine and alanine, between aspartic acid and glutamic acid, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005).

[0026] Furthermore, substitution of cysteine ​​at position 274 of SEQ ID NO: 1 with any of glutamic acid, histidine and serine is preferred in terms of further improving productivity.

[0027] The Fc-binding protein may further have an oligopeptide attached to its N- or C-terminus, which is useful for separating the Fc-binding protein from a solution containing contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Furthermore, a cysteine-containing oligopeptide, which is useful for immobilizing the Fc-binding protein on a solid phase such as a chromatographic support, may be attached to the N- or C-terminus of the Fc-binding protein. The length of the oligopeptide attached to the N- or C-terminus of the Fc-binding protein is not particularly limited, as long as it does not impair the antibody-binding ability or stability of the Fc-binding protein. When attaching the oligopeptide to the Fc-binding protein, a polynucleotide encoding the oligopeptide may be prepared and then attached to the N- or C-terminus of the Fc-binding protein by genetic engineering using methods well known to those skilled in the art. Alternatively, the chemically synthesized oligopeptide may be added to the N-terminus or C-terminus of the Fc-binding protein by chemically binding.

[0028] Furthermore, a signal peptide may be added to the N-terminus of the Fc-binding protein to promote efficient expression in the host. When the host is Escherichia coli, examples of such signal peptides include signal peptides that direct protein secretion into the periplasm, such as PelB, DsbA, MalE (the region from amino acids 1 to 26 of the amino acid sequence described in UniProt No. P0AEX9), and TorT (Japanese Patent Application Laid-Open No. 2011-097898). In particular, the use of: (A) a polynucleotide encoding the native OmpA signal peptide (the region from amino acids 1 to 21 of UniProt No. P0A910); or (B) an oligonucleotide encoding a polypeptide in which one or more residues of the signal peptide described in (A) have been substituted, deleted, inserted, or added (modified) is preferred, as this allows for more efficient production.

[0029] Examples of methods for producing a polynucleotide encoding an Fc-binding protein of the present disclosure (hereinafter also simply referred to as a polynucleotide of the present disclosure) include: <1> a method in which the amino acid sequence of the Fc-binding protein of the present disclosure is converted into a nucleotide sequence, and a polynucleotide containing the nucleotide sequence is artificially synthesized; and <2> a method in which a polynucleotide containing the entire or partial sequence of the Fc-binding protein is artificially prepared directly, or prepared from the cDNA of the Fc-binding protein or the like using a DNA amplification method such as PCR, and the prepared polynucleotides are ligated by an appropriate method.

[0030] In the method <1>, when converting an amino acid sequence to a nucleotide sequence, it is preferable to consider the codon usage frequency in the host to be transformed. For example, when the host is Escherichia coli, AGA, AGG, CGG, and CGA are used for arginine (R), ATA for isoleucine (I), CTA for leucine (L), GGA for glycine (G), and CCC for proline (P), and these codons are used infrequently (so-called rare codons), so conversion can be performed to avoid these codons. Analysis of codon usage frequency can also be performed using public databases (e.g., the Codon Usage Database on the Kazusa DNA Research Institute website).

[0031] When transforming a host using a polynucleotide of the present disclosure, the polynucleotide itself may be used, but it is more preferable to use an expression vector, such as a bacteriophage, cosmid, or plasmid, typically used for transforming prokaryotic or eukaryotic cells, into which the polynucleotide 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 Escherichia coli is used as the host, examples include pET plasmid vectors, pUC plasmid vectors, pTrc plasmid vectors, pCDF plasmid vectors, and pBBR plasmid vectors. The appropriate position refers to a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or regions involved in transmissibility. When inserting the polynucleotide into the expression vector, it is preferable to insert it in a state linked to a functional polynucleotide, such as a promoter, required for expression. When the host is Escherichia coli, examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and further the λPL promoter and λPR promoter of λ phage.

[0032] The polynucleotide prepared by the above method can be inserted into an expression vector containing the polynucleotide (hereinafter referred to as the expression vector of the present disclosure) to transform a host using a method commonly used by those skilled in the art. For example, when a microorganism belonging to the genus Escherichia (such as E. coli JM109, E. coli BL21(DE3), or E. coli W3110) is selected as the host, transformation can be performed using a method described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). Transformants obtained by transformation using the above method can be screened by an appropriate method to obtain a transformant capable of expressing the Fc-binding protein (hereinafter referred to as the transformant of the present disclosure). There are no particular limitations on the host in which the Fc-binding protein is expressed, and examples include animal cells (CHO (Chinese Hamster Ovary) cells, HEK cells, HeLa cells, COS cells, etc.), yeast (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus, Schizosaccharomyces pombe, etc.), insect cells (Sf9, Sf21, etc.), Escherichia coli (JM109 strain, BL21(DE3) strain, W3110 strain, etc.), and Bacillus subtilis. In terms of productivity, it is preferable to use animal cells or E. coli as a host, and it is more preferable to use E. coli as a host.

[0033] To prepare an expression vector of the present disclosure from a transformant of the present disclosure, the vector can be prepared from the culture obtained by culturing the transformant using an alkaline extraction method or a commercially available extraction kit such as the QIAprep Spin Miniprep kit (Qiagen). The Fc-binding protein can be produced by culturing the transformant and recovering the Fc-binding protein from the resulting culture. As used herein, the term "culture" may include not only the cultured cells of the transformant of the present disclosure, but also the medium used for the culture. The transformant used in the protein production method of the present disclosure may be cultured in a medium suitable for culturing the target host. When the host is Escherichia coli, LB (Luria-Bertani) medium supplemented with necessary nutrients is one example of a preferred medium.

[0034] To selectively grow the transformant of the present disclosure depending on whether or not the vector of the present disclosure has been introduced, it is preferable to culture the transformant in a medium containing a drug corresponding to the drug resistance gene contained in the vector. For example, if the vector contains a kanamycin resistance gene, kanamycin can be added to the medium. The medium may also contain appropriate nutrient sources in addition to carbon, nitrogen, and inorganic salt sources, and may optionally contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. Furthermore, a reagent such as glycine that promotes protein secretion from the transformant into the culture medium may be added. Specifically, when the host is Escherichia coli, it is preferable to add glycine to the medium at 2% (w / v) or less.

[0035] When the host is E. coli, the culture temperature is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but this can be selected depending on the properties of the protein to be expressed. When the host is E. coli, the pH of the medium is 6.8 to 7.4, preferably around pH 7.0. Furthermore, when an inducible promoter is contained in the vector of the present disclosure, induction is preferably carried out under conditions that allow good expression of the Fc-binding protein of the present disclosure. An example of an inducer 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 culture, to induce expression of the Fc-binding protein. The concentration of IPTG to be added may be appropriately selected from the range of 0.005 mmol / L to 1.0 mmol / L, preferably from 0.01 mmol / L to 0.5 mmol / L. IPTG induction may be carried out under various conditions well known in the art.

[0036] As used herein, "improved productivity" means that the amount of Fc-binding protein that can be recovered from the same amount of culture medium is increased compared to the conventional amount.

[0037] To recover the Fc-binding protein of the present disclosure from a culture obtained by culturing a transformant of the present disclosure, the Fc-binding protein can be recovered by isolation and purification from the culture using a method appropriate for the expression form of the Fc-binding protein in the transformant. For example, if the protein is expressed in the culture supernatant, the cells can be separated by centrifugation, and the Fc-binding protein can be purified from the resulting culture supernatant. Alternatively, if the protein is expressed intracellularly (including the periplasm), the cells can be collected by centrifugation, then disrupted by adding an enzyme treatment agent, surfactant, or the like, to extract the Fc-binding protein. The Fc-binding protein can then be purified. Purification of the Fc-binding protein can be achieved using methods known in the art, including separation and purification using liquid chromatography. Liquid chromatography includes ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, and the like. Highly purified Fc-binding proteins can be obtained by combining these chromatographic methods for purification. The productivity of the Fc-binding protein may be evaluated by SDS-PAGE or by measuring absorbance at 280 nm.

