Modified fc-binding protein and method for producing same

Abstract

The present disclosure addresses the problem of providing an Fc-binding protein that has been modified so as to have improved productivity.　The problem is solved by an Fc-binding protein in which an amino acid residue at a specific position among amino acids constituting the Fc-binding protein is substituted with another specific amino acid residue.

Description

Modified Fc-binding protein and method for producing the same

[0001] This disclosure relates to a modified Fc-binding protein. One aspect of this disclosure relates to a modified human neonatal Fc receptor (human FcRn) for improved productivity, and a method for producing said human FcRn.

[0002] Fc-binding proteins such as Fc receptors (FcRs) are receptor proteins that bind to the Fc region of immunoglobulin molecules and transmit signals into cells by binding to immune complexes between antigens and immunoglobulins (Non-Patent Literature 1). Each molecule recognizes a single or same group of immunoglobulin isotypes via a recognition domain belonging to the immunoglobulin superfamily on the FcR. This determines which accessory cells are mobilized in the immune response.

[0003] FcR can be further classified into several subtypes, including the Fcγ receptor (FcγR), which is a receptor for immunoglobulin G (IgG), as well as the Fcα receptor (FcαR), the Fcε receptor (FcεR), and others. Each receptor is further classified into subtypes, and in the case of FcγR, it can be classified into FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIIa (CD16a), and FcγRIIIIb (CD16b) (Non-Patent Documents 1 and 2).

[0004] On the other hand, human neonatal Fc receptor (human FcRn) is a major histocompatibility complex (MHC) class I-related molecule, and is composed of a heavy chain (α-chain) and 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 a public database such as UniProt (Accession number: P55899). Also, the amino acid sequence of the β-chain (SEQ ID NO: 2) has been published in UniProt (Accession number: P61769). Fig. 1 shows a schematic diagram of the structure of the α-chain of human FcRn, and Fig. 2 shows a schematic diagram of the structure of the β-chain. Note that the amino acid numbers in Fig. 1 correspond to the amino acid numbers described in SEQ ID NO: 1. That is, from the first methionine (M) to the 23rd glycine (G) in SEQ ID NO: 1 is the signal sequence (S), from the 24th alanine (A) to the 297th serine (S) is the extracellular region (EC), from the 298th valine (V) to the 321st tryptophan (W) is the transmembrane region (TM), and from the 322nd arginine (R) to the 365th alanine (A) is the intracellular region (C). Also, the amino acid numbers in Fig. 2 correspond to the amino acid numbers described in SEQ ID NO: 2. That is, from the first methionine (M) to the 20th alanine (A) in SEQ ID NO: 2 is the signal sequence (S), and from the 21st isoleucine (I) to the 119th methionine (M) is β2-microglobulin (B2M).<> <>

[0005] The binding of human FcRn and 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 mechanism and transport mechanism of IgG in the body by human FcRn, and it is known that IgG and Fc fusion proteins that bind and dissociate to human FcRn in a pH-dependent manner have a long in vivo lifespan (Non-Patent Document 4). A method for evaluating the in vivo lifespan of human IgG using an affinity column with recombinant human FcRn as a ligand has been known (Non-Patent Document 5).<> <>

[0006] As mentioned above, recombinant human FcRn possesses the property of functioning as a ligand for affinity columns. Recombinant human FcRn with improved functions and methods for producing them have been disclosed to date (Patent Documents 1 to 5).

[0007] Japanese Patent Publication No. 2018-183087, Japanese Patent Publication No. 2021-073883, Japanese Patent Publication No. 2021-136967, Japanese Patent Publication No. 2022-076998, WO2025 / 105466

[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] As mentioned above, Patent Documents 1 to 5 disclose Fc-binding proteins with improved functions and methods for producing them, but there was still room for further improvement in productivity.

[0010] Furthermore, the inventors' research has revealed that some of the Fc-binding proteins disclosed to date are degraded during the production process, which includes the cultivation of the Fc-binding protein-producing bacteria. Therefore, the inventors believe that the productivity of Fc-binding proteins can be further improved by suppressing this degradation.

[0011] Therefore, an object of this disclosure is to provide a modified Fc-binding protein that improves productivity. Another object of one aspect of this disclosure is to provide a modified human neonatal Fc receptor (human FcRn) that improves productivity.

[0012] Furthermore, an object of one aspect of this disclosure is to provide a modified Fc-binding protein that suppresses the degradation (also called degradation) of the Fc-binding protein during culture in the production of the Fc-binding protein, which includes a culture step of the Fc-binding protein-producing bacteria. Another object of one aspect of this disclosure is to provide a modified human FcRn that suppresses degradation.

[0013] Furthermore, an object of one aspect of this disclosure is to provide a modified Fc-binding protein that has improved productivity and suppressed degradation. Another object of one aspect of this disclosure is to provide a modified human FcRn that has improved productivity and suppressed degradation.

[0014] As a result of diligent research to solve the above-mentioned problems, the inventors have found that the productivity of Fc-binding proteins 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.

[0015] In other words, the present disclosure includes the embodiments described in [1] to [9] below. [1] An Fc-binding protein selected from any of the following to <c>: An Fc-binding protein comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) below occurs at the said amino acid residue; (1) The 205th methionine in SEQ ID NO: 1 is replaced with valine (2) The 206th arginine in SEQ ID NO: 1 is replaced with threonine (13) The 25th glutamic acid in SEQ ID NO: 1 is replaced with glycine (14) The 28th leucine in SEQ ID NO: 1 is replaced with histidine (15) The 31st leucine in SEQ ID NO: 1 is replaced with arginine (16) The 65th arginine in SEQ ID NO: 1 is replaced with histidine (17) Alanine at position 73 of SEQ ID NO: 1 is replaced with threonine. (18) Threonine at position 89 of SEQ ID NO: 1 is replaced with glutamine. (19) Lysine at position 132 of SEQ ID NO: 1 is replaced with arginine. (20) Lysine at position 173 of SEQ ID NO: 1 is replaced with arginine. (21) Aspartic acid at position 248 of SEQ ID NO: 1 is replaced with glycine. (22) Phenylalanine at position 249 of SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 of SEQ ID NO: 1 is replaced with leucine. (24) Serine at position 253 of SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 of SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 of SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 of SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 of SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 of SEQ ID NO: 2 is replaced with arginine. (30) Asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) Valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) Lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) Methionine at position 119 of SEQ ID NO: 2 is replaced with threonine. An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs at the amino acid residues described in SEQ ID NO: 1 and at least one amino acid substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, in addition to at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33); and <c> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and in the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) remains, and which is an Fc-binding protein having antibody-binding activity. [1'] Fc-binding proteins selected from any of the following <a'> to <c'>: <a'> Fc-binding proteins comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein the following amino acid substitution (1) occurs at the amino acid residue; (1) The 205th methionine in Sequence ID No. 1 is substituted with valine<b'> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which the amino acid substitution described in SEQ ID NO: 1 is performed from the 24th alanine to the 297th serine and the amino acid substitution described in SEQ ID NO: 2 is performed from the 21st isoleucine to the 119th methionine, wherein the amino acid substitution described in (1) occurs at said amino acid residues, and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitution described in (1); and <c'> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitution described in (1) occurs at the amino acid residues from the 24th alanine to the 297th serine and the amino acid residues from the 21st isoleucine to the 119th methionine, wherein the amino acid substitution described in (1) remains; and an Fc-binding protein having antibody-binding activity. [1''] An Fc-binding protein according to [1'], selected from any of the following <a''> to <c''>: <a''> An Fc-binding protein comprising an amino acid sequence in which the amino acid substitution described in <a''> occurs, and further comprising at least one amino acid substitution selected from the group consisting of (2) and (13) to (33) below; (2) Arginine at position 206 of SEQ ID NO: 1 is replaced with threonine (13) Glutamic acid at position 25 of SEQ ID NO: 1 is replaced with glycine (14) Leucine at position 28 of SEQ ID NO: 1 is replaced with histidine (15) Leucine at position 31 of SEQ ID NO: 1 is replaced with arginine (16) Arginine at position 65 of SEQ ID NO: 1 is replaced with histidine (17) Alanine at position 73 of SEQ ID NO: 1 is replaced with threonine (18) Threonine at position 89 of SEQ ID NO: 1 is replaced with glutamine (19) Lysine at position 132 of SEQ ID NO: 1 is replaced with arginine (20) Lysine at position 173 of SEQ ID NO: 1 is replaced with arginine. (21) Aspartic acid at position 248 of SEQ ID NO: 1 is replaced with glycine. (22) Phenylalanine at position 249 of SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 of SEQ ID NO: 1 is replaced with leucine.(24) Serine at position 253 of SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 of SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 of SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 of SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 of SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 of SEQ ID NO: 2 is replaced with arginine. (30) Asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) Valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) Lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) Methionine at position 119 of SEQ ID NO: 2 is replaced with threonine. <b''> An amino acid sequence in which the amino acid substitution described in <a'> occurs, further comprising at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitution in (1) and at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and an Fc-binding protein having antibody-binding activity; and <c''> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitution described in <a'> occurs, further comprising an amino acid sequence in which the amino acid substitution described in <a'> occurs, further comprising at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and further comprising an amino acid sequence in which the amino acid substitution in (1) and at least one amino acid substitution selected from the group consisting of (2) and (13) to (33) remain, and an Fc-binding protein having antibody-binding activity. [2] An Fc-binding protein as described in [1], selected from any of the following <d> to <f>:<d> An Fc-binding protein comprising an amino acid sequence in which the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) above, as well as the amino acid substitutions shown in (3) to (12) below, occur at the said amino acid residues; (3) The 71st cysteine ​​in SEQ ID NO: 1 is substituted with arginine (4) The 78th asparagine in SEQ ID NO: 1 is substituted with aspartic acid (5) The 80th valine in SEQ ID NO: 1 is substituted with aspartic acid (6) The 96th lysine in SEQ ID NO: 1 is substituted with glutamic acid (7) The 172nd asparagine in SEQ ID NO: 1 is substituted with aspartic acid (8) The 192nd arginine in SEQ ID NO: 1 is substituted with leucine (9) The 196th asparagine in SEQ ID NO: 1 is substituted with aspartic acid (10) The 232nd glutamine in SEQ ID NO: 1 is replaced with leucine. (11) The 274th cysteine ​​in SEQ ID NO: 1 is replaced with serine. (12) The 295th lysine in SEQ ID NO: 1 is replaced with glutamic acid. <e> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) and the amino acid substitutions shown in (3) to (12) occur at said amino acid residues, and further comprising an amino acid sequence in which at least one substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions occurs in addition to the amino acid substitutions selected from the group consisting of (1), (2) and (13) to (33) and the amino acid substitutions shown in (3) to (12), and<f> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33) occurs, and the amino acid substitutions shown in (3) to (12) occur, in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, and in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33), and the amino acid substitutions shown in (3) to (12) remain, and an Fc-binding protein having antibody-binding activity. [2'] An Fc-binding protein as described in [1'], selected from any of the following <d'> to <f'>: <d'> An Fc-binding protein comprising an amino acid sequence in which the amino acid substitutions described in <a'> above have been further replaced by the amino acid substitutions shown in (3) to (12) below; (3) Cysteine ​​at position 71 of SEQ ID NO: 1 is replaced with arginine (4) Asparagine at position 78 of SEQ ID NO: 1 is replaced with aspartic acid (5) Valine at position 80 of SEQ ID NO: 1 is replaced with aspartic acid (6) Lysine at position 96 of SEQ ID NO: 1 is replaced with glutamic acid (7) Asparagine at position 172 of SEQ ID NO: 1 is replaced with aspartic acid (8) Arginine at position 192 of SEQ ID NO: 1 is replaced with leucine (9) Asparagine at position 196 of SEQ ID NO: 1 is replaced with aspartic acid (10) Glutamine at position 232 of SEQ ID NO: 1 is replaced with leucine (11) The 274th cysteine ​​in SEQ ID NO: 1 is replaced with serine. (12) The 295th lysine in SEQ ID NO: 1 is replaced with glutamic acid. <e'> An amino acid sequence in which the amino acid substitutions described in <a'> above occur is further modified by the amino acid substitutions shown in (3) to (12) above, and furthermore, in addition to the amino acid substitutions shown in (1) and (3) to (12) above, one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions occur, and an Fc-binding protein having antibody-binding activity; and<f'>An amino acid sequence having the amino acid substitution described in <a'> above, further having 70% or more identity with the entire amino acid sequence having the amino acid substitutions shown in (3) to (12), and containing an amino acid sequence in which the amino acid substitutions shown in (1) and (3) to (12) remain, and having antibody-binding activity, and an Fc-binding protein. [2''] Fc-binding proteins according to [1''] selected from any of the following <d''> to <f''>: <d''> Fc-binding proteins comprising an amino acid sequence in which the amino acid substitution described in <a''> has occurred, and further comprising an amino acid sequence in which the amino acid substitutions shown in (3) to (12) have occurred; <e''> Fc-binding proteins having antibody-binding activity comprising an amino acid sequence in which the amino acid substitution described in <a''> has occurred, and further comprising an amino acid sequence in which the amino acid substitutions shown in (1), at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and one or more substitutions, deletions, insertions and additions of one or more amino acid residues at one or more positions other than the amino acid substitutions shown in (3) to (12); and <f''> An amino acid sequence having the amino acid substitution described in <a''> above, further having 70% or more identity with the entire amino acid sequence having the amino acid substitutions shown in (3) to (12), comprising the amino acid substitution of (1), at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and the amino acid substitutions shown in (3) to (12), and having antibody-binding activity, and an Fc-binding protein. [3] A polynucleotide encoding the Fc-binding protein described in [1], [1'], [1''], [2], [2'] or [2'']. [4] An expression vector comprising the polynucleotide described in [3]. [5] A transformant obtained by transforming a host with the expression vector described in [4]. [6] The transformant according to [5], wherein the host is Escherichia coli.A method for producing an Fc-binding protein, comprising the steps of: culturing a transformant according to [7], [5], or [6] to express an Fc-binding protein; and recovering the expressed Fc-binding protein from the obtained culture. [8] An antibody adsorbent comprising an insoluble carrier and an Fc-binding protein according to [1], [1'], [1''], [2], [2'], or [2''] immobilized on the carrier. [9] A method for purifying an antibody, comprising the steps of: contacting the antibody adsorbent according to [8] with a solution containing a substance having an Fc region to adsorb the substance having an Fc region onto the antibody adsorbent; and eluting the substance having an Fc region adsorbed onto the antibody adsorbent using an eluate.