[0038] The binding activity of the obtained Fc-binding protein toward an antibody can be measured, for example, by measuring the binding activity toward an antibody using an enzyme-linked immunosorbent assay (hereinafter referred to as ELISA). The antibody is preferably immunoglobulin G (IgG). Furthermore, the IgG used to measure binding activity is preferably human IgG. The Fc-binding activity of the Fc-binding protein may be measured by ELISA not only for antibodies, but also for any substance having an Fc region. Even Fc-binding activity measured using any substance having an Fc region other than an antibody may be considered to substantially exhibit binding activity toward an antibody.

[0039] As used herein, "improved alkali resistance" may mean that the binding activity to an antibody remaining after treatment with an alkali such as NaOH is greater than that of conventional Fc-binding proteins.

[0040] Alkali resistance may be evaluated, for example, by treating the Fc-binding protein with alkali and then measuring its binding activity to an antibody. An example of a method for treating the Fc-binding protein with alkali is to place the Fc-binding protein in an aqueous sodium hydroxide (NaOH) solution, more specifically, in a 0.01 mol / L to 0.05 mol / L NaOH solution. Methods for measuring the binding activity of an Fc-binding protein to an antibody include the methods described above.

[0041] An antibody adsorbent can be produced by binding (immobilizing) the Fc-binding protein of the present disclosure to an insoluble carrier. The insoluble carrier is not particularly limited, and examples include carriers made from polysaccharides such as agarose, alginate (alginic acid salts), 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 synthetic polymers are preferred as insoluble carriers. Examples of preferred carriers include polymethacrylate gels with introduced hydroxy groups, such as Toyopearl (Tosoh Corporation), agarose gels such as Sepharose (Cytiva), and cellulose gels such as Cellufine (JNC). The shape of the insoluble carrier is not particularly limited, and it may be granular or non-granular, porous or non-porous.

[0042] To immobilize an Fc-binding protein of the present disclosure on an insoluble support, an active group such as an N-hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetamide (iodoacetamide, bromoacetamide, etc.) may be attached to the insoluble support, and the human Fc-binding protein may be covalently bonded to the insoluble support via the active group. Commercially available supports to which active groups have been attached may be used as they are, or they may be prepared by introducing active groups onto the support surface under appropriate reaction conditions. Examples of commercially available carriers to which active groups have been added include TOYOPEARL AF-Epoxy-650M and TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, and Epoxy-activated Sepharose 6B (all manufactured by Cytiva), and SulfoLink Coupling Resin (Thermo Fisher Scientific).

[0043] On the other hand, an example of a method for introducing active groups onto the support surface is a method in which one of a compound having two or more active sites is reacted with a hydroxy group, epoxy group, carboxy group, amino group, or the like present on the support surface. Examples of such compounds that introduce epoxy groups into hydroxy groups or amino groups on the support surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of compounds that introduce carboxy groups onto the support surface after introducing epoxy groups onto the support surface using the above-mentioned compounds include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0044] Compounds that introduce maleimide groups into hydroxy groups, epoxy groups, carboxy groups, and amino groups present on the surface of a support include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, 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.

[0045] Examples of compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the support surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester. Another example of a method involves reacting an ω-alkenylalkane glycidyl ether with hydroxy groups or amino groups present on the support surface, followed by halogenating and activating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenylalkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0046] Another example of a method for introducing active groups onto the support surface is to introduce active groups onto carboxy 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-hydroxybenzotriazole.

[0047] Examples of buffer solutions that can be used when immobilizing an Fc-binding protein of the present disclosure on an insoluble carrier include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffer, Tris buffer, borate buffer, and bis-trispropane (1,3-bis[tris(hydroxymethyl)methylamino]propane) buffer. The reaction temperature during immobilization may be appropriately set within the range of 5°C to 50°C, taking into consideration the reactivity of the active group and the stability of the Fc-binding protein, and is preferably within the range of 10°C to 35°C.

[0048] To purify an antibody using an antibody adsorbent obtained by immobilizing an Fc-binding protein of the present disclosure on an insoluble carrier, for example, a buffer solution containing the antibody is added to a column packed with the adsorbent using a liquid delivery means such as a pump, thereby allowing the antibody to be specifically adsorbed to the adsorbent, and then an appropriate eluent is added to the column to elute the antibody.

[0049] The adsorbent can be used to separate substances having an Fc region. Examples of substances having an Fc region include antibodies, Fc fusion proteins, and complexes of an Fc region and a drug. The antibody may be any type, as long as it contains at least the Fc region of an antibody that has affinity for an Fc-binding protein. Examples of antibodies include chimeric antibodies, humanized antibodies, human antibodies, and their amino acid-substituted derivatives, as well as bispecific antibodies, which are commonly used as antibodies for antibody drugs. Examples of Fc fusion proteins include those in which a peptide, nucleic acid, or protein is fused to an Fc region. Examples of complexes of an antibody Fc region and a drug include antibody-drug conjugates (ADCs). The Fc region of a substance having an Fc region may contain at least the Fc region of IgG. Furthermore, the Fc region of a substance having an Fc region may contain at least the Fc region of human IgG.

[0050] As used herein, the term "Fc region" specifically refers to a region of a protein having an amino acid sequence set forth in any one of SEQ ID NOS: 101 to 117, or a region having an amino acid sequence in which one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in the amino acid sequence set forth in any one of SEQ ID NOS: 101 to 117 have occurred, or a region having an amino acid sequence having 70% or more, 80% or more, 85% or more, or 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOS: 101 to 117. Note that "one or several" may mean, for example, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 amino acids.

[0051] Examples of substances having a region having the amino acid sequence set forth in any one of SEQ ID NOs: 101 to 117 include Rituxan, Synagis, Avastin, Erbitux, Herceptin, Kadcyla, Remicade, Humira, Actemra, Vectibix, Ranmark, Emgaity, Zaltrap, Trulicity, Orencia, Romiplate, and Ultomiris. In other words, these substances may be examples of substances having an Fc region. The amino acid sequence set forth in SEQ ID NO: 117 is the amino acid sequence of the Fc region of Ultomiris.

[0052] Furthermore, it is preferable to equilibrate the column with an appropriate buffer before adding the antibody-containing buffer to the column, since this allows for the antibody to be purified to a higher purity. Examples of the buffer include buffers containing inorganic salts, such as phosphate buffer, and the pH of the buffer is from 3.0 to 10.0, preferably from 5.0 to 8.0.

[0053] The antibody adsorbed to the adsorbent can be eluted by weakening the interaction between the antibody and the ligand (the Fc-binding protein of the present disclosure). Specifically, the interaction between the antibody and the ligand may be weakened, for example, by changing the pH using a buffer, a counter peptide, a temperature change, or a salt concentration change. Specific examples of elution solutions for eluting the antibody adsorbed to the adsorbent include buffers that are more basic than the solution used to adsorb the antibody to the adsorbent. Examples of buffer solutions include phosphate buffers, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) buffers, Tris buffers, borate buffers, and bis-trispropane (1,3-Bis[tris(hydroxymethyl)methylamino]propane) buffers, which have a buffering capacity from neutral to basic. The pH of the buffer solution may be set within a range that does not impair the functions of the antibody, and may be preferably from pH 7.0 to 10.0, more preferably from pH 7.0 to 9.0.