[0016] According to this disclosure, a modified Fc-binding protein can be provided to improve productivity. Furthermore, according to one aspect of this disclosure, a modified human neonatal Fc receptor (human FcRn) can be provided to improve productivity.

[0017] Furthermore, according to one aspect of this disclosure, a modified Fc-binding protein can be provided in which the degradation (also called degradation) of the Fc-binding protein during culture is suppressed in the production of the Fc-binding protein, which includes a culture step of the Fc-binding protein-producing bacteria. Furthermore, according to one aspect of this disclosure, a modified human FcRn can be provided in which degradation is suppressed.

[0018] Furthermore, according to one aspect of this disclosure, a modified Fc-binding protein can be provided that has improved productivity and suppressed degradation. Furthermore, according to one aspect of this disclosure, a modified human FcRn can be provided that has improved productivity and suppressed degradation. Therefore, the Fc-binding protein of this disclosure is also suitable for industrial production.

[0019] This is a schematic diagram of the α-chain of human FcRn. The numbers in the diagram indicate the amino acid sequence numbers described in Sequence ID No. 1. In the diagram, S indicates the signal sequence, EC indicates the extracellular region, TM indicates the transmembrane region, and C indicates the intracellular region. This is a schematic diagram of the β-chain of human FcRn. The numbers in the diagram indicate the amino acid sequence numbers described in Sequence ID No. 2. In the diagram, S indicates the signal sequence, and B2M indicates β2-microglobulin. This figure shows the results of quantifying the expression levels (intensity) and degradation product levels (intensity) of Fc-binding protein (FcRn-m11a) in which the 205th methionine of Sequence ID No. 1 is substituted with valine (Mα205V) and Fc-binding protein (FcRn-m11b) in which the 204th serine of Sequence ID No. 1 is substituted with lysine (Sα204K). The brightness is expressed as a relative value with the value for Fc-binding protein (FcRn-m10) without amino acid substitutions in Sα204 and Mα205 set to 1. Fc-binding protein (FcRn-m11a) with methionine at position 205 of SEQ ID NO: 1 substituted with valine (Mα205V), Fc-binding protein (FcRn-m12a) with methionine at position 205 of SEQ ID NO: 1 substituted with valine (Mα205V) and arginine at position 206 substituted with threonine (Rα206T), and Fc-binding protein (FcRn-m12a) with methionine at position 205 of SEQ ID NO: 1 substituted with valine (Mα205V) and arginine at position 206 substituted with histidine (Rα206H). This figure shows the results of quantifying the expression levels (brightness) of the binding protein (FcRn-m12b), the Fc-binding protein (FcRn-m12c) obtained by substituting methionine at position 205 of SEQ ID NO: 1 with valine (Mα205V) and arginine at position 206 with serine (Rα206S), or the Fc-binding protein (FcRn-m12d) obtained by substituting methionine at position 205 of SEQ ID NO: 1 with valine (Mα205V) and arginine at position 206 with lysine (Rα206K). Brightness is expressed as a relative value with the value for FcRn-m11a set to 1. This figure shows the results of separating monoclonal antibodies (Zenyaku Kogyo Co., Ltd., Rituxan) using a column packed with FcRn-m11a immobilized gel. This figure shows the results of separating various antibodies using a column packed with FcRn-m11a immobilized gel. <No. > corresponds to the antibodies listed in Table 6.The first figure shows the results of comparing the purified yield of Fc-binding proteins in which one amino acid substitution from among Lα28H, Lα31R, Tα89Q, and Kα132R was introduced to EC_m10 in FcRn-m10. The purified yield is expressed as a relative value with the value for FcRn-m10 set to 1. The second figure shows the results of comparing the purified yield of Fc-binding proteins in which one amino acid substitution from among Kβ95R, Kβ114R, Eβ56S, Hβ71Q, Nβ103E, and Vβ105S was introduced to B2M in FcRn-m10. The purified yield is expressed as a relative value with the value for FcRn-m10 set to 1. This figure shows a comparison of the purified yield of Fc-binding proteins in which one amino acid substitution from the following groups was introduced relative to FcRn-m10: Eα25G, Rα65H, Aα73T, Kα173R, Dα248G, Fα249S, Pα251L, Sα253P, Hα271Y, Kβ61E, and Mβ119T. The purified yield is expressed as a relative value with the value for FcRn-m10 set to 1.

[0020] The present disclosure is described in detail below. In this specification and the attached claims, the singular forms "a," "an," and "the" include plural nouns unless the context clearly indicates otherwise.

[0021] The Fc-binding protein of this disclosure is a protein that binds to the Fc region of an antibody, such as an immunoglobulin, and contains at least the amino acid residues shown in (I) and (II) below, wherein specific amino acid substitutions have occurred at the amino acid residues: (I) Amino acid residues from the 24th alanine (A) to the 297th serine (S), corresponding to the extracellular region (region EC in Figure 1) of a human FcRnα chain consisting of the amino acid sequence described in SEQ ID NO: 1. (II) Amino acid residues from the 21st isoleucine (I) to the 119th methionine (M), corresponding to the β2 microglobulin region (region B2M in Figure 2) of a human FcRnβ chain consisting of the amino acid sequence described in SEQ ID NO: 2.

[0022] Therefore, the Fc-binding protein of this disclosure may include all or part of the signal peptide region (region S in Figures 1 and 2) located at the N-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain and the β2-microglobulin region (region B2M in Figure 2) of the human FcRnβ chain. The Fc-binding protein may also include all or part of the transmembrane region (region TM in Figure 1) and intracellular region (region C in Figure 1) located at the C-terminal end of the extracellular region (EC region in Figure 1) of the human FcRnα chain.

[0023] In this specification, an Fc-binding protein containing at least the amino acid residues shown in (I) and (II) above is defined as a protein whose amino acid sequence contains at least the amino acid residues shown in (I) and (II), and the order of the amino acid residues shown in (I) and (II) is irrelevant. That is, the amino acid residue shown in (II) may be on the N-terminal side or the C-terminal side of the amino acid residue shown in (I). Furthermore, the amino acid residues shown in (I) and (II) may be directly linked, or they may be linked via a known linker such as a GS linker (for example, a linker consisting of a repeat of glycine (G) 4 residues - serine (S) 1 residue).

[0024] The amino acid substitutions at the specific positions mentioned above refer specifically to those shown in (1), (2), and (13) through (33) below. We have found that having at least one of the amino acid substitutions shown below improves the productivity of Fc-binding proteins. (1) The methionine at position 205 in SEQ ID NO: 1 is replaced with valine. (2) The arginine at position 206 in SEQ ID NO: 1 is replaced with threonine. (13) The glutamic acid at position 25 in SEQ ID NO: 1 is replaced with glycine. (14) The leucine at position 28 in SEQ ID NO: 1 is replaced with histidine. (15) The leucine at position 31 in SEQ ID NO: 1 is replaced with arginine. (16) The arginine at position 65 in SEQ ID NO: 1 is replaced with histidine. (17) The alanine at position 73 in SEQ ID NO: 1 is replaced with threonine. (18) The threonine at position 89 in SEQ ID NO: 1 is replaced with glutamine. (19) The lysine at position 132 in SEQ ID NO: 1 is replaced with arginine. (20) The lysine at position 173 in SEQ ID NO: 1 is replaced with arginine. (21) The aspartic acid at position 248 in SEQ ID NO: 1 is replaced with glycine. (22) The phenylalanine at position 249 in SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 of SEQ ID NO: 1 is replaced with leucine. (24) Serine at position 253 of SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 of SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 of SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 of SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 of SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 of SEQ ID NO: 2 is replaced with arginine. (30) Asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) Valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) Lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) Methionine at position 119 of SEQ ID NO: 2 is replaced with threonine.

[0025] In another embodiment, the Fc-binding protein may be one of the proteins shown below. An Fc-binding protein comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33) occurs at the said amino acid residues; (1) The 205th methionine in SEQ ID NO: 1 is substituted with valine (2) The 206th arginine in SEQ ID NO: 1 is substituted with threonine (13) The 25th glutamic acid in SEQ ID NO: 1 is substituted with glycine (14) The 28th leucine in SEQ ID NO: 1 is substituted with histidine (15) The 31st leucine in SEQ ID NO: 1 is substituted with arginine (16) The 65th arginine in SEQ ID NO: 1 is substituted with histidine (17) The 73rd alanine in SEQ ID NO: 1 is substituted with threonine (18) Threonine at position 89 in SEQ ID NO: 1 is replaced with glutamine. (19) Lysine at position 132 in SEQ ID NO: 1 is replaced with arginine. (20) Lysine at position 173 in SEQ ID NO: 1 is replaced with arginine. (21) Aspartic acid at position 248 in SEQ ID NO: 1 is replaced with glycine. (22) Phenylalanine at position 249 in SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 in SEQ ID NO: 1 is replaced with leucine. (24) Serine at position 253 in SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 in SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 in SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 in SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 in SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 in SEQ ID NO: 2 is replaced with arginine. (30) The asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) The valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) The lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) The methionine at position 119 of SEQ ID NO: 2 is replaced with threonine. An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs at the amino acid residues described in Sequence ID No. 1 and at least one amino acid substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions in addition to at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33), and wherein at least one amino acid substitution, deletion, insertion, and addition of one or more amino acid residues occurs at one or more positions. <c> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and in the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) remains, and which is an Fc-binding protein having antibody-binding activity.