[0054] According to one embodiment, the Fc-binding protein of the present disclosure has excellent alkali resistance. Therefore, any buffer solution used in the antibody adsorbent obtained by immobilizing the Fc-binding protein of the present disclosure on an insoluble carrier may have a high pH and is not particularly limited. Furthermore, even if the buffer solution has a high pH, ​​the adsorbent may be reused.

[0055] Hereinafter, the present disclosure will be described in detail using examples and comparative examples, but the present disclosure is not limited to these examples.

[0056] Example 1 Preparation of Plasmid Vector (pETMalE-p7) A plasmid vector was prepared by the following method.

[0057] (1) A polynucleotide encoding the MalE signal peptide (an oligopeptide consisting of 26 residues from the N-terminus of UniProt No. P0AEX9, SEQ ID NO: 3) was prepared by the following two-step PCR. (1-1) Using the reaction solution composition shown in Table 1, the first step of PCR was carried out by repeating five cycles 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. The oligonucleotides used were those consisting of the base sequences set forth in SEQ ID NO: 4 (5'-TATACATATGAAAATAAAAAAACAGGTGCACGCATCC-3'), SEQ ID NO: 5 (5'-GCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCC-3'), SEQ ID NO: 6 (5'-ATCGTCGTTAATGCGGATAATGCGAGGATGCGTGCACCTG-3'), and SEQ ID NO: 7 (5'-TTGTCCCATGGCTTCTTCGATTTTGGCGAGAGCCG-3').

[0058]

[0059] (1-2) Using the reaction solution composition shown in Table 2, the second-stage PCR was carried out by repeating 30 cycles 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. The template DNA was the first-stage PCR product obtained in (1-1), and the PCR primers used were an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 8 (5'-TATACATATGAAAATAAAAAAACAGGTGCACGCATCC-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 9 (5'-GCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCC-3').

[0060]

[0061] (2) The polynucleotide encoding the MalE signal peptide prepared in (1) was digested with restriction enzymes NdeI and NcoI and then ligated to the plasmid vector pET-26b(+) (Novagen) previously digested with restriction enzymes NdeI and NcoI. This was used to transform Escherichia coli BL21(DE3) strain (Novagen) by the calcium chloride method. (3) The resulting transformant was cultured in LB (Luria-Bertani) medium containing 50 μg / mL kanamycin sulfate (Nacalai Tesque), and the plasmid vector (designated pETMalE) was extracted using a QIAprep Spin Miniprep kit (Qiagen). (4) PCR was performed in the same manner as in (1-2), except that the plasmid vector pETMalE obtained in (3) was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 10 (5'-AGTAGTAGGTTGAGGCCGTTGAG-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 11 (5'-TTTTCATATGTATTATGTATATCTCCTTCTTAAA-3') were used as PCR primers. (5) The PCR product obtained in (4) was digested with restriction enzymes SphI and NdeI and then ligated to pETMalE previously digested with restriction enzymes SphI and NdeI, and used to transform E. coli BL21(DE3) strain. (6) After culturing the transformant of (5), a plasmid was extracted (designated pETMalE-p7).

[0062] Example 2 Preparation of Fc-binding protein expression plasmid (MalE) A polynucleotide encoding the Fc-binding protein FcRn(B2M-GS-EC_m7a)-6H consisting of the amino acid sequence set forth in SEQ ID NO: 12 was inserted into the expression vector pETMalE-p7 prepared in Example 1 by the method described below to prepare a plasmid capable of expressing the protein. In FcRn(B2M-GS-EC_m7a)-6H, positions 1 to 99 represent the β2-microglobulin region of the human FcRn β-chain (the region from positions 21 to 119 of SEQ ID NO: 2, B2M), positions 100 to 124 represent the GS linker sequence (a sequence consisting of five repeats of an oligopeptide consisting of four glycine (G) residues and one serine (S) residue, GS), positions 125 to 398 represent an amino acid substitution region of the extracellular region of the human FcRn α-chain (designated EC_m7a, SEQ ID NO: 13), and positions 399 to 404 represent the histidine tag (6H) sequence. EC_m7a is a polypeptide in which the following seven amino acid substitutions have been introduced into the extracellular region of the human FcRn α-chain (region from positions 24 to 297 in SEQ ID NO: 1, EC); Cysteine ​​(C) at position 71 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 13) is substituted with arginine (R); Asparagine (N) at position 78 in SEQ ID NO: 1 (position 55 in SEQ ID NO: 13) is substituted with aspartic acid (D); Glycine (G) at position 151 in SEQ ID NO: 1 (position 128 in SEQ ID NO: 13) is substituted with aspartic acid (D); Arginine (R) at position 192 in SEQ ID NO: 1 (position 169 in SEQ ID NO: 13) is substituted with leucine (L); Asparagine (N) at position 196 in SEQ ID NO: 1 (position 173 in SEQ ID NO: 13) is substituted with aspartic acid (D). Glutamine (Q) at position 232 of SEQ ID NO: 1 (position 209 of SEQ ID NO: 13) is substituted with leucine (L). Lysine (K) at position 295 of SEQ ID NO: 1 (position 272 of SEQ ID NO: 13) is substituted with glutamic acid (E).

[0063] (1) In order to add a cysteine ​​tag (SEQ ID NO: 15) for immobilization to an insoluble carrier to the C-terminus of FcRn (B2M-GS-EC_m7a)-6H, a plasmid containing a polynucleotide (SEQ ID NO: 14) encoding an Fc-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 12 was used as a template DNA. PCR primers were used: an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 16 (5'-GCCTCGGCTCTCGCCATTCAACGTACGCCAAAAATC-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 17 (5'-AGTGCGGCCGCAAGCTTATCCGCAGGTATCGTTGCGGCAGTGATGATGATGATGATGAGAGGATTCGGC-3'). PCR was carried out in the same manner as in Example 1 (1-2), except that the following primers were used: (1) PCR primer (primer) (2) PCR product obtained in (1) was subjected to agarose gel electrophoresis, and purified from the gel using a QIAquick Gel Extraction kit (Qiagen). (3) PCR was performed in the same manner as in Example 1(1-2), except that the template DNA used was pETMalE-p7 prepared in Example 1, and the PCR primers used were an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 18 (5'-GCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTGAGA-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 19 (5'-GGCGAGAGCCGAGGCGGAAAACATCATCGTCGTTAA-3'). (4) The PCR product obtained in (3) was subjected to agarose gel electrophoresis and purified by the method described in (2). (5) The purified PCR products obtained in (2) and (4) were ligated using In-Fusion HD Cloning Kit (Takara Bio Inc.), and the ligation product was then used to transform Escherichia coli JM109 strain (Takara Bio Inc.). (6) A plasmid was extracted from the obtained transformant by the method described in Example 1(3) (designated pETMalE-FcRn(B2M-GS-EC_m7a)-6HC-p7).(7) Of the plasmid pETMalE-FcRn(B2M-GS-EC_m7a)-6HC-p7 extracted in (6), the polynucleotide encoding the Mal signal peptide and FcRn(B2M-GS-EC_m7a) and the base sequence of the surrounding region were analyzed using a fully automated DNA sequencer, Genetic Analyzer 3500 (Thermo Fisher Scientific), and it was confirmed that they were the desired sequences. In this analysis, oligonucleotides consisting of the sequences set forth in SEQ ID NO: 20 (5'-GGATCTCGACGCTCTCCCT-3'), SEQ ID NO: 21 (5'-ATGGGCATTCAACGTACGCCAAA-3'), SEQ ID NO: 22 (5'-CTGCGCATCAAGGAAAAACTGTTC-3'), SEQ ID NO: 23 (5'-CACGCTGGTCTCGCGCAACC-3'), and SEQ ID NO: 24 (5'-ATGCTAGTTATTGCTCAGCGGG-3') were used as sequencing primers.

[0064] The amino acid sequence of the polypeptide MalE-FcRn(B2M-GS-EC_m7a)-6HC expressed by the plasmid pETMalE-FcRn(B2M-GS-EC_m7a)-6HC-p7 is shown in SEQ ID NO: 25, and the nucleotide sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26. In SEQ ID NO: 25, the portion from the 1st methionine (M) to the 26th alanine (A) is the MalE signal peptide (SEQ ID NO: 3), the portion from the 27th isoleucine (I) to the 125th methionine (M) is the B2M region, the portion from the 126th glycine (G) to the 150th serine (S) is the GS linker sequence, the portion from the 151st alanine (A) to the 424th serine (S) is EC_m7a (SEQ ID NO: 13), the 425th to 430th histidines (H) are the histidine tag sequence, and the portion from the 431st cysteine ​​(C) to the 437th glycine (G) is the cysteine ​​tag sequence (SEQ ID NO: 15).

[0065] Example 3 Preparation of Fc-binding protein expression plasmid (OmpA) A polypeptide (SEQ ID NO: 28) was designed in which the signal peptide of MalE-FcRn(B2M-GS-EC_m7a)-6HC (SEQ ID NO: 25) was substituted for the MalE signal peptide (SEQ ID NO: 3) with the OmpA signal peptide (an oligopeptide consisting of 21 residues on the N-terminal side of UniProt No. P0A910, SEQ ID NO: 27), and a plasmid capable of expressing this polypeptide was prepared.

[0066] (1) PCR was performed in the same manner as in Example 1 (1-2), except that the plasmid pETMalE-FcRn(B2M-GS-EC_m7a)-6HC-p7 prepared in Example 2 was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence described in SEQ ID NO: 29 (5'-GCTGGTTTCGCTACCGTAGCGCAGGCCATTCAACGTACGCCAAAAATCCAAGTATACTCACG-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence described in SEQ ID NO: 30 (5'-GGTAGCGAAACCAGCCAGTGCCACTGCAATCGCGATAGCTGTCTTTTCATATGTATTATGTATATC-3') were used as PCR primers. (2) The PCR product obtained in (1) was subjected to agarose gel electrophoresis and purified by the method described in Example 2(2). (3) The purified PCR product obtained in (2) was ligated and transformed by the method described in Example 2(5). The resulting transformant was cultured by the method described in Example 2(6) to prepare a plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m7a)-6HC-p7). (4) In the plasmid pETOmpA-FcRn(B2M-GS-EC_m7a)-6HC-p7 prepared in (3), the nucleotide sequences of the polynucleotides encoding the OmpA signal peptide and FcRn(B2M-GS-EC_m7a) and their surrounding regions were analyzed by the method described in Example 2(7), and it was confirmed that they were the desired sequences. The amino acid sequence of the polypeptide expressed by the plasmid pETOmpA-FcRn(B2M-GS-EC_m7a)-6HC-p7 is shown in SEQ ID NO: 28, and the nucleotide sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 31.In SEQ ID NO: 28, the portion from the 1st methionine (M) to the 21st alanine (A) is the OmpA signal peptide (SEQ ID NO: 27), the portion from the 22nd isoleucine (I) to the 120th methionine (M) is the B2M region, the portion from the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the portion from the 146th alanine (A) to the 419th serine (S) is EC_m7a (SEQ ID NO: 13), the portion from the 420th to 425th histidines (H) is the histidine tag sequence, and the portion from the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag sequence (SEQ ID NO: 15).

[0067] Example 4 Site-specific amino acid substitution in Fc-binding protein Site-specific amino acid substitution was performed on EC_m7a at the cysteine ​​at position 251 of SEQ ID NO: 13 (position 274 in SEQ ID NO: 1, position 396 in SEQ ID NO: 28) (hereinafter also referred to as "Cα274").

[0068] (1) PCR was performed in the same manner as in Example 1 (1-2), except that the plasmid pETOmpA-FcRn(B2M-GS-EC_m7a)-6HC-p7 prepared in Example 3 was used as the template DNA, and a combination of oligonucleotides having the sequences set forth in the SEQ ID NOs shown in Table 3 was used as PCR primers for amino acid substitution.

[0069]

[0070] (2) After preparing a plasmid by the method described in Example 3(2) to (3), the base sequence of the amino acid substitution site and its surrounding region was analyzed by the method described in Example 2(7), and it was confirmed that each sequence was the desired sequence.

[0071] Example 5 Expression of Fc-binding protein (part 1) An Fc-binding protein was expressed by the following method.

[0072] (1) E. coli strain BL21 (DE3) was transformed with each of the plasmids prepared in Example 3 or Example 4, and the resulting transformants capable of expressing Fc-binding proteins were inoculated into 10 mL of TB liquid medium (12 g / L tryptone, 24 g / L yeast extract, 10 g / L glycerol, 9.4 g / L dipotassium hydrogen phosphate, 2.2 g / L potassium dihydrogen phosphate) containing 50 μg / mL kanamycin, and precultured overnight at 37° C. under aerobic shaking. (2) 3 mL of the preculture solution from (1) was inoculated into 200 mL of TB liquid medium containing 50 μg / mL kanamycin in a 1-L baffled flask, and the precultured medium was aerobic and shaken at 30° C. (3) Two hours after the start of cultivation, IPTG (Isopropyl β-D-1-Thiogalactopyranoside) was added to the culture at a final concentration of 0.05 mmol / L while the culture was cooled on ice, and expression was induced by aerobically culturing overnight at 25°C with shaking. (4) After cultivation, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells. (5) After recovering the bacterial cells from the culture medium of the transformant, 100 mmol / L Tris-HCl buffer (pH 8.0) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 5000 Units / L Benzonase (Merck), 0.006% (w / v) lysozyme, and 0.6% (w / v) Triton X-100 (trade name) was added, and the mixture was allowed to stand at room temperature for 2 hours to extract the bacterial cells. This was then centrifuged at 15,000 rpm at 4°C for 20 minutes to obtain an extract containing the expressed Fc-binding protein. (6) The extract obtained in (5) was applied to an Econopack column (Bio-Rad) packed with 1 mL of Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd.) that had been previously equilibrated with 100 mmol / L Tris buffer (pH 8.0) containing 150 mmol / L sodium chloride.(7) The column was washed with 100 mmol / L Tris-HCl buffer (pH 8.0) containing 20 mmol / L imidazole and 150 mmol / L sodium chloride, followed by elution with 100 mmol / L Tris buffer (pH 8.0) containing 300 mmol / L imidazole and 150 mmol / L sodium chloride to obtain a purified solution containing Fc-binding protein. (8) The eluted fraction (purified solution) obtained in (7) was subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and stained with CBB (Coomassie Brilliant Blue) staining solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The intensity of the band corresponding to the Fc-binding protein was quantified using image analysis software (ImageQuant TL 10.0, Cytiva).

[0073] The results are shown in Figure 3. In Figure 3, the brightness is expressed as a relative value, with the value for unsubstituted (i.e., Cα274 as is) being set at 1. It can be seen that substituting Cα274 with other amino acids increases the band brightness, i.e., improves the expression level of Fc-binding protein. In particular, substituting Cα274 with glutamic acid (E) (Cα274E, SEQ ID NO: 38), histidine (H) (Cα274H, SEQ ID NO: 47), or serine (S) (Cα274S, SEQ ID NO: 74) significantly improves the expression level of Fc-binding protein (Cα274E: 5.97-fold, Cα274H: 4.72-fold, Cα274S: 4.70-fold).