[0026] In another embodiment, the Fc-binding protein may be the following proteins: <a'> An Fc-binding protein comprising an amino acid sequence that includes amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein the following amino acid substitution (1) occurs at said amino acid residues; (1) The 205th methionine in SEQ ID NO: 1 is replaced with valine <b'> An Fc-binding protein comprising an amino acid sequence that includes amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein the above amino acid substitution (1) occurs at said amino acid residues, and further comprises 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 addition to the above amino acid substitution (1), and has antibody-binding activity; and <c'> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitution described in (1) occurs in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, and which includes an amino acid sequence in which the amino acid substitution described in (1) remains, and which is an Fc-binding protein having antibody-binding activity.

[0027] In another embodiment, the Fc-binding protein may be the following protein: <a''> An Fc-binding protein comprising an amino acid sequence in which the amino acid substitution described in <a''> occurs, and further includes at least one amino acid substitution selected from the group consisting of (2) and (13) to (33);(2) Arginine at position 206 of SEQ ID NO: 1 is replaced with threonine. (13) Glutamic acid at position 25 of SEQ ID NO: 1 is replaced with glycine. (14) Leucine at position 28 of SEQ ID NO: 1 is replaced with histidine. (15) Leucine at position 31 of SEQ ID NO: 1 is replaced with arginine. (16) Arginine at position 65 of SEQ ID NO: 1 is replaced with histidine. (17) Alanine at position 73 of SEQ ID NO: 1 is replaced with threonine. (18) Threonine at position 89 of SEQ ID NO: 1 is replaced with glutamine. (19) Lysine at position 132 of SEQ ID NO: 1 is replaced with arginine. (20) Lysine at position 173 of SEQ ID NO: 1 is replaced with arginine. (21) Aspartic acid at position 248 of SEQ ID NO: 1 is replaced with glycine. (22) Phenylalanine at position 249 of SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 of SEQ ID NO: 1 is replaced with leucine. (24) Serine at position 253 of SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 of SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 of SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 of SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 of SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 of SEQ ID NO: 2 is replaced with arginine. (30) Asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) Valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) Lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) Methionine at position 119 of SEQ ID NO: 2 is replaced with threonine. <b''>An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which the amino acid substitution described in <a'> above occurs, further comprising at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitution in (1) and at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and having antibody-binding activity;and <c''> an amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitution described in <a'> has occurred, and in which at least one amino acid substitution selected from the group consisting of (2) and (13) to (33) has occurred, wherein the amino acid substitution of (1) and at least one amino acid substitution selected from the group consisting of (2) and (13) to (33) remain, and an Fc-binding protein having antibody-binding activity.

[0028] In another embodiment, the Fc-binding protein may be the following protein: <d> An Fc-binding protein comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) above, as well as the amino acid substitutions shown in (3) to (12) below, occurs at said amino acid residues. (3) The cysteine ​​at position 71 in SEQ ID NO: 1 is replaced with arginine. (4) The asparagine at position 78 in SEQ ID NO: 1 is replaced with aspartic acid. (5) The valine at position 80 in SEQ ID NO: 1 is replaced with aspartic acid. (6) The lysine at position 96 in SEQ ID NO: 1 is replaced with glutamic acid. (7) The asparagine at position 172 in SEQ ID NO: 1 is replaced with aspartic acid. (8) The arginine at position 192 in SEQ ID NO: 1 is replaced with leucine. (9) The asparagine at position 196 in SEQ ID NO: 1 is replaced with aspartic acid. (10) The glutamine at position 232 in SEQ ID NO: 1 is replaced with leucine. (11) The cysteine ​​at position 274 in SEQ ID NO: 1 is replaced with serine. (12) The lysine at position 295 in SEQ ID NO: 1 is replaced with glutamic acid. <e> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which at least one of the following occurs: an amino acid residue from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and an amino acid residue from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33), as well as the amino acid substitutions shown in (3) to (12), and further comprising an amino acid sequence in which at least one of the following occurs: substitution, deletion, insertion, or addition of one or more amino acid residues at one or more positions, in addition to the amino acid substitutions selected from the group consisting of (1), (2), and (13) to (33), and the amino acid substitutions shown in (3) to (12).<f> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33) occurs, and the amino acid substitutions shown in (3) to (12) occur, in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, and in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33), and the amino acid substitutions shown in (3) to (12) remain, and an Fc-binding protein having antibody-binding activity.

[0029] In another embodiment, the Fc-binding protein may be one of the proteins shown below. <d'> An Fc-binding protein comprising an amino acid sequence in which the amino acid substitutions described in <a'> above have further occurred, as shown in (3) to (12) below: (3) Cysteine ​​at position 71 of SEQ ID NO: 1 is replaced with arginine (4) Asparagine at position 78 of SEQ ID NO: 1 is replaced with aspartic acid (5) Valine at position 80 of SEQ ID NO: 1 is replaced with aspartic acid (6) Lysine at position 96 of SEQ ID NO: 1 is replaced with glutamic acid (7) Asparagine at position 172 of SEQ ID NO: 1 is replaced with aspartic acid (8) Arginine at position 192 of SEQ ID NO: 1 is replaced with leucine (9) Asparagine at position 196 of SEQ ID NO: 1 is replaced with aspartic acid (10) Glutamine at position 232 of SEQ ID NO: 1 is replaced with leucine (11) Cysteine ​​at position 274 of SEQ ID NO: 1 is replaced with serine (12) Lysine at position 295 of SEQ ID NO: 1 is replaced with glutamic acid <e'> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which the amino acid substitution described in <a'> has occurred, further comprising the amino acid substitutions shown in (3) to (12), and further comprising one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions in addition to the amino acid substitutions shown in (1) and (3) to (12); and <f'> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which the amino acid substitution described in <a'> has occurred, further comprising an amino acid sequence having 70% or more identity with the entire amino acid sequence in which the amino acid substitutions shown in (3) to (12) have occurred, and in which the amino acid substitutions shown in (1) and (3) to (12) remain.

[0030] In another embodiment, the Fc-binding protein may be the following proteins: <d''> An Fc-binding protein comprising an amino acid sequence in which the amino acid substitution described in <a''> occurs, and further comprising an amino acid sequence in which the amino acid substitution described in (3) to (12) occurs; <e''> An Fc-binding protein having antibody-binding activity comprising an amino acid sequence in which the amino acid substitution described in <a''> occurs, and further comprising an amino acid sequence in which the amino acid substitution described in (1), at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and one or more substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions other than the amino acid substitution described in (3) to (12); and <f''>An amino acid sequence having the amino acid substitution described in <a''> above, further having 70% or more identity with the entire amino acid sequence having the amino acid substitutions shown in (3) to (12), comprising the amino acid substitution of (1), at least one amino acid substitution selected from the group consisting of (2) and (13) to (33), and an amino acid sequence in which the amino acid substitutions shown in (3) to (12) remain, and having antibody-binding activity, and an Fc-binding protein.

[0031] The Fc-binding protein of this disclosure only needs to have at least the amino acid substitution at the aforementioned specific position, and may further have one or more of the following in addition to the amino acid substitution at the aforementioned specific position: substitution, deletion, insertion, and addition of amino acid residues (hereinafter collectively referred to as "modification").

[0032] In the above , <b'>, ​​<b''>, <e>, <e'>, and <e''>, "one or several" means one or several amino acids, although this can vary depending on the position and type of amino acid residues in the three-dimensional structure of the protein. For example, it means one to 50, one to 40, one to 30, one to 25, one to 20, one to 15, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, or one to 2. Furthermore, modifications of "one or more" amino acid residues may occur at locations other than those disclosed in Japanese Patent Publication Nos. 2018-183087, 2021-073883, 2021-136967, 2022-076998, and WO2025 / 105466, as long as they maintain antibody-binding activity. Also, "one or more of substitutions, deletions, insertions, and additions" includes naturally occurring mutations (mutants or variants) based on individual differences in the microorganisms from which the gene originates, species differences, etc. Furthermore, the Fc-binding proteins of this disclosure may further have conservative substitutions, which are substitutions between amino acids that are similar in physical and / or chemical properties. It is known to those skilled in the art that, not limited to Fc-binding proteins, conservative substitutions generally maintain the function of a protein between those with and without substitutions. Examples of conservative substitutions include those occurring between glycine and alanine, aspartic acid and glutamic acid, serine and proline, or glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005).

[0033] The amino acid sequence identity in <c>, <c'>, <c''>, <f>, <f'>, and <f''> only needs to be 70% or more, and may have a higher degree of identity, for example, 80% or more, 85% or more, 90% or more, or 95% or more.

[0034] In this specification, "identity" of amino acid sequences is expressed as a percentage obtained by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning the sequences as necessary, gaps are inserted into one or both of the two sequences to be compared as appropriate. The method of aligning sequences is not particularly limited, but it can be done using well-known sequence comparison programs such as BLAST (Basic Local Alignment Search Tool), FASTA, or CLUSTALW. When gaps are inserted, the total number of amino acid residues is the number of residues counted with 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 matching amino acid residues by the total number of amino acid residues of the longer sequence.

[0035] In the above , <b'>, ​​<b''>, <c>, <c'>, <c''>, <e>, <e'>, <e''>, <f>, <f'>, and <f''>, "antibody binding activity" may also mean the binding activity of the antibody to the Fc region.

[0036] The Fc-binding protein of this disclosure may have an oligopeptide added to its N-terminus or C-terminus that is useful for separation from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid.

[0037] Furthermore, a cysteine-containing oligopeptide, useful for immobilizing the Fc-binding protein of this disclosure onto an insoluble support for chromatography, may be further added to the N-terminal or C-terminal side of the Fc-binding protein. The length of the oligopeptide added to the N-terminal or C-terminal side 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.

[0038] When attaching the oligopeptide to the Fc-binding protein, the polynucleotide encoding the oligopeptide may be prepared and then genetically engineered to attach it to the N-terminus or C-terminus of the Fc-binding protein using a method well known to those skilled in the art. Alternatively, the oligopeptide may be chemically synthesized and attached to the N-terminus or C-terminus of the Fc-binding protein. An example of a cysteine-containing oligopeptide is the oligopeptide consisting of the amino acid sequence described in Sequence ID No. 15.

[0039] Furthermore, a signal peptide may be added to the N-terminus of the Fc-binding protein to promote efficient expression in the host. Examples of the signal peptide when the host is E. coli include signal peptides that induce protein secretion into the periplasm, such as Pelb, DsbA, MalE (the region from the 1st to the 26th amino acid sequence described in UniProt No. P0AEX9, SEQ ID NO: 3), and TorT (Japanese Patent Publication No. 2011-097898). In particular, it is preferable to use (A) a polynucleotide encoding the natural OmpA signal peptide (the region from the 1st to the 21st amino acid sequence of UniProt No. P0A910, SEQ ID NO: 27), or (B) an oligonucleotide encoding a polypeptide in which one or more residues of the signal peptide described in (A) have been modified by substitution, deletion, insertion, or addition. This allows for more efficient production and is therefore preferable.

[0040] Examples of methods for producing a polynucleotide encoding the Fc-binding protein of this disclosure (hereinafter also referred to as "the polynucleotide of this disclosure") include: (1) a method of converting the amino acid sequence of the Fc-binding protein of this disclosure into a nucleotide sequence and artificially synthesizing a polynucleotide containing the nucleotide sequence; and (2) a method of directly and artificially preparing a polynucleotide containing the whole or partial sequence of the Fc-binding protein, or preparing it from the cDNA of the Fc-binding protein using a DNA amplification method such as PCR, and then linking the prepared polynucleotide by an appropriate method.