[0074] Comparative Example 1 A polypeptide (designated OmpA-FcRn(B2M-GS-EC_m6)-6HC, SEQ ID NO: 89) was designed in which the aspartic acid (D) at position 273 of SEQ ID NO: 28 (position 151 of SEQ ID NO: 1) of the Fc-binding protein OmpA-FcRn(B2M-GS-EC_m7a)-6HC was restored to the native sequence (glycine (G)), and a plasmid capable of expressing this polypeptide was prepared.

[0075] (1) PCR was performed in the same manner as in Example 1 (1-2), except that the plasmid pETOmpA-FcRn(B2M-GS-EC_m7a)-6HC-p7 prepared in Example 3 was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 90 (5'-CCTGGGGCGGCGATTGGCCAGAGGC-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 91 (5'-TCGCCGCCCCAGGTGCCCTGTTTCA-3') were used as PCR primers. (2) A plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m6)-6HC-p7) was prepared by the method described in Example 3(2) to (3), and the nucleotide sequence of the amino acid substitution site and its surrounding region was analyzed by the method described in Example 2(7) to confirm that it was the desired sequence.

[0076] Example 6 Site-Specific Amino Acid Substitution into OmpA-FcRn(B2M-GS-EC_m6)-6HC As a result of the site-specific amino acid substitution into Cα274 ( FIG. 3 ), a substitution with serine (Cα274S) was selected from among the amino acid substitutions that significantly improved the expression level of the Fc-binding protein, and this amino acid substitution was introduced into OmpA-FcRn(B2M-GS-EC_m6)-6HC (SEQ ID NO: 89) to prepare a plasmid for expressing an Fc-binding protein (designated OmpA-FcRn(B2M-GS-EC_m7b)-6HC; SEQ ID NO: 92).

[0077] (1) PCR was performed in the same manner as in Example 1 (1-2), except that the plasmid pETOmpA-FcRn(B2M-GS-EC_m6)-6HC-p7 prepared in Comparative Example 1 was used as the template DNA, and an oligonucleotide (forward primer) consisting of the base sequence set forth in SEQ ID NO: 75 (5'-ACTACAGCTGCATCGTGCAGCACGC-3') and an oligonucleotide (reverse primer) consisting of the base sequence set forth in SEQ ID NO: 76 (5'-ATGCAGCTGTAGTGATGTTCGTCGC-3') were used as PCR primers. (2) A plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m7b)-6HC-p7) was prepared by the method described in Example 3(2) to (3), and the nucleotide sequence of the amino acid substitution site and its surrounding region was analyzed by the method described in Example 2(7) to confirm that it was the desired sequence.

[0078] Example 7 Expression of Fc-binding protein (part 2) An Fc-binding protein was expressed by the following method.

[0079] (1) E. coli BL21 strain (DE3) was transformed with any of the plasmids capable of expressing OmpA-FcRn(B2M-GS-EC_m7a)-6HC (SEQ ID NO: 28) prepared in Example 3, the plasmid capable of expressing the polypeptide consisting of the sequence set forth in SEQ ID NO: 74 prepared in Example 4, the plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m6)-6HC (SEQ ID NO: 89) prepared in Comparative Example 1, and the plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m7b)-6HC (SEQ ID NO: 92) prepared in Example 6. The resulting transformants capable of expressing Fc-binding proteins were inoculated into 30 mL of TB liquid medium containing 50 μg / mL kanamycin. Subsequently, the transformants were pre-cultured overnight at 37 ° C. under aerobic shaking. (2) 30 mL of the preculture solution from (1) was inoculated into 1 L of TB liquid medium supplemented with 50 μg / mL kanamycin in a 5 L baffled flask, and the mixture was cultured aerobically at 30°C with shaking. (3) Expression induction with IPTG was performed using the method described in Example 5(3), and bacterial cells (transformants) were recovered using the method described in Example 5(4). (4) After recovering bacterial cells from the transformant culture, 50 mmol / L of bis-tris propane buffer (pH 10.0) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 5000 Units / L Benzonase (Merck), 0.006% (w / v) lysozyme, and 0.6% (w / v) Triton X-100 (trade name) was added. The extract was left at room temperature for 2 hours to extract bacterial cells. The mixture was then centrifuged at 15,000 rpm for 20 minutes at 4°C to obtain an extract containing the expressed Fc-binding protein. (5) The extract containing the Fc-binding protein obtained in (4) was applied to an Econopack column (Bio-Rad) packed with 5 mL of Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries) that had been equilibrated in advance with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 150 mmol / L sodium chloride.After washing with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 20 mmol / L imidazole and 150 mmol / L sodium chloride, a crude solution containing Fc-binding protein was obtained by elution with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 300 mmol / L imidazole and 150 mmol / L sodium chloride. (6) The crude solution obtained in (5) was adjusted to pH 6.5 using 1 M hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) and then applied to a PolyPrep column (Bio-Rad) packed with 2 mL of IgG-Sepharose (Cytiva) that had been equilibrated with 50 mmol / L bis-tris propane buffer (pH 6.5) containing 150 mmol / L sodium chloride. After washing with the buffer used for equilibration, the column was eluted with 50 mmol / L bis-tris propane buffer (pH 8.5) containing 150 mmol / L sodium chloride to obtain a purified Fc-binding protein. The purified Fc-binding protein obtained using a plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m7a)-6HC (SEQ ID NO: 28) is also referred to as FcRnm7a (corresponding to the 22nd isoleucine (I) through the 432nd glycine (G) of SEQ ID NO: 28). The purified Fc-binding protein obtained using a plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m7b)-6HC (SEQ ID NO: 92) is also referred to as FcRnm7b (corresponding to the 22nd isoleucine (I) through the 432nd glycine (G) of SEQ ID NO: 92). (7) The absorbance (wavelength: 280 nm) of the purified protein solution obtained in (6) was measured, and the purification yield was calculated. The results of comparing the purification yields are shown in Tables 4 and 5. In Tables 4 and 5, the purification yields are expressed as relative values, with the purification yield for an Fc-binding protein without the Cα274S amino acid substitution set at 1.00. In both cases, it can be seen that the purification yield is improved by introducing the Cα274S amino acid substitution.

[0080]

[0081]

[0082] Example 8 Introduction of Amino Acid Substitutions into OmpA-FcRn(B2M-GS-EC_m7b)-6HC The following three amino acid substitutions were introduced into the Fc-binding protein OmpA-FcRn(B2M-GS-EC_m7b)-6HC (SEQ ID NO: 92), to prepare a plasmid for expressing an Fc-binding protein (designated OmpA-FcRn(B2M-GS-EC_m10)-6HC, SEQ ID NO: 93). A substitution of valine (V) at position 202 of SEQ ID NO:92 (position 80 in SEQ ID NO:1) with aspartic acid (D) (hereinafter also referred to as "Vα80D") A substitution of lysine (K) at position 218 of SEQ ID NO:92 (position 96 in SEQ ID NO:1) with glutamic acid (E) (hereinafter also referred to as "Kα96E") A substitution of asparagine (N) at position 294 of SEQ ID NO:92 (position 172 in SEQ ID NO:1) with aspartic acid (D) (hereinafter also referred to as "Nα172D")