[0041] In the method described in <1> above, when converting from an amino acid sequence to a nucleotide sequence, it is preferable to consider the frequency of codon use in the host being transformed. For example, if the host is Escherichia coli, AGA, AGG, CGG, and CGA are used infrequently in arginine (R), ATA in isoleucine (I), CTA in leucine (L), GGA in glycine (G), and CCC in proline (P). These are all rare codons, so the conversion should be done in a way that avoids these codons. Codon usage frequency can also be analyzed using public databases (for example, the Codon Usage Database on the Kazusa DNA Research Institute website).

[0042] When transforming a host using the polynucleotides of this disclosure, the polynucleotides themselves may be used, but it is more preferable to use an expression vector, such as a bacteriophage, cosmid, or plasmid commonly used for the transformation of prokaryotic or eukaryotic cells, in which the polynucleotides have 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 being transformed. Examples of such expression vectors when using E. coli as the host include pET plasmid vectors, pUC plasmid vectors, pTrc plasmid vectors, pCDF plasmid vectors, and pBBR plasmid vectors. The aforementioned appropriate position means a position that does not disrupt the replication function of the expression vector, the desired antibiotic marker, or the region involved in transduction. When inserting the polynucleotides into the expression vector, it is preferable to insert them in a state where they are linked to a functional polynucleotide, such as a promoter, necessary for expression. Examples of such promoters include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, lpp promoter, and also the λPL promoter and λPR promoter of λ phages, when the host is E. coli.

[0043] To transform a host using an expression vector containing the polynucleotide prepared by the above method (hereinafter referred to as the expression vector of this disclosure), a method commonly used by those skilled in the art may be used. For example, when selecting a microorganism belonging to the genus Escherichia (such as Escherichia coli strain JM109, Escherichia coli strain BL21 (DE3), or Escherichia coli strain W3110) as the host, transformation may be carried out by methods described in known literature (e.g., Molecular Cloning, Cold Spring Harbor Laboratory, 256, 1992). The transformants obtained by the above method can be screened by an appropriate method to obtain transformants capable of expressing the Fc-binding protein (hereinafter referred to as the transformants of this disclosure). There are no particular restrictions on the host organisms that express Fc-binding proteins. 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. Furthermore, using animal cells or E. coli as hosts is preferable in terms of productivity, and using E. coli as a host is even preferable.

[0044] To prepare the expression vector of the present disclosure from the transformant of the present disclosure, it may be prepared from a culture obtained by culturing the transformant using an alkaline extraction method or a commercially available extraction kit such as QIAprep Spin Miniprep kit (Qiagen). By culturing the transformant and recovering the Fc-binding protein from the obtained culture, the Fc-binding protein can be produced. In the present specification, the culture may include not only the cells of the cultured transformant of the present disclosure but also the medium used for the culture. The transformant used in the method for producing the protein 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 nutrient sources is mentioned as an example of a preferable medium.

[0045] In addition, in order to selectively grow the transformant of the present disclosure depending on the presence or absence of introduction of the expression vector of the present disclosure, it is preferable to add a drug corresponding to the drug resistance gene contained in the expression vector to the medium and culture. For example, when the expression vector contains a kanamycin resistance gene, kanamycin may be added to the medium. In addition to carbon, nitrogen, and inorganic salt supply sources, an appropriate nutrient source may be added to the medium, and if desired, it may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol. Furthermore, a reagent that promotes protein secretion from the transformant to the culture solution, such as glycine, may be added.

[0046] The culture temperature when the host is E. coli is generally 10°C to 40°C, preferably 20°C to 37°C, and more preferably around 25°C, but it should be selected according to the characteristics of the protein to be expressed. The pH of the culture medium when the host is E. coli is pH 6.8 to pH 7.4, preferably around pH 7.0. Furthermore, if the expression vector disclosed herein contains an inducible promoter, it is preferable to induce it under conditions that allow for good expression of the Fc-binding protein disclosed herein. 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, and then the culture is continued to induce the expression of the Fc-binding protein. The concentration of IPTG added can be appropriately selected from the range of 0.005 mmol / L to 1.0 mmol / L, but a range of 0.01 mmol / L to 0.5 mmol / L is preferred. Various conditions for IPTG induction can be carried out under conditions well known in the art.

[0047] To recover the Fc-binding protein of this disclosure from a culture obtained by culturing the transformant of this disclosure, the Fc-binding protein can be recovered by separating and purifying it from the culture using a method suitable for the expression mode of the Fc-binding protein in the transformant. For example, if the protein is expressed in the culture supernatant, the bacterial cells can be separated by centrifugation, and the Fc-binding protein can be purified from the resulting culture supernatant. If the protein is expressed intracellularly (including in the periplasm), after collecting the bacterial cells by centrifugation, the bacterial cells can be disrupted by adding an enzyme treatment agent or surfactant to extract the Fc-binding protein, and then the protein can be purified. To purify the Fc-binding protein, any method known in the art can be used, and one example is separation and purification using liquid chromatography. Liquid chromatography may be, for example, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, affinity chromatography, etc., and these chromatography methods may be combined to perform the purification operation.

[0048] In this specification, "improvement in productivity" means that the amount of undegraded Fc-binding protein that can be recovered from the same amount of culture broth is increased as compared to the conventional amount. The productivity of the Fc-binding protein can be evaluated by SDS-PAGE and absorbance measurement at 280 nm. In this specification, "degradability" may mean an index indicating how much the Fc-binding protein degrades during culture in the production of the Fc-binding protein including the culture step of the Fc-binding protein-producing bacterium. In this specification, "suppression of degradability" means that the ratio of the Fc-binding protein that has been degraded into low molecular weight forms is decreased as compared to the conventional amount. The degradability of the Fc-binding protein can be evaluated by SDS-PAGE, and by quantifying the luminance of each of the band corresponding to the Fc-binding protein (complete form) and the band corresponding to the degradation product that occurs on the lower molecular weight side of that band by image processing using image analysis software (ImageQuant TL 10.0, Cytiva).

[0049] As a method for measuring the antibody-binding activity of the obtained Fc-binding protein, for example, the binding activity to human IgG may be measured by the Enzyme-Linked Immunosorbent Assay (hereinafter referred to as ELISA) method.

[0050] An antibody adsorbent can be produced by binding (immobilizing) the Fc-binding protein of this disclosure to an insoluble carrier. The insoluble carrier is not particularly limited, and examples include carriers made from polysaccharides such as agarose, alginate (alginate salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch, carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane, and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and carriers made from synthetic polymers are preferred as insoluble carriers. Examples of preferred carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (Tosoh Corporation), agarose gels such as Sepharose (Cytiva Corporation), and cellulose gels such as Cellfine (JNC Corporation). There are no particular limitations on the shape of the insoluble carrier; it may be granular or non-granular, porous or non-porous.

[0051] To immobilize the Fc-binding protein of this disclosure onto an insoluble carrier, an active group such as an N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, or haloacetamide (iodoacetamide, bromoacetamide, etc.) is attached to the insoluble carrier, and the human Fc-binding protein is immobilized by covalent bonding between the insoluble carrier and the active group via this active group. The carrier to which the active group is attached may be a commercially available carrier as is, or it may be prepared by introducing the active group to the surface of the carrier under appropriate reaction conditions. Examples of commercially available carriers conferred with active groups include TOYOPEARL AF-Epoxy-650M and TOYOPEARL AF-Tresyl-650M (both from Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, and Epoxy-activated Sepharose 6B (all from Cytiva Corporation), and SulfoLink Coupling Resin (Thermo Fisher Scientific).

[0052] On the other hand, as a method for introducing active groups to the surface of a support, one example is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the surface of the support. Examples of such compounds that introduce epoxy groups to hydroxyl groups or amino groups on the surface of the support include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether. Examples of compounds that introduce epoxy groups to the surface of the support using the above compound and then introduce carboxyl groups to the surface of the support include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0053] Compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and amino groups present on the carrier surface include N-(ε-maleimidocaproic acid)hydrazide, N-(ε-maleimidopropionic acid)hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. Examples include pionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-6-aminohexanoic acid, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0054] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the carrier surface include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide. Another example is a method in which hydroxyl or amino groups present on the carrier surface are reacted with an ω-alkenyl alkane glycidyl ether, and then the ω-alkenyl moiety is halogenated and activated with a halogenating agent. Examples of ω-alkenyl alkane 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.

[0055] Another method for introducing active groups to a support surface involves introducing active groups to carboxyl groups present on the support surface using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0056] Examples of buffers used when immobilizing the Fc-binding protein of this disclosure onto an insoluble carrier include acetate buffer, phosphate buffer, MES (2-Morpholinoethanesulfonic acid) buffer, HEPES (2-[4-(2-Hydroxymethyl)-1-piperazinyl]ethanesulfonic acid) buffer, Tris (Tris(Hydroxymethyl)aminomethyl) buffer, borate buffer, and bis-trispropane (1,3-Bis[tris(Hydroxymethyl)methylamino]propane) buffer. The reaction temperature for immobilization can be set appropriately within the temperature range of 5°C to 50°C, taking into consideration the reactivity of the active group and the stability of the Fc-binding protein, and is preferably in the range of 10°C to 35°C.

[0057] To purify a substance having an Fc region using an antibody adsorbent obtained by immobilizing the Fc-binding protein of this disclosure on an insoluble carrier, for example, the procedure may include the steps of: contacting the substance having an Fc region with the adsorbent to adsorb the substance having an Fc region onto the adsorbent; and eluting the substance having an Fc region adsorbed on the adsorbent using an eluent.

[0058] This 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 can be of any form, as long as it contains at least an Fc region of an antibody that has affinity for an Fc-binding protein.

[0059] As an example, antibodies include chimeric antibodies, humanized antibodies, human antibodies and their amino acid substitutions, and bispecific antibodies, which are commonly used as antibodies in antibody drugs. Fc fusion proteins include, for example, peptides, nucleic acids, and proteins fused with an Fc region. A complex of the Fc region of an antibody and a drug is, for example, an antibody-drug conjugate (ADC). 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.

[0060] Contact between an antibody adsorbent obtained by immobilizing the Fc-binding protein of this disclosure on an insoluble carrier and a substance having an Fc region is conveniently and preferably carried out, for example, by adding the substance to a column packed with the adsorbent.

[0061] Substances having an Fc region can be added to the column, for example, using a liquid delivery means such as a pump. It is preferable to equilibrate the column with an appropriate buffer before adding the buffer containing the substance having an Fc region, as this allows for higher purity purification of the substance having an Fc region. Examples of buffers include phosphate buffers and other buffers containing inorganic salts, and the pH of the buffer is between 3.0 and 10.0, preferably between 5.0 and 8.0.

[0062] To elute a substance having an Fc region adsorbed onto the adsorbent, the interaction between the substance having an Fc region and its ligand (the Fc-binding protein in this disclosure) can be weakened. Specifically, the interaction between the substance having an Fc region and its ligand can be weakened by, for example, a change in pH due to a buffer, a counterpeptide, a change in temperature, or a change in salt concentration. A specific example of an elution solution for elutering a substance having an Fc region adsorbed onto the adsorbent is a buffer solution that is more basic than the solution used to adsorb the substance having an Fc region onto the adsorbent.

[0063] Examples of buffer types include phosphate buffer, HEPES (2-[4-(2-Hydroxymethyl)-1-piperazinyl]ethanesulfonic acid) buffer, Tris buffer, borate buffer, and bis-trispropane (1,3-Bis[tris(Hydroxymethyl)methylamino]propane) buffer, all of which have buffering capacity from neutral to basic. The pH of the buffer should be set within a range that does not impair the function of the substance having the Fc region, preferably pH 7.0 to 10.0, and more preferably pH 7.0 to 9.0.