[0083] (1) PCR was performed in the same manner as in Example 1 (1-2), except that the plasmid pETOmpA-FcRn(B2M-GS-EC_m7b)-6HC-p7 prepared in Example 6 was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence described in SEQ ID NO: 94 (5'-ACCAGGATTCTTGGTACTGGGAGAA-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence described in SEQ ID NO: 95 (5'-CAAGAATCCTGGTCTTCCCAAACCC-3') were used as PCR primers. (2) A plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m8)-6HC-p7) was prepared by the methods described in Example 3 (2) to (3). (3) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m8)-6HC-p7 prepared in (2) was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence described in SEQ ID NO: 96 (5'-AGGAAGAACTGTTCCTGGAAGCCTT-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence described in SEQ ID NO: 97 (5'-AACAGTTCTTCCTTGATGCGCAGAT-3') were used as PCR primers. (4) A plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m9)-6HC-p7) was prepared by the methods described in Example 3 (2) to (3). (5) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m9)-6HC-p7 prepared in (4) was used as the template DNA, and an oligonucleotide (forward primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 98 (5'-CTGCGGATAAAGAACTGACGTTCCT-3') and an oligonucleotide (reverse primer) consisting of the nucleotide sequence set forth in SEQ ID NO: 99 (5'-TCTTTATCCGCAGCCTTATCTTGCT-3') were used as PCR primers.(6) A plasmid (designated pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7) was prepared by the method described in Example 3(2) to (3), and the nucleotide sequence of the site where the amino acid substitution was introduced and its surrounding region was analyzed by the method described in Example 2(7). It was confirmed that the sequence was the desired one (i.e., the polynucleotide (SEQ ID NO: 100) encoding OmpA-FcRn(B2M-GS-EC_m10)-6HC (SEQ ID NO: 93) was inserted into the plasmid).

[0084] Example 9 Expression of Fc-binding protein (part 3) An Fc-binding protein was expressed by the following method.

[0085] (1) The E. coli W3110 strain was transformed with the plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m7b)-6HC (SEQ ID NO: 92) prepared in Example 6 or the plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m10)-6HC (SEQ ID NO: 93) prepared in Example 8, and the resulting recombinant E. coli capable of expressing an Fc-binding protein was inoculated into 2xYT medium (tryptone: 16 g / L, yeast extract: 10 g / L, sodium chloride: 5 g / L, kanamycin sulfate: 50 mg / L) and pre-cultured at 30°C for 16 hours. (2) To 1.2 L of the initial culture medium (18 g / L disodium hydrogen phosphate dodecahydrate, 6 g / L trisodium phosphate dodecahydrate, 40 g / L Difco Soytone (Thermo Fisher Scientific), 1 g / L ammonium chloride, 10 g / L glucose, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L iron (II) sulfate heptahydrate, 0.005 g / L manganese (II) chloride tetrahydrate, 200 μL / L Adekanol (ADEKA), 50 mg / L kanamycin sulfate), 36 mL of the preculture solution (1) was added, and the main culture was performed. The culture was performed using a BMS-03PI culture apparatus manufactured by Able, with an agitation speed of 400 to 700 rpm, an air flow rate of 1.5 L / min, a culture temperature of 30°C, and a pH of 6.8 to 7.2. Fluctuations in pH during culture were controlled within the above range by adding 14% (w / v) aqueous ammonia or 50% (w / v) phosphoric acid. (3) When the DO (dissolved oxygen concentration) measured using a DO electrode attached to the BMS-03PI exceeded 40% saturation, the feed pump was started and the feed medium (425 g / L D(+)-glucose, 124 g / L Red Label Yeast Extract (Oriental Yeast Co., Ltd.), 12.5 g / L magnesium sulfate heptahydrate, and 50 mg / L kanamycin sulfate) was continuously supplied until the DO returned to 40% saturation or below. This operation was continued until the end of the culture. (4) 19 to 21 hours after the start of culture, the culture temperature was changed to 25°C, the stirring speed to 600 rpm, and IPTG was added to a final concentration of 0.1 mmol / L to induce expression of the Fc-binding protein. (5) 48 hours after the start of culture, the culture was terminated, and the cultured cells were collected by centrifugation of the culture medium.(6) The collected bacterial cells were extracted for 2 hours at room temperature using 50 mmol / L bis-tris propane buffer (pH 10.0) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 5000 Units / L Benzonase (Merck), 0.006% (w / v) lysozyme, and 0.6% (w / v) Triton X-100 (trade name). The bacterial extract was then centrifuged at 15,000 rpm at 4°C for 20 minutes to obtain an extract containing Fc-binding protein. (7) The extract containing the Fc-binding protein obtained in (6) was applied to an Econopack column (Bio-Rad) packed with 5 mL of Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries, Ltd.) previously equilibrated with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 150 mmol / L sodium chloride. The column was washed with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 20 mmol / L imidazole and 150 mmol / L sodium chloride. Subsequently, a crude protein solution containing the Fc-binding protein was obtained by elution with 50 mmol / L bis-tris propane buffer (pH 10.0) containing 300 mmol / L imidazole and 150 mmol / L sodium chloride. (8) The crude protein solution containing the Fc-binding protein obtained in (7) was adjusted to pH 6.5 using 1 mol / L hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.). (9) The crude protein solution containing the pH-adjusted Fc-binding protein obtained in (8) was applied to a PolyPrep column (Bio-Rad) packed with 2 mL of IgG-Sepharose (Cytiva) that had been previously equilibrated with 50 mmol / L bis-tris propane buffer (pH 6.5) containing 150 mmol / L sodium chloride. After washing with the buffer used for equilibration, the column was eluted with 50 mmol / L bis-tris propane buffer (pH 8.5) containing 150 mmol / L sodium chloride to obtain a purified Fc-binding protein.The purified Fc-binding protein obtained using a plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m7b)-6HC (SEQ ID NO: 92) is also referred to as FcRnm7b-2 (corresponding to the sequence from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 92). The purified Fc-binding protein obtained using a plasmid capable of expressing OmpA-FcRn(B2M-GS-EC_m10)-6HC (SEQ ID NO: 93) is also referred to as FcRnm10 (corresponding to the sequence from isoleucine (I) at position 22 to glycine (G) at position 432 in SEQ ID NO: 93). (10) The absorbance at a wavelength of 280 nm of the purified protein solution obtained in (9) was measured, and the purification yield was calculated.

[0086] The results are shown in Figure 4. In Figure 4, the purification yield is expressed as a relative value, with the purification yield for FcRnm7b-2 set at 1. It can be seen that the purification yield is further improved by introducing at least one or more amino acid substitutions among Vα80D, Kα96E, and Nα172D.

[0087] Example 10 Measurement of Binding Activity of Fc-binding Protein to IgG The binding activity of Fc-binding protein to IgG was measured by the method described below.

[0088] (1) The purified Fc-binding protein FcRnm10 solution obtained in Example 9 was diluted with 50 mmol / L MES buffer (pH 6.5) containing 150 mmol / L sodium chloride to prepare solutions with the following Fc-binding protein concentrations: 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.3 μg / mL, and 15.6 μg / mL. (2) A human antibody gamma globulin preparation (KM Biologics) was immobilized at 10 μg / well to the wells of a 96-well microplate (4°C for 18 hours). (3) The plate was washed with a washing buffer (50 mmol / L MES buffer (pH 6.5) containing 150 mmol / L sodium chloride) and then blocked with a buffer (pH 7.4) containing 2% (w / v) SKIM MILK (Becton Dickinson), 137 mmol / L sodium chloride, 8.1 mmol / L disodium hydrogen phosphate, 2.7 mmol / L potassium chloride, and 1.5 mmol / L potassium dihydrogen phosphate. (4) After washing with the washing buffer, a solution containing an Fc-binding protein whose antibody binding activity was to be evaluated was added, and the Fc-binding protein was allowed to react with the immobilized gamma globulin (at 30°C for 1 hour). (5) After washing with the washing buffer, 100 μL / well of anti-FcRn monoclonal antibody (mouse) (SANTA CRUZ BIOTECHNOLOGY: sc-271745) diluted 10,000-fold with the washing buffer was added and allowed to react (30°C for 1 hour). (6) After washing with the washing buffer, 100 μL / well of anti-mouse IgG-heavy and light chain, HRP (goat) (BETHYL LABOLATORIES: A90-216P) diluted 10,000-fold with the washing buffer was added and allowed to react (30°C for 1 hour). (7) After washing with the washing buffer, TMB Peroxidase Substrate (KPL) was added at 50 μL / well. 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).

[0089] The results are shown in Figure 5. Note that the "no IgG immobilization" in Figure 5 represents control data in which a buffer solution (pH 7.4) containing 137 mmol / L sodium chloride, 8.1 mmol / L disodium hydrogen phosphate, 2.7 mmol / L potassium chloride, and 1.5 mmol / L potassium dihydrogen phosphate was added instead of the gamma globulin preparation in (2). Compared to the wells without IgG immobilization, the wells with IgG immobilization had higher absorbance, and this absorbance depended on the concentration of the Fc-binding protein, indicating that the Fc-binding protein of the present disclosure has binding activity to IgG.

[0090] Example 11 Immobilization of Fc-binding protein on an insoluble carrier Two grams of a porous hydrophilic polymer for adsorbents (Tosoh Corporation, G5000PW) was collected as a suction-dried gel, and the hydroxy groups on the gel surface were activated with iodoacetyl groups. 10 mg of FcRnm10 obtained in Example 9 was then reacted with the gel to obtain an OmpA-FcRn(B2M-GS-EC_m10)-6HC-immobilized gel.

[0091] Example 12 Antibody Purification Using an Antibody Adsorbent Column (Part 1) (1) An antibody adsorbent column was prepared by packing 0.83 mL of the OmpA-FcRn(B2M-GS-EC_m10)-6HC-immobilized gel (hereinafter also referred to as "antibody adsorbent") obtained in Example 11 into a 4.6 mm diameter, 50 mm long stainless steel column (Tosoh Corporation). (2) After connecting the antibody adsorbent column prepared in (1) to a Nexera system (Shimadzu Corporation), the column was equilibrated by pumping 5 column volumes (hereinafter referred to as CV, 1 CV = 0.83 mL) of 50 mmol / L MES buffer (pH 6.5) containing 150 mmol / L sodium chloride (hereinafter also referred to as "equilibration solution") into the column at a flow rate of 0.4 mL / min. (3) A monoclonal antibody (Zenyaku Kogyo Co., Ltd., Rituxan) was diluted to 1.0 mg / mL with the equilibration solution used in (2), and 30 μL of this diluted solution was pumped through the antibody adsorbent column at a flow rate of 0.4 mL / min (30 μg antibody load), thereby adsorbing the antibody to the antibody adsorbent. (4) Subsequently, the equilibration solution was pumped through the antibody adsorbent column at a flow rate of 0.4 mL / min for 10 minutes, and then an eluate (50 mmol / L Tris buffer (pH 8.5) containing 150 mmol / L sodium chloride) was pumped through the antibody adsorbent column with a linear gradient such that the eluate reached 100% in 20 minutes, thereby eluting the antibody adsorbed to the antibody adsorbent. After antibody elution, the eluate was pumped through the antibody adsorbent column at a flow rate of 0.4 mL / min for 10 minutes, and then the buffer used for equilibration was pumped through the antibody adsorbent column for 10 minutes to equilibrate it. A chromatogram of the separated monoclonal antibody is shown in Figure 6. In Figure 6, the monoclonal antibody was loaded onto the antibody adsorbent column at time 0 minutes. The monoclonal antibody eluted between 10 and 30 minutes during the linear gradient run, confirming that the antibody can be separated in a column packed with a gel onto which the Fc-binding protein of the present disclosure has been immobilized.

[0092] Example 13: Antibody purification using an antibody adsorbent column (part 2) Separation tests were conducted not only for the monoclonal antibody described in Example 12(3) but also for other substances having an Fc region. In these tests, in addition to the monoclonal antibody, antibody-drug conjugates (ADCs) and Fc fusion proteins were used as substances having an Fc region.

[0093] (1) An antibody adsorbent column was prepared by the method described in Example 12(1), and the column was equilibrated by the method described in Example 12(2). (2) The antibody was adsorbed onto the antibody adsorbent in the same manner as in Example 12(3), except that one of the substances shown in Table 6 was used as the substance having an Fc region, and the antibody adsorbed onto the adsorbent was eluted by the method described in Example 12(4).

[0094]

[0095] The amino acid sequence of the Fc region of the monoclonal antibody described in Example 12 (3), i.e., Rituxan, is shown in SEQ ID NO: 101. Among the substances having an Fc region in Table 6, the amino acid sequence of the Fc region of Synagis is shown in SEQ ID NO: 102, the amino acid sequence of the Fc region of Avastin is shown in SEQ ID NO: 103, the amino acid sequence of the Fc region of Erbitux is shown in SEQ ID NO: 104, the amino acid sequence of the Fc region of Herceptin is shown in SEQ ID NO: 105, the amino acid sequence of the Fc region of Kadcyl a is shown in SEQ ID NO: 106, the amino acid sequence of the Fc region of Remicade is shown in SEQ ID NO: 107, the amino acid sequence of the Fc region of Humira is shown in SEQ ID NO: 108, and the amino acid sequence of the Fc region of Actemra is shown in SEQ ID NO: 109. The amino acid sequence of the Fc region of Vectibix is ​​shown in SEQ ID NO: 110, the amino acid sequence of the Fc region of Ranmark is shown in SEQ ID NO: 111, the amino acid sequence of the Fc region of Emgaity is shown in SEQ ID NO: 112, the amino acid sequence of the Fc region of Zaltrap is shown in SEQ ID NO: 113, the amino acid sequence of the Fc region of Trulicity is shown in SEQ ID NO: 114, the amino acid sequence of the Fc region of Orencia is shown in SEQ ID NO: 115, and the amino acid sequence of the Fc region of Romiplate is shown in SEQ ID NO: 116.

[0096] Figure 7 shows a chromatogram obtained by separating substances having an Fc region shown in Table 6. As in Figure 6, each antibody was loaded onto the antibody adsorbent column at time 0. Each protein eluted between 10 and 30 minutes into the linear gradient run, confirming that substances having an Fc region, other than Rituxan (Example 12), can be separated in a column packed with a gel onto which an Fc-binding protein of the present disclosure has been immobilized, regardless of the type (monoclonal antibody, ADC, Fc fusion protein) of the substance.

[0097] Example 14 Evaluation of Alkaline Tolerance of Fc-Binding Protein (1) Protein Preparation (1-1) An Escherichia coli BL21(DE3) strain transformed with the expression vector pET-eFcRn was prepared using the method disclosed in Example 1 of JP 2018-183087 A. (1-2) A purified Fc-binding protein, FcRnWT (corresponding to the isoleucine (I) at position 34 to the histidine (H) at position 437 of SEQ ID NO: 118), was obtained by the same procedure as in Example 9, except that the E. coli strain prepared in (1-1) was used. The amino acid sequence of the polypeptide expressed by the expression vector pET-eFcRn is shown in SEQ ID NO: 118, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 119. In SEQ ID NO: 118, the portion from methionine (Met) at position 1 to alanine (Ala) at position 26 represents the MalE signal peptide; the portion from lysine (Lys) at position 27 to glycine (Gly) at position 33 represents the linker sequence; the portion from isoleucine (Ile) at position 34 to methionine (Met) at position 132 represents the β2 microglobulin region of the human FcRn β chain (the region from positions 21 to 119 of SEQ ID NO: 2); the portion from glycine (Gly) at position 133 to serine (Ser) at position 157 represents the GS linker sequence; the portion from alanine (Ala) at position 158 to serine (Ser) at position 431 represents the extracellular region of the human FcRn α chain (the region from positions 24 to 297 of SEQ ID NO: 1); and the portion from histidine (His) at positions 432 to 437 represents the tag sequence.