[0064] The present disclosure will be described in more detail below using examples and comparative examples, but the present disclosure is not limited to these examples.

[0065] Example 1 Plasmid vector preparation (pETMalE-p7) A plasmid vector was prepared by the following method. (1) A polynucleotide encoding the MalE signal peptide (an oligopeptide consisting of the N-terminal 26 residues of UniProt No. P0AEX9, SEQ ID NO: 3) was prepared by the following two-step PCR. (1-1) The first step of PCR was performed by repeating a reaction cycle consisting of the first step at 98°C for 10 seconds, the second step at 55°C for 5 seconds, and the third step at 72°C for 1 minute, five times under the reaction mixture composition shown in Table 1. The substituents used were those consisting of the nucleotide sequences described in SEQ ID NO: 4 (5'-TATACATATGAAAAATAAAAAACAGGTGCACGCCATCC-3'), SEQ ID NO: 5 (5'-GCATTAACGACGATGAATGTTTTTCCGCCCTCCGGCTCTCCC-3'), SEQ ID NO: 6 (5'-ATCGTCGTTAATGCGGGAATATGCGAGGATGCGTGCACCTG-3'), and SEQ ID NO: 7 (5'-TTGTCCCATGGGCTTTCTCGAATTTTTGGGCGAGAGCCG-3').

[0066]

[0067] (1-2) Using the reaction mixture composition shown in Table 2, the second stage of PCR was performed by repeating a reaction cycle consisting of a first step of 10 seconds at 98°C, a second step of 5 seconds at 55°C, and a third step of 1 minute at 72°C for 30 cycles. The template DNA was the PCR product obtained in the first stage in (1-1), and the PCR primers used were oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 8 (5'-TATACATATGAAAAATAAAAAAACAGGTGCACGCCATCC-3') (Forward primer) and oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 9 (5'-GCATTAACGACGATGAATGTTTTTCCGCCCTCCGGCTCTCCGCC-3') (Reverse primer).

[0068]

[0069] (2) The polynucleotide encoding the MalE signal peptide prepared in (1) was digested with restriction enzymes NdeI and NcoI, and then ligated into the plasmid vector pET-26b(+) (Novagen), which had been previously digested with restriction enzymes NdeI and NcoI. This was then used to transform Escherichia coli BL21 (DE3) strain (Novagen) by the calcium chloride method.

[0070] (3) The obtained transformants were cultured in LB (Luria-Bertani) medium containing 50 μg / mL kanamycin sulfate (Nacalai Tesque), and the plasmid vector was extracted using QIAprep Spin Miniprep kit (Qiagen) (named pETMale).

[0071] (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 the oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 10 (5'-AGTAGTTAGGTTGAGGCCCGTTGAG-3') (Forward primer) and SEQ ID NO: 11 (5'-TTTTCATATGTTATTATGTTAATCTCCCTCTTTAAA-3') (Reverse primer) were used as PCR primers.

[0072] (5) The PCR products obtained in (4) were digested with restriction enzymes SphI and NdeI, and then ligated to pETMaLE that had been previously digested with restriction enzymes SphI and NdeI. This was then used to transform E. coli BL21 (DE3) strain. (6) After culturing the transformants from (5), the plasmid was extracted (named pETMaLE-p7).

[0073] Example 2: Preparation of an Fc-binding protein expression plasmid (MalE) A polynucleotide encoding the Fc-binding protein FcRn(B2M-GS-EC_m10)-6H, consisting of the amino acid sequence described in Sequence ID No. 12, was inserted into the expression vector pETMalE-p7 prepared in Example 1 by the method shown below to prepare a plasmid capable of expressing the protein. Of the FcRn(B2M-GS-EC_m10)-6H sequence, positions 1 through 99 are the β2 microglobulin region of the human FcRn β chain (regions 21 through 119 in SEQ ID NO: 2, B2M), positions 100 through 124 are the GS linker sequence (a sequence consisting of five repetitions of an oligopeptide composed of four glycine (G) residues and one serine (S) residue, GS), positions 125 through 398 are the amino acid substitution region of the extracellular domain of the human FcRn α chain (named EC_m10, SEQ ID NO: 13), and positions 399 through 404 are the histidine tag (6H) sequence. Furthermore, EC_m10 is a polypeptide obtained by introducing the following 10 amino acid substitutions into the extracellular domain of the human FcRn α chain (regions 24 through 297 in SEQ ID NO: 1, EC); The cysteine ​​(C) at position 71 in SEQ ID NO: 1 (position 48 in SEQ ID NO: 13) is substituted with arginine (R). The asparagine (N) at position 78 in SEQ ID NO: 1 (position 55 in SEQ ID NO: 13) is substituted with aspartic acid (D). The valine (V) at position 80 in SEQ ID NO: 1 (position 57 in SEQ ID NO: 13) is substituted with aspartic acid (D). The lysine (K) at position 96 in SEQ ID NO: 1 (position 73 in SEQ ID NO: 13) is substituted with glutamic acid (E). The asparagine (N) at position 172 in SEQ ID NO: 1 (position 149 in SEQ ID NO: 13) is substituted with aspartic acid (D). The arginine (R) at position 192 in SEQ ID NO: 1 (position 169 in SEQ ID NO: 13) is substituted with leucine (L). The asparagine (N) at position 196 in SEQ ID NO: 1 (position 173 in SEQ ID NO: 13) is substituted with aspartic acid (D). The glutamine (Q) at position 232 in SEQ ID NO: 1 (position 209 in SEQ ID NO: 13) is substituted with leucine (L). The cysteine ​​(C) at position 274 in SEQ ID NO: 1 (position 251 in SEQ ID NO: 13) is substituted with serine (S). The lysine (K) at position 295 in SEQ ID NO: 1 (position 272 in SEQ ID NO: 13) is substituted with glutamic acid (E).

[0074] (1) In order to attach a cysteine ​​tag (SEQ ID NO: 15) for immobilization on an insoluble carrier to the C-terminus of FcRn(B2M-GS-EC_m10)-6H, a plasmid containing a polynucleotide (SEQ ID NO: 14) encoding an Fc-binding protein consisting of the amino acid sequence described in SEQ ID NO: 12 is used as the template DNA, and an oligonucleotide (Forward primer) consisting of the base sequence described in SEQ ID NO: 16 (5'-GCCTCGGCTCTCGCCATTCAACGTACGCCCAAAAAATC-3') and an oligonucleotide (Reverse primer) consisting of the base sequence described in SEQ ID NO: 17 (5'-AGTGCGGCGCGCCAAGCTTATTCCGCCAAGGTTATTGCGCCAAGGTGATTGATTGATTGATTCGGC-3') are used as PCR primers. PCR was performed in the same manner as in Example 1 (1-2), except that the respective primers were used.

[0075] (2) The PCR product obtained in (1) was subjected to agarose gel electrophoresis, and the gel was purified using QIAquick Gel Extraction kit (Qiagen). (3) PCR was performed in the same manner as in Example 1 (1-2), except that pETMalE-p7 prepared in Example 1 was used as the template DNA, and oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 18 (5'-GCTTGCGCGCGCACTCGAGCAACCACACCACACCACACCACTGAGA-3') (Forward primer) and SEQ ID NO: 19 (5'-GGCGAGAGCCCGAGGCGGGAAAAACATCATCGTCCGTTAA-3') (Reverse primer) were used as PCR primers.

[0076] (4) The PCR products obtained in (3) were 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 the In-Fusion HD Cloning Kit (Takara Bio Inc.), and Escherichia coli strain JM109 (Takara Bio Inc.) was transformed using the ligated products. (6) Plasmids were extracted from the transformed cells by the method described in Example 1 (3) (named pETMalE-FcRn(B2M-GS-EC_m10)-6HC-p7).

[0077] (7) The base sequences of the Mal signal peptide and the polynucleotide encoding FcRn(B2M-GS-EC_m10)-6HC-p7 plasmid extracted in (6), as well as the surrounding regions, were analyzed using a fully automated DNA sequencer Genetic Analyzer 3500 (Thermo Fisher Scientific) to confirm that they were the desired sequences. During the analysis, oligonucleotides consisting of the sequences described in SEQ ID NO 20 (5'-GGATCTCGACGCTCTCCCCT-3'), SEQ ID NO 21 (5'-ATGGGCATTCAACGTACGCCAAAA-3'), SEQ ID NO 22 (5'-CTGCGCATCAAGGAAAAACTGTTC-3'), SEQ ID NO 23 (5'-CAGCCTGGTCTCGCGCCAACC-3'), and SEQ ID NO 24 (5'-ATGCTAGTTATTGCTCCAGCGG-3') were used as sequencing primers.

[0078] The amino acid sequence of the polypeptide MalE-FcRn(B2M-GS-EC_m10)-6HC expressed by the plasmid pETMalE-FcRn(B2M-GS-EC_m10)-6HC-p7 is shown in SEQ ID NO: 25, and the base sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 26. Of the sequence number 25, the first methionine (M) to the 26th alanine (A) is the MalE signal peptide (sequence number 3), the 27th isoleucine (I) to the 125th methionine (M) is the B2M region, the 126th glycine (G) to the 150th serine (S) is the GS linker sequence, the 151st alanine (A) to the 424th serine (S) is EC_m10 (sequence number 13), the 425th to the 430th histidine (H) is the histidine tag sequence, and the 431st cysteine ​​(C) to the 437th glycine (G) is the cysteine ​​tag sequence (sequence number 15).

[0079] Example 3 Preparation of Fc-binding protein expression plasmid (OmpA) A polypeptide (SEQ ID NO: 28) was designed by substituting the signal peptide of MalE-FcRn(B2M-GS-EC_m10)-6HC (SEQ ID NO: 25) from MalE signal peptide (SEQ ID NO: 3) with OmpA signal peptide (an oligopeptide consisting of the N-terminal 21 residues of UniProt No. P0A910, SEQ ID NO: 27), and a plasmid capable of expressing this polypeptide was prepared.

[0080] (1) PCR was performed in the same manner as in Example 1 (1-2), except that plasmid pETMalE-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 2 was used as the template DNA, and oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 29 (5'-GCTGGTTTTCGCCTACCGTTAGCGCCAAGGCCATTCCAACGTTAACGCCAAAAATCCCAAGTAACTACCG-3') (Forward primer) and oligonucleotides consisting of the nucleotide sequence described in SEQ ID NO: 30 (5'-GGTAGCGAAAAACCCAGCCAAGTGCCACACTGCCAATCCGCGATAGCCTGTCTTTTTTCCATATGTTATTATGTTATTC-3') (Reverse primer) were used as PCR primers.

[0081] (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), and the resulting transformants were cultured by the method described in Example 2 (6) to prepare a plasmid (named pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7). (4) Of the plasmid pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in (3), the nucleotide sequences of the polynucleotide encoding the OmpA signal peptide and FcRn(B2M-GS-EC_m10), as well as the surrounding region, were analyzed by the method described in Example 2 (7) to confirm that it was the desired sequence.

[0082] The amino acid sequence of the polypeptide OmpA-FcRn(B2M-GS-EC_m10)-6HC (hereinafter also referred to as "FcRn-m10") expressed by plasmid pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 is shown in SEQ ID NO: 28, and the base sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 31. Of the sequence number 28, the first methionine (M) to the 21st alanine (A) is the OmpA signal peptide (sequence number 27), the 22nd isoleucine (I) to the 120th methionine (M) is the B2M region, the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the 146th alanine (A) to the 419th serine (S) is EC_m10 (sequence number 13), the 420th to the 425th histidine (H) is the histidine tag sequence, and the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag sequence (sequence number 15).