[0098] (2) Evaluation of Alkaline Tolerance (2-1) FcRnWT obtained in (1), FcRnm7a and FcRnm7b obtained in Example 7, and FcRnm10 obtained in Example 9 were adjusted to a final concentration of 0.1 mg / mL. (2-2) NaOH was added to the Fc-binding protein solution prepared in (2-1) to a final concentration of 0.04 mol / L or 0.08 mol / L, and the solution was allowed to stand at room temperature for 1.5 hours. (2-3) After standing in (2-2), 50 mmol / L MES buffer (pH 5.5) containing 150 mmol / L sodium chloride was added to adjust the pH to 6.0. (2-4) The protein solution containing the pH-adjusted Fc-binding protein obtained in (2-3) was applied to a PolyPrep column (Bio-Rad) packed with 0.2 mL of IgG-Sepharose (Cytiva) that had been previously equilibrated with 50 mmol / L bis-tris propane buffer (pH 6.0) containing 150 mmol / L sodium chloride. After washing with the buffer used for equilibration, the Fc-binding protein was obtained by elution with 50 mmol / L bis-tris propane buffer (pH 8.5) containing 150 mmol / L sodium chloride. The same procedure was also performed on the Fc-binding protein solution prepared in (2-1). (2-5) The eluted fraction (purified solution) obtained in (2-4) was subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and stained with CBB (Coomassie Brilliant Blue) staining solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The brightness of the band corresponding to the Fc-binding protein was then quantified using image analysis software (ImageQuant TL 10.0, Cytiva).

[0099] The results are shown in Figure 8. Note that in Figure 8, the brightness is expressed as a relative value, with the value for the elution fraction of each Fc-binding protein to which NaOH was not added being set at 1. It was found that the IgG-binding activity of FcRnWT treated with 0.04 mol / L NaOH was inactivated, whereas IgG-binding activity remained in FcRnm7a, FcRnm7b, and FcRnm10. Furthermore, FcRnm7b and FcRnm10 retained more IgG-binding activity than FcRnm7a. In other words, it was revealed that FcRnm7b and FcRnm10 have improved alkali resistance compared to FcRnm7a. Furthermore, it was revealed that FcRnm10 has improved alkali resistance compared to FcRnm7b.

[0100] That is, it was demonstrated that an Fc-binding protein in which cysteine ​​at position 274 of SEQ ID NO: 1 was substituted with another amino acid not only had improved productivity but also had improved alkaline resistance.

[0101] The Fc-binding protein of the present disclosure is a protein in which an amino acid residue at a specific position in the extracellular region of the human neonatal Fc receptor α-chain is substituted with another amino acid residue. This Fc-binding protein has significantly improved productivity compared to conventional Fc-binding proteins. This Fc-binding protein is useful as a ligand for an adsorbent for separating substances that have an Fc region, such as immunoglobulins, and its improved productivity makes it suitable for industrial production. Furthermore, in one aspect, the Fc-binding protein of the present disclosure has significantly improved alkali resistance compared to conventional Fc-binding proteins. By using this Fc-binding protein as a ligand for an antibody adsorbent, an adsorbent with high resistance to alkaline washing can be obtained. This can therefore contribute to cost reduction in the industrial production of antibody pharmaceuticals.

Claims

1. An Fc binding protein selected from any of the following to <c>: An Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the amino acid substitutions shown in (1) to (7) below has occurred in said amino acid residues; (1) cysteine ​​at position 71 in SEQ ID NO: 1 is replaced by arginine; (2) asparagine at position 78 in SEQ ID NO: 1 is replaced by aspartic acid; (3) arginine at position 192 in SEQ ID NO: 1 is replaced by leucine; (4) asparagine at position 196 in SEQ ID NO: 1 is replaced by aspartic acid; (5) glutamine at position 232 in SEQ ID NO: 1 is replaced by leucine; (6) lysine at position 295 in SEQ ID NO: 1 is replaced by glutamic acid; (7) cysteine ​​at position 274 in SEQ ID NO: 1 is replaced by another amino acid; an Fc-binding protein comprising the amino acid residues from alanine at position 24 to serine at position 297 of the amino acid sequence set forth 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 set forth in SEQ ID NO: 2, with the proviso that the amino acid substitutions shown in (1) to (7) have occurred in said amino acid residues, and further comprising one or more of substitutions, deletions, insertions, and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (7), and having antibody-binding activity; <c> an Fc-binding protein comprising the amino acid sequence from alanine at position 24 to serine at position 297 of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from isoleucine at position 21 to methionine at position 119 of the amino acid sequence set forth in SEQ ID NO: 2, which has an identity of 70% or more to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (7) have occurred, and in which the amino acid substitutions shown in (1) to (7) remain, and having antibody-binding activity.

2. The Fc binding protein according to claim 1, selected from any of the following <d> to <f>: <d> An Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO:1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO:2, with the proviso that the amino acid residues have the amino acid substitutions shown in (1) to (7) above and any one or more of the amino acid substitutions shown in (8) to (10) below; (8) Substitution of valine at position 80 in SEQ ID NO:1 with aspartic acid; (9) Substitution of lysine at position 96 in SEQ ID NO:1 with glutamic acid; (10) Substitution of asparagine at position 172 in SEQ ID NO:1 with aspartic acid. <e> an Fc-binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least one of the amino acid substitutions shown in (1) to (7) and (8) to (10) has occurred in said amino acid residues, and further comprising at least one of substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (10), and having antibody-binding activity; <f> An Fc binding protein comprising an amino acid sequence having an identity of 70% or more to the entire amino acid sequence in which the amino acid sequence from the 24th alanine to the 297th serine in the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence set forth in SEQ ID NO: 2 has the amino acid substitutions shown in (1) to (7) and at least one of the amino acid substitutions shown in (8) to (10), and in which the amino acid substitutions shown in (1) to (10) remain, and which has antibody binding activity.

3. The Fc binding protein according to claim 2, selected from any of the following <g> to : <g> an Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least the amino acid substitutions shown in (1) to (10) have occurred in said amino acid residues; <h> an Fc binding protein comprising amino acid residues from alanine at position 24 to serine at position 297 in the amino acid sequence set forth in SEQ ID NO: 1 and amino acid residues from isoleucine at position 21 to methionine at position 119 in the amino acid sequence set forth in SEQ ID NO: 2, with the proviso that at least the amino acid substitutions shown in (1) to (10) have occurred in said amino acid residues, and further comprising any one or more of substitutions, deletions, insertions and additions of one or several amino acid residues at one or several positions other than the amino acid substitutions shown in (1) to (10), and having antibody binding activity; An Fc-binding protein comprising an amino acid sequence that has 70% or more identity to the entire amino acid sequence in which the amino acid substitutions shown in (1) to (10) have occurred in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence set forth in SEQ ID NO: 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence set forth in SEQ ID NO: 2, and in which the amino acid substitutions shown in (1) to (10) remain, and which has antibody-binding activity.

4. The Fc binding protein according to claim 1, wherein the other amino acid in (7) is any one of serine, glutamic acid and histidine.

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

6. An expression vector comprising the polynucleotide of claim 5.

7. A transformant capable of producing an Fc-binding protein, obtained by transforming a host with the expression vector according to 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 the transformant described in claim 7, and recovering the Fc-binding protein produced from the resulting culture.

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

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

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