[0083] Example 4 Expression of Fc-binding protein (Part 1) The Fc-binding protein FcRn-m10 was expressed by the method shown below. (1) A transformant capable of expressing the Fc-binding protein FcRn-m10, obtained by transforming Escherichia coli BL21 strain (DE3) with pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 3, was inoculated into 10 mL of TB liquid medium containing 50 μg / mL kanamycin (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), and pre-cultured by aerobic shaking overnight at 37°C.

[0084] (2) 3 mL of the pre-culture solution from (1) was inoculated into 200 mL of TB liquid medium containing 50 μg / mL of kanamycin in a 1 L baffled flask, and aerobic shaking culture was performed at 30°C. (3) Two hours after the start of culture, IPTG (IsoPropyl β-D-1-ThioGalactopyranoside) was added to a final concentration of 0.05 mmol / L while the culture was cooled on ice, and expression was induced by continuing aerobic shaking culture at 25°C overnight. (4) After the end of culture, the culture solution was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0085] (5) After recovering the bacterial cells from the culture medium of the transformed cells, 100 mmol / L Tris hydrochloride buffer (pH 8.0) containing 150 mmol / L sodium chloride, 2.4 mmol / L magnesium sulfate, 5000 Unit / L Benzonase (Merck), 0.006% (w / v) lysozyme, and 0.6% (w / v) Triton X-100 (trade name) was added, and the mixture was left at room temperature for 2 hours to extract the bacterial cells. The mixture was then centrifuged at 15000 rpm for 20 minutes at 4°C to obtain an extract containing the expressed Fc-binding protein FcRn-m10.

[0086] (6) The extract containing the Fc-binding protein obtained in (5) was adjusted to pH 6 using 1 mol / L hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and then applied to a polyprep column (Bio-Rad) packed with 1 mL of IgG-Sepharose (Cytiva) that had been pre-equilibrated with 50 mmol / L bis-trispropane (1,3-Bis[tris(hydroxymethyl)methylamino]propane) buffer (pH 6.0) containing 150 mmol / L sodium chloride. After washing with the buffer used for equilibration, a purified solution containing the Fc-binding protein FcRn-m10 (hereinafter also referred to as "FcRn-m10 purified solution") was obtained by eluting with 4 mL of 50 mmol / L bis-trispropane buffer (pH 8.5) containing 150 mmol / L sodium chloride.

[0087] Example 5 Site-Specific Amino Acid Substitution in Fc-Binding Proteins (Part 1) Mass spectrometry was used to identify the sites of degradation of FcRn-m10 in the purified FcRn-m10 solution obtained in Example 4. It was found that degradation occurs at serine at position 204 of SEQ ID NO: 1 (position 181 in SEQ ID NO: 13) (hereinafter also referred to as "Sα204") and methionine at position 205 (position 182 in SEQ ID NO: 13) (hereinafter also referred to as "Mα205"). Therefore, an amino acid substitution was introduced for Mα205. Specifically, Mα205 was substituted with valine (V) using the method shown below (hereinafter also referred to as "Mα205V").

[0088] (1) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 3 was used as the template, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 32 (5'-CGTCCTGTGCGCCTGAAAAGCGCGCTC-3') (Forward primer) and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 33 (5'-AGGCGCCACAGAACGGGAGGCTCCCTTC-3') (Reverse primer) were used as PCR primers.

[0089] (2) Except for subjecting the PCR product obtained in (1) to agarose gel electrophoresis, a plasmid (named pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7) was prepared by the method described in Example 3(2) to (3), and the base sequences of the amino acid substitution sites and surrounding regions were analyzed by the method described in Example 2(7) to confirm that they were the desired sequences.

[0090] The amino acid sequence of the polypeptide OmpA-FcRn(B2M-GS-EC_m11a)-6HC (hereinafter also referred to as "FcRn-m11a") expressed by plasmid pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7 is shown in SEQ ID NO: 34, and the base sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 35. Of the sequence numbers 34, the first methionine (M) to the 21st alanine (A) is the OmpA signal peptide (sequence number 27), the 22nd isoleucine (I) to the 120th methionine (M) is the B2M region, the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the 146th alanine (A) to the 419th serine (S) is EC_m11a (EC_m10 with Mα205V introduced, sequence number 36), the 420th to the 425th histidine (H) is the histidine tag sequence, and the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag sequence (sequence number 15). Mα205V is located at position 327 in sequence number 34 and at position 182 in EC_m11a (sequence number 36).

[0091] Example 6 Expression of Fc-binding protein (Part 2) The Fc-binding protein FcRn-m11a was expressed by the following method. (1) E. coli BL21 strain (DE3) was transformed with pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7 prepared in Example 5, and the transformant capable of expressing the Fc-binding protein was inoculated into 30 mL of TB liquid medium containing 50 μg / mL kanamycin. Pre-culture was then performed by aerobic shaking culture at 37°C overnight.

[0092] (2) 1 mL of the pre-culture solution from (1) was inoculated into 100 mL of TB liquid medium containing 50 μg / mL of kanamycin in a 500 mL baffled flask, and aerobic shaking culture was performed at 30°C. (3) Expression induction with IPTG was performed according to the method of Example 5 (3), and the bacterial cells (transformers) were collected according to the method of Example 5 (4). (4) After collecting the bacterial cells from the culture solution of the transformants, 50 mmol / L of bis-trispropane buffer (pH 10.0) containing 150 mmol / L of sodium chloride, 2.4 mmol / L of magnesium sulfate, 5000 Unit / L of Benzonase (Merck), 0.006% (w / v) of lysozyme, and 0.6% (w / v) of Triton X-100 (trade name) was added. The extract was left at room temperature for 2 hours to extract the bacterial cells. Subsequently, the mixture was centrifuged at 15,000 rpm for 20 minutes at 4°C to obtain an extract containing the expressed Fc-binding protein FcRn-m11a.

[0093] (5) A purified solution containing the Fc-binding protein FcRn-m11a (hereinafter also referred to as "FcRn-m11a purified solution") was obtained by the same method as in Example 4 (6), except that the extract containing the Fc-binding protein obtained in (4) was used as the extract.

[0094] Comparative Example 1 Site-Specific Amino Acid Substitution in Fc-Binding Proteins (Part 2) As described in Example 5, when the site of degradation of FcRn-m10 was identified, it was found to be degraded between Sα204 and Mα205. Therefore, using the same method as in Example 5, Sα204 was substituted with lysine (K) (hereinafter also referred to as "Sα204K").

[0095] (1) PCR was performed in the same manner as in Example 5, except that an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 37 (5'-CTCCGAAAAATGCGCCTGAAAAGCGCCGTC-3') (Forward primer) and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 38 (5'-CGCATTTTCGGGAGGCTCCTCTCCATTCGAGG-3') (Reverse primer) were used as PCR primers.

[0096] (2) After preparing a plasmid (named pETOmpA-FcRn(B2M-GS-EC_m11b)-6HC-p7) using the methods described in Examples 3(2) to (3), the base sequences of the amino acid substitution sites and surrounding regions were analyzed using the method described in Example 2(7) to confirm that they were the desired sequences.

[0097] The amino acid sequence of the polypeptide OmpA-FcRn(B2M-GS-EC_m11b)-6HC (hereinafter also referred to as "FcRn-m11b") expressed by plasmid pETOmpA-FcRn(B2M-GS-EC_m11b)-6HC-p7 is shown in Sequence ID No. 39, and the base sequence of the polynucleotide encoding the polypeptide is shown in Sequence ID No. 40. Of the sequence numbers 39, the first methionine (M) to the 21st alanine (A) is the OmpA signal peptide (sequence number 27), the 22nd isoleucine (I) to the 120th methionine (M) is the B2M region, the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the 146th alanine (A) to the 419th serine (S) is EC_m11b (EC_m10 with Sα204K introduced, sequence number 41), the 420th to the 425th histidine (H) is the histidine tag sequence, and the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag sequence (sequence number 15). Sα204K is located at position 326 in sequence number 39 and at position 181 in EC_m11b (sequence number 41).

[0098] Comparative Example 2 Expression of Fc-binding protein (Part 3) The Fc-binding protein was expressed by the method shown below. (1) Except that instead of using pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7 prepared in Example 5 in Example 6 (1), pETOmpA-FcRn(B2M-GS-EC_m11b)-6HC-p7 prepared in Comparative Example 1 was used, a purified solution containing the Fc-binding protein FcRn-m11b (hereinafter also referred to as "FcRn-m11b purified solution") was obtained by the same method as in Example 6.

[0099] Example 7 Quantification of purified yield and degradation products (1) The absorbance (wavelength: 280 nm) of the purified FcRn-m10 solution obtained in Example 4, the purified FcRn-m11a solution obtained in Example 6, and the purified FcRn-m11b solution obtained in Comparative Example 2 was measured, and the purified yield of each Fc-binding protein was calculated. The amount of purified solution in each case was 4 mL.

[0100] (2) The purified solutions of FcRn-m10, FcRn-m11a, and FcRn-m11b were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), stained with CBB (Coomassie Brilliant Blue) staining solution (Fujifilm Wako Pure Chemical Industries), and the brightness of the bands corresponding to the Fc-binding protein (complete form) and its degradation products was quantified using image analysis software (ImageQuant TL 10.0, Cytiva).

[0101] Table 3 shows the results of comparing the refined yields. The refined yield of FcRn-m11a was equivalent to that of FcRn-m10. On the other hand, the refined yield of FcRn-m11b was less than 40% of that of FcRn-m10.

[0102]

[0103] Figure 3 shows the results of quantifying the brightness of the complete protein and its degradation products. In Figure 3, the brightness is expressed as a relative value, with the brightness of the band corresponding to the complete protein (FcRn) and the brightness of the band corresponding to the degradation product set to 1 in FcRn-m10. The band corresponding to the Fc-binding protein (complete protein) was located at approximately 45 kDa. The band corresponding to the degradation product was located on the lower molecular weight side than the band corresponding to the Fc-binding protein (complete protein). Furthermore, since there were multiple bands corresponding to the degradation product, the brightness of the band corresponding to the degradation product represents the sum of the brightness of the bands corresponding to each degradation product.

[0104] Introducing the Mα205V amino acid substitution increased the expression level of the complete (i.e., undegraded) Fc-binding protein, and also reduced the amount of degradation products (i.e., degradation was suppressed). On the other hand, when the Sα204K amino acid substitution was introduced, the amount of degradation products decreased compared to FcRn-m10, but the expression level of the complete Fc-binding protein also decreased.

[0105] Example 8 Site-Specific Amino Acid Substitution in Fc-Binding Proteins (Part 3) In the purified FcRn-m11a solution obtained in Example 6, the degradation site of FcRn-m11a was identified as described in Example 5. Since degradation occurred between Vα205 and Rα206, Rα206 was substituted with threonine (T) (hereinafter also referred to as "Rα206T") using the same method as in Example 5.

[0106] (1) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7 obtained in Example 5 was used as the template, and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 42 (5'-GAGCCTCCGTCTGTGAACCCTGAAAAGCG-3') (Forward primer) and an oligonucleotide consisting of the nucleotide sequence described in SEQ ID NO: 43 (5'-CACAGAACGGGAGGCTCCTCTCCATTCCGAG-3') (Reverse primer) were used as PCR primers.

[0107] (2) After preparing a plasmid (named pETOmpA-FcRn(B2M-GS-EC_m12a)-6HC-p7) using the methods described in Examples 3(2) to (3), the base sequences of the amino acid substitution sites and surrounding regions were analyzed using the method described in Example 2(7) to confirm that they were the desired sequences.

[0108] The amino acid sequence of the polypeptide OmpA-FcRn(B2M-GS-EC_m12a)-6HC (hereinafter also referred to as "FcRn-m12a") expressed by plasmid pETOmpA-FcRn(B2M-GS-EC_m12a)-6HC-p7 is shown in SEQ ID NO: 44, and the base sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 45. Of the sequence number 44, the first methionine (M) to the 21st alanine (A) is the OmpA signal peptide (sequence number 27), the 22nd isoleucine (I) to the 120th methionine (M) is the B2M region, the 121st glycine (G) to the 145th serine (S) is the GS linker sequence, the 146th alanine (A) to the 419th serine (S) is EC_m12a (EC_m10 with Mα205V and Rα206T introduced, sequence number 46), the 420th to the 425th histidine (H) is the histidine tag sequence, and the 426th cysteine ​​(C) to the 432nd glycine (G) is the cysteine ​​tag sequence (sequence number 15). Rα206T is located at position 328 in sequence number 44 and at position 183 in EC_m12a (sequence number 46).

[0109] Example 9 Expression of Fc-binding protein (Part 4) An Fc-binding protein was expressed by the method shown below. (1) An extract containing the Fc-binding protein FcRn-m12a was obtained by the same method as in Examples 6(1) to (4), except that a transformant capable of expressing an Fc-binding protein was used, obtained by transforming Escherichia coli BL21 strain (DE3) with pETOmpA-FcRn(B2M-GS-EC_m12a)-6HC-p7 prepared in Example 8 as the transformant.

[0110] (2) The extract containing the Fc-binding protein obtained in (1) was applied to an Econopack column (Bio-Rad) packed with 5 mL of Ni-NTA agarose (Fujifilm Wako Pure Chemical Industries) that had been pre-equilibrated with 50 mM bis-trispropane buffer (pH 10.0) containing 150 mmol / L sodium chloride. After washing with 50 mmol / L bis-trispropane buffer (pH 10.0) containing 20 mmol / L imidazole and 150 mmol / L sodium chloride, a crude purified solution containing the Fc-binding protein FcRn-m12a was obtained by eluting with 50 mmol / L bis-trispropane buffer (pH 10.0) containing 300 mmol / L imidazole and 150 mmol / L sodium chloride.

[0111] (3) The crude purified solution obtained in (2) was adjusted to pH 6.5 using 1 mol / L 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 pre-equilibrated with 50 mmol / L bis-trispropane buffer (pH 6.5) containing 150 mmol / L sodium chloride. After washing with the buffer used for equilibration, a purified solution containing FcRn-m12a (hereinafter also referred to as "FcRn-m12a purified solution") was obtained by eluting with 50 mmol / L bis-trispropane buffer (pH 8.5) containing 150 mmol / L sodium chloride.

[0112] Comparative Example 3 Site-Specific Amino Acid Substitution in Fc-Binding Proteins (Part 4) In the purified FcRn-m11a solution obtained in Example 6, the degradation site of FcRn-m11a was identified as described in Example 5. Since degradation occurred between Vα205 and Rα206, Rα206 was substituted with the amino acids shown in Table 4 using the same method as in Example 5.

[0113]

[0114] (1) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m11a)-6HC-p7 obtained in Example 5 was used as the template, and the sequences (sequence numbers) shown in Table 4 were used as PCR primers (Forward / Reverse).

[0115] (2) After preparing plasmids using the methods described in Examples 3(2) to (3), the base sequences of the amino acid substitution sites and surrounding regions were analyzed using the method described in Example 2(7) to confirm that they were the desired sequences. The amino acid sequences of the Fc-binding proteins that can be expressed using the plasmids (expression vectors) prepared in this example (i.e., FcRn-m12b, FcRn-m12c, or FcRn-m12d) are the same as FcRn-m11a (SEQ ID NO: 34), except for the amino acid-substituted EC_m11a region (i.e., EC_m12b, EC_m12c, or EC_m12d).

[0116] Comparative Example 4 Expression of Fc-binding protein (Part 5) (1) A purified solution containing Fc-binding protein was obtained in the same manner as in Example 9, except that the expression vector prepared in Comparative Example 3 was used as the transformant.

[0117] Comparative Example 5 Determination of Purified Yield (1) The absorbance (wavelength: 280 nm) of the purified solutions of each Fc-binding protein obtained in Comparative Example 4, the purified solution of FcRn-m11a obtained in Example 6, and the purified solution of FcRn-m12a obtained in Example 8 was measured, and the purified yield of each Fc-binding protein was calculated. The amount of purified solution in each case was 3 mL.

[0118] (2) The brightness of the band corresponding to the Fc-binding protein (complete form) was quantified using the method described in Example 7 (2) for each of the purified Fc-binding protein solutions obtained in Comparative Example 4, FcRn-m11a, FcRn-m12a, and Comparative Example 4.

[0119] Table 5 shows the results of comparing the refined yields. The refined yields of FcRn-m12b, FcRn-m12c, and FcRn-m12d were 0.18, 0.28, and 0.75 times, respectively, the refined yield of FcRn-m11a, indicating that productivity was lower for all of them compared to FcRn-m11a.

[0120]

[0121] Figure 4 shows the results of quantifying the brightness of the Fc-binding protein (complete form). In Figure 4, the brightness is expressed as a relative value with the brightness value of the band corresponding to the complete form (FcRn, at approximately 45 kDa) in FcRn-m11a set to 1. It can be seen that when amino acid substitutions Rα206H (FcRn-m12b), Rα206S (FcRn-m12c), or Rα206K (FcRn-m12d) are introduced into FcRn-m11a, the production volume decreases compared to FcRn-m11a.

[0122] Example 10 A porous hydrophilic polymer for immobilizing Fc-binding proteins onto an insoluble carrier (TSKgel G5000PW, Tosoh Corporation) was collected as a suction dry gel at a rate of 2 g. The hydroxyl groups on the surface of the gel were activated with iodoacetyl groups, and then 10 mg of FcRn-m11a obtained in Example 6 was reacted with it to obtain an FcRn-m11a immobilized gel.

[0123] Example 11 Antibody purification using an antibody adsorbent-packed column (Part 1) (1) An antibody adsorbent column was prepared by packing 0.83 mL of the FcRn-m11a immobilized gel obtained in Example 10 (hereinafter also referred to as "antibody adsorbent") into a stainless steel column (Tosoh Corporation) with a diameter of 4.6 mm and a length of 50 mm.

[0124] (2) After connecting the antibody adsorbent column prepared in (1) to the Nexera system (Shimadzu Corporation), the column was equilibrated by delivering 50 mmol / L MES (2-Morphorinoethanesulfonic acid) buffer (pH 6.0) containing 150 mmol / L sodium chloride (hereinafter also referred to as "equilibrium solution") at a flow rate of 0.4 mL / min in 5 column volumes (hereinafter also referred to as "CV", 1 CV = 0.83 mL).

[0125] (3) A monoclonal antibody (Zenyaku Kogyo Co., Ltd., Rituxan, amino acid sequence of the Fc region: SEQ ID NO: 53) was diluted to 1.0 mg / mL with the equilibration solution used in (2), and 10 μL of this diluted solution was delivered to an antibody adsorbent column at a flow rate of 0.4 mL / min (10 μg of antibody was loaded) to adsorb the antibody onto the antibody adsorbent.

[0126] (4) The equilibration solution was then delivered to the antibody adsorbent column at a flow rate of 0.4 mL / min for 10 minutes. Then, the eluate (50 mmol / L Tris buffer (pH 8.5) containing 150 mmol / L sodium chloride) was delivered to the antibody adsorbent column in a linear gradient until the eluate reached 100% in 20 minutes, and the antibodies adsorbed on the antibody adsorbent were eluted. After antibody elution, the eluate was then delivered to the antibody adsorbent column at a flow rate of 0.4 mL / min for 10 minutes, and then the equilibration solution was flowed through the antibody adsorbent column for 10 minutes to equilibrate it.

[0127] Figure 5 shows the chromatogram of the separated monoclonal antibody. In Figure 5, the monoclonal antibody is loaded onto the antibody adsorbent column at 0 minutes. The monoclonal antibody elutes between 10 and 30 minutes during the linear gradient, confirming that the antibody can be separated using a column packed with the gel immobilized with the Fc-binding protein of this disclosure.

[0128] Example 12 Antibody purification using an antibody adsorbent column (part 2) Separation tests were performed not only on the monoclonal antibody described in Example 11(2), but also on other substances having an Fc region. In this test, in addition to the monoclonal antibody, substances having an Fc region (hereinafter also referred to as "Fc fusion protein") were used as substances having an Fc region.

[0129] (1) The antibody adsorbent column prepared by the method described in Example 11(1) was equilibrated by the method described in Example 11(2). (2) The Fc fusion protein was adsorbed onto the antibody adsorbent in the same manner as in Example 11(3), except that one of the Fc fusion proteins shown in Table 6 was used, and the Fc fusion protein adsorbed onto the adsorbent was eluted by the method described in Example 11(4). Among the substances having an Fc region in Table 6, the amino acid sequence of the Fc region of Herceptin® is shown as SEQ ID NO: 54, the amino acid sequence of the Fc region of Vectibix® is shown as SEQ ID NO: 55, the amino acid sequence of the Fc region of Zaltrap® is shown as SEQ ID NO: 56, and the amino acid sequence of the Fc region of Trulicity® is shown as SEQ ID NO: 57.

[0130]

[0131] Figure 6 shows chromatograms of substances having the Fc region shown in Table 6. Similar to Figure 5, each Fc fusion protein was loaded onto the antibody adsorbent column at 0 minutes. Each Fc fusion protein was eluted between 10 and 30 minutes during the linear gradient process. This confirms that substances having the Fc region, even those other than Rituxan (Example 11), can be separated using a column packed with a gel immobilized with the Fc-binding protein of this disclosure, regardless of their form (monoclonal antibody, Fc fusion protein).

[0132] Example 13 Site-Specific Amino Acid Substitution to Fc-Binding Protein (Part 5) A plasmid (expression vector) capable of expressing an Fc-binding protein was prepared using the same method as in Example 5, by introducing the amino acid substitution Lα28H, Lα31R, Tα89Q, or Kα132R into the EC_m10 region (amino acid sequence from position 146 to 419 in SEQ ID NO: 13 and SEQ ID NO: 28) of OmpA-FcRn(B2M-GS-EC_m10)-6HC(FcRn-m10, SEQ ID NO: 28).

[0133] (1) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 3 was used as the template, and the sequences (sequence numbers) shown in Table 7 were used as PCR primers (Forward / Reverse).

[0134]

[0135] (2) After preparing plasmids (expression vectors) using the methods described in Example 3(2) to (3), the base sequences of the amino acid substitution sites and surrounding regions were analyzed using the method described in Example 2(7) to confirm that they were the desired sequences. Table 7 shows the amino acid sequences (sequence numbers) of Fc-binding proteins that can be expressed with the plasmids prepared in this example.

[0136] Example 14 Expression of Fc-binding proteins (Part 6) (1) A purified solution containing Fc-binding proteins was obtained in the same manner as in Example 9, except that the expression vector prepared in Example 13 was used as the transformant. (2) The absorbance (wavelength: 280 nm) of the obtained purified solution was measured, and the purified yield of each Fc-binding protein was calculated. The amount of purified solution was 3 mL in all cases.

[0137] Figure 7 shows the results of comparing the purified yields. For Fc-binding proteins in which one of the following amino acid substitutions was introduced into the EC_m10 region—Lα28H, Lα31R, Tα89Q, and Kα132R—the productivity was improved compared to the Fc-binding protein before the amino acid substitution (FcRn-m10).

[0138] Example 15 Site-Specific Amino Acid Substitution to Fc-Binding Protein (Part 6) A plasmid (expression vector) capable of expressing an Fc-binding protein was prepared using the same method as in Example 5, by introducing amino acid substitutions of Kβ95R, Kβ114R, Eβ56S, Hβ71Q, Nβ103E, or Vβ105S into the B2M region (amino acid sequence from position 22 to 120 in SEQ ID NO: 2 and SEQ ID NO: 28) of OmpA-FcRn(B2M-GS-EC_m10)-6HC(FcRn-m10, SEQ ID NO: 28).

[0139] (1) PCR was performed in the same manner as in Example 1 (1-2), except that pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 3 was used as the template, and the sequences (sequence numbers) shown in Table 8 were used as PCR primers (Forward / Reverse).

[0140]

[0141] (2) After preparing plasmids (expression vectors) using the methods described in Example 3(2) to (3), the base sequences of the amino acid substitution sites and surrounding regions were analyzed using the method described in Example 2(7) to confirm that they were the desired sequences. Table 8 shows the amino acid sequences (sequence numbers) of Fc-binding proteins that can be expressed with the plasmids prepared in this example.

[0142] Example 16 Expression of Fc-binding proteins (Part 7) (1) A purified solution containing Fc-binding proteins was obtained in the same manner as in Example 9, except that the expression vector prepared in Example 15 was used as the transformant. (2) The absorbance (wavelength: 280 nm) of the obtained purified solution was measured, and the purified yield of each Fc-binding protein was calculated. The amount of purified solution was 3 mL in all cases.

[0143] Figure 8 shows the results of comparing the purified yields. For the B2M region, Fc-binding proteins with one amino acid substitution from among Kβ95R, Kβ114R, Eβ56S, Hβ71Q, Nβ103E, and Vβ105S showed improved productivity compared to the Fc-binding protein before amino acid substitution (FcRn-m10).

[0144] Example 17: Using the Fc-binding protein screening expression vector pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 as a template, random mutations were introduced into the polynucleotide portion encoding the protein using error-prone PCR.

[0145] (1) Error-prone PCR was performed using pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 as a template. Error-prone PCR was performed by preparing a reaction solution with the composition shown in Table 9, heat-treating the reaction solution at 98°C for 2 minutes, performing a reaction consisting of a first step of 30 seconds at 98°C, a second step of 20 seconds at 55°C, and a third step of 90 seconds at 72°C for 30 cycles, and finally heat-treating at 72°C for 5 minutes. The above error-prone PCR successfully introduced mutations into the polynucleotide encoding the Fc-binding protein.

[0146]

[0147] (2) The PCR product obtained in (1) was purified and ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.), and then the ligated product was used to transform E. coli BL21 (DE3) strain (Takara Bio Inc.).

[0148] (3) After the ligation reaction was complete, Escherichia coli BL21 (DE3) was transformed with the reaction solution and cultured in LB plate medium containing 50 μg / mL kanamycin (at 37°C for 18 hours). The colonies formed on the plate were used as a random mutant library. (4) The random mutant library (transformed organisms) prepared in (3) was inoculated into 200 μL of 2YT liquid medium (16 g / L Tryptone, 10 g / L Yeast Extract, 5 g / L sodium chloride) containing 50 μg / mL kanamycin, and cultured overnight with shaking at 37°C using a 96-well deep-well plate.

[0149] (5) The culture media from (4) were centrifuged, and the binding activity between Fc-binding proteins and antibodies was evaluated using the ELISA (Enzyme-Linked Immuno Sorbent Assay) method described below. (5-1) A human antibody, gamma globulin preparation (Japan Blood Products Organization), was immobilized at 10 μg / well in the wells of a 96-well microplate (at 4°C for 18 hours). After immobilization, the cells were blocked with MES buffer (pH 6.0) containing 2% (w / v) SKIM MILK (Becton, Dickinson and Company) and 150 mM sodium chloride. (5-2) After washing with washing buffer (MES buffer containing 150 mM sodium chloride (pH 6.0)), the solution containing human FcRn prepared in (4) was reacted with immobilized gamma globulin (at 30°C for 2 hours). (5-3) After the reaction was complete, the sample was washed with the washing buffer and 100 μL / well of mouse anti-FcRn antibody (Santacruz) diluted to 100 μg / mL with blocking solution was added. (5-4) After the reaction was complete, the sample was washed with the washing buffer and 100 μL / well of HRP-modified anti-mouse antibody (Bethyl Laboratories) diluted to 100 μg / mL with blocking solution was added. (5-5) The mixture was reacted at 30°C for 1 hour, washed with the washing buffer, and then 50 μL / well of TMB Peroxidase Substrate (Kirkegard and Perry Laboratories) was added. The color development was stopped by adding 50 μL / well of 1 mol / L phosphoric acid, and the absorbance at 450 nm was measured using a microplate reader (Tecan).

[0150] (6) A random mutant library of approximately 2000 strains was evaluated using the method described in (5), and transformants expressing an Fc-binding protein with improved residual activity compared to the parent molecule FcRn-m10 were selected from among them. The selected transformants were cultured, and expression vectors were prepared using the QIAprep Spin Miniprep kit (Qiagen).

[0151] Example 18 Evaluation of Fc-binding protein (1) A purified solution containing Fc-binding protein was obtained in the same manner as in Example 9, except that pETOmpA-FcRn(B2M-GS-EC_m10)-6HC-p7 prepared in Example 3 and the expression vector prepared in Example 17 were used as transformants. (2) The sequence of the polynucleotide region encoding the Fc-binding protein inserted into the obtained expression vector was analyzed by the method described in Example 2(7). Table 10 shows the amino acid substitution positions and amino acid sequences (sequence numbers) of the Fc-binding protein obtained in this screening relative to FcRn-m10 (sequence number 28).

[0152]

[0153] (3) The absorbance (wavelength: 280 nm) of the purified solution obtained in (1) was measured, and the purified yield of each Fc-binding protein was calculated. The volume of the purified solution was 1.6 mL in all cases. The results of comparing the purified yields are shown in Figure 9. All of the Fc-binding proteins into which any of the amino acid substitutions shown in Table 10 were introduced had a relative production of 1 or more, indicating improved productivity compared to FcRn-m10.

Claims

1. An Fc-binding protein selected from any of the following to <c>: An Fc-binding protein comprising an amino acid sequence that includes the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1 and the amino acid residues from the 21st isoleucine to the 119th methionine in the amino acid sequence described in SEQ ID NO: 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) below occurs at said amino acid residues; (1) The 205th methionine in SEQ ID NO: 1 is replaced with valine (2) The 206th arginine in SEQ ID NO: 1 is replaced with threonine (13) The 25th glutamic acid in SEQ ID NO: 1 is replaced with glycine (14) The 28th leucine in SEQ ID NO: 1 is replaced with histidine (15) The 31st leucine in SEQ ID NO: 1 is replaced with arginine (16) The 65th arginine in SEQ ID NO: 1 is replaced with histidine (17) Alanine at position 73 of SEQ ID NO: 1 is replaced with threonine. (18) Threonine at position 89 of SEQ ID NO: 1 is replaced with glutamine. (19) Lysine at position 132 of SEQ ID NO: 1 is replaced with arginine. (20) Lysine at position 173 of SEQ ID NO: 1 is replaced with arginine. (21) Aspartic acid at position 248 of SEQ ID NO: 1 is replaced with glycine. (22) Phenylalanine at position 249 of SEQ ID NO: 1 is replaced with serine. (23) Proline at position 251 of SEQ ID NO: 1 is replaced with leucine. (24) Serine at position 253 of SEQ ID NO: 1 is replaced with proline. (25) Histidine at position 271 of SEQ ID NO: 1 is replaced with tyrosine. (26) Glutamic acid at position 56 of SEQ ID NO: 2 is replaced with serine. (27) Lysine at position 61 of SEQ ID NO: 2 is replaced with glutamic acid. (28) Histidine at position 71 of SEQ ID NO: 2 is replaced with glutamine. (29) Lysine at position 95 of SEQ ID NO: 2 is replaced with arginine. (30) Asparagine at position 103 of SEQ ID NO: 2 is replaced with glutamic acid. (31) Valine at position 105 of SEQ ID NO: 2 is replaced with serine. (32) Lysine at position 114 of SEQ ID NO: 2 is replaced with arginine. (33) Methionine at position 119 of SEQ ID NO: 2 is replaced with threonine. An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs at the amino acid residues described in SEQ ID NO: 1 and at least one amino acid substitution, deletion, insertion, and addition of one or more amino acid residues at one or more positions, in addition to at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33); and <c> An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs in the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in Sequence ID No. 1 and in the amino acid sequence from the 21st isoleucine to the 119th methionine in the amino acid sequence described in Sequence ID No. 2, wherein at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) remains, and which is an Fc-binding protein having antibody-binding activity.

2. An Fc-binding protein according to claim 1, selected from any of the following <d> to <f>: <d> An Fc-binding protein comprising an amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) occurs at the amino acid residues from the 24th alanine to the 297th serine in the amino acid sequence described in SEQ ID NO: 1, and at least one amino acid substitution selected from the group consisting of (1), (2) and (13) to (33) above, as well as the amino acid substitutions shown in (3) to (12) below, occurs at the amino acid residues; (3) The 71st cysteine ​​in SEQ ID NO: 1 is substituted with arginine; (4) The 78th asparagine in SEQ ID NO: 1 is substituted with aspartic acid; (5) The 80th valine in SEQ ID NO: 1 is substituted with aspartic acid; (6) The 96th lysine in SEQ ID NO: 1 is substituted with glutamic acid; (7) The 172nd asparagine in SEQ ID NO: 1 is substituted with aspartic acid; (8) The 192nd arginine in SEQ ID NO: 1 is substituted with leucine (9) The 196th asparagine molecule in SEQ ID NO: 1 is replaced with aspartic acid. (10) The 232nd glutamine molecule in SEQ ID NO: 1 is replaced with leucine. (11) The 274th cysteine ​​molecule in SEQ ID NO: 1 is replaced with serine. (12) The 295th lysine molecule in SEQ ID NO: 1 is replaced with glutamic acid. <e> An Fc-binding protein having antibody-binding activity, comprising an amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33), and at least one of the substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions, in addition to the amino acid substitutions selected from the group consisting of (1), (2), and (13) to (33), occurs at the amino acid residues, and further comprising an amino acid sequence in which at least one of the substitutions, deletions, insertions, and additions of one or more amino acid residues at one or more positions occurs;and <f>An amino acid sequence having 70% or more identity with the entire amino acid sequence in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33) occurs, as well as the amino acid substitutions shown in (3) to (12), in the amino acid sequence from the 24th alanine to the 297th serine of the amino acid sequence described in Sequence ID No. 1 and the amino acid sequence from the 21st isoleucine to the 119th methionine of the amino acid sequence described in Sequence ID No. 2, and in which at least one amino acid substitution selected from the group consisting of (1), (2), and (13) to (33), as well as the amino acid substitutions shown in (3) to (12), remains, and an Fc-binding protein having antibody-binding activity.

3. A polynucleotide encoding an Fc-binding protein according to claim 1 or 2.

4. An expression vector comprising the polynucleotide described in claim 3.

5. A transformant obtained by transforming a host with the expression vector described in claim 4.

6. The transformant according to claim 5, wherein the host is Escherichia coli.

7. A method for producing an Fc-binding protein, comprising the steps of: culturing the transformant described in claim 5 to express an Fc-binding protein; and recovering the expressed Fc-binding protein from the obtained culture.

8. An antibody adsorbent comprising an insoluble carrier and an Fc-binding protein according to claim 1 or 2 immobilized on the carrier.

9. A method for purifying an antibody, comprising the steps of: contacting an antibody adsorbent according to claim 8 with a substance having an Fc region, thereby adsorbing the substance having the Fc region onto the antibody adsorbent; and eluting the substance having the Fc region adsorbed onto the antibody adsorbent using an eluent.

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