Polypeptide complex whose antigen-binding activity changes depending on plasma protein concentration

Abstract

The present application relates to a polypeptide conjugate comprising a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen other than a plasma protein, wherein the first antigen-binding moiety and the second antigen-binding moiety are directly linked without a linker or are linked by a non-cleavable linker consisting of four or fewer amino acid residues. The polypeptide of the present application does not bind to the target antigen in the presence of plasma protein, but binds to the target antigen in the absence of plasma protein. TIFF2026502811000027.tif121170

Description

[Technical Field]

[0001] The present invention relates to a polypeptide complex whose antigen-binding activity changes depending on the concentration of a plasma protein, a pharmaceutical composition comprising the polypeptide complex, etc. The present invention also relates to a method for producing the polypeptide complex, a screening method, etc. [Background technology]

[0002] Monoclonal antibodies have great therapeutic potential in the treatment of neurological or central nervous system (CNS) disorders, and monoclonal antibodies targeting endogenous brain-active proteins are being developed for the treatment of CNS disorders. Generally, therapeutic monoclonal antibodies exert their activity by binding to their target antigen. Therefore, when the target antigen is expressed in both the diseased site (CNS) and normal tissues (non-CNS), systemic administration of a therapeutic monoclonal antibody may result in undesirable effects in normal tissues (non-CNS).

[0003] In the field of cancer treatment, protease-dependent antigen-binding molecules are known as molecules that act specifically at cancer pathological sites. Protease-dependent antigen-binding molecules are composed of a peptide that inhibits an antigen-binding site, a protease cleavage site, and a target antigen-binding site. Prior to protease treatment, binding of the target antigen-binding site to the target antigen is inhibited by the peptide that inhibits the antigen-binding site. When a protease-dependent antigen-binding molecule migrates to a cancer pathological site, the protease cleavage site of the molecule is cleaved by a protease whose expression is specifically elevated at the cancer pathological site. As a result, the peptide that inhibits the target antigen-binding site dissociates from the protease-dependent antigen-binding molecule, exposing the target antigen-binding site and allowing it to bind to the target antigen. Known peptides that inhibit antigen-binding sites include peptides that bind to the target antigen-binding site and peptides that bind to the plasma protein albumin (e.g., WO2019222282 (Patent Document 1) and WO2013192546 (Patent Document 2)). In either case, the protease cleavage site is an essential component: in the absence of the protease cleavage site and cleavage by the protease, the peptide that inhibits antigen binding does not dissociate, and therefore binding to the target antigen by the target antigen-binding site is not restored.

[0004] Unlike the above, the use of protease-dependent antigen-binding molecules is difficult when tissue- or disease-site-specific proteases are difficult to utilize. Furthermore, when using protease-dependent antigen-binding molecules, protease cleavage is irreversible, and if the protease-cleaved molecules are redistributed throughout the body, they may act on normal tissues and cause side effects. In the treatment of CNS diseases, even if therapeutic molecules reach the diseased site (CNS), they inevitably redistribute throughout the body as they are excreted from brain interstitial fluid and cerebrospinal fluid (CSF). Therefore, as the therapeutic molecules move from the CNS to non-CNS tissues, their binding to the target antigen must be reduced again. However, no therapeutic molecules are known that exert their effects only in the diseased site (CNS) and not systemically in normal tissues (non-CNS) or blood. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication Number WO2019222282 [Patent Document 2] International Publication No. WO2013192546 [Patent Document 3] International Publication No. WO2012004384 [Non-patent literature]

[0006] [Non-Patent Document 1] Comput Struct Biotechnol J. 2013;6:e201303009 [Non-patent document 2] J Biol Chem. 2002 Sep 20;277(38):35035-43 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a pharmaceutical composition useful for treating CNS diseases, an active ingredient thereof, and a method for screening and producing the pharmaceutical composition and the active ingredient. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to achieve the above-mentioned object and have discovered a polypeptide complex whose binding activity to a target antigen changes depending on plasma protein concentration. Furthermore, the present inventors have devised a method for screening and producing the polypeptide complex, thereby completing the present invention.

[0009] The polypeptide complexes of the present invention comprise a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen other than a plasma protein. In the polypeptide complexes of the present invention, binding of the first antigen-binding moiety to the plasma protein restricts the binding of the second antigen-binding moiety to the target antigen in the presence of plasma protein (or in the presence of a high concentration of plasma protein), but does not restrict the binding of the second antigen-binding moiety to the target antigen in the absence of plasma protein (or in the presence of a low concentration of plasma protein). Surprisingly, the polypeptide complexes of the present invention regain their binding to the target antigen upon transition from the presence of plasma protein (or in the presence of a high concentration of plasma protein) to the absence of plasma protein (or in the presence of a low concentration of plasma protein), without requiring dissociation of the first antigen-binding moiety by protease cleavage. Such polypeptide complexes, or pharmaceutical compositions comprising the polypeptide complexes, are expected to be useful for treating CNS diseases.

[0010] The present invention is based on such findings, and includes the following in one non-limiting specific embodiment. [A1] A polypeptide complex comprising a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, the target antigen is not a plasma protein, the first antigen-binding portion and the second antigen-binding portion are linked without a linker; Polypeptide complexes. [A2] The polypeptide complex according to [A1], which does not bind to a target antigen when bound to a plasma protein. [A3] The polypeptide complex according to [A1], which has a lower binding activity to the target antigen in the presence of plasma proteins than in the absence of plasma proteins. [A4] The polypeptide complex according to [A1], wherein the binding activity to the target antigen in the presence of a plasma protein at a first concentration is different from the binding activity to the target antigen in the presence of a plasma protein at a second concentration. [A5] The polypeptide complex according to [A1], which has a lower binding activity to the target antigen in a human plasma sample than the binding activity to the target antigen in the absence of human plasma proteins. [A6] The polypeptide complex according to [A1], which has a lower binding activity to the target antigen in a human plasma sample than in a human cerebrospinal fluid (CSF) sample. [A7] The polypeptide complex according to [A1], which has a lower binding activity to a target antigen in the presence of 50 mg / ml of plasma protein than in the presence of 0.25 mg / ml of plasma protein. [A8] The polypeptide complex according to [A1], whose binding activity to a target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is lower than its binding activity to a target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [A9] The polypeptide complex according to [A1], wherein the KD value for the target antigen in the presence of 50 mg / ml of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of 0.25 mg / ml of plasma protein. [A10] The polypeptide complex according to [A1], wherein the KD value for the target antigen in the presence of approximately 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of approximately 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [A11] The polypeptide complex according to any one of [A1] to [A10], wherein the plasma protein is albumin. [A12] The polypeptide complex according to [A11], wherein the albumin is human albumin. [A13] Two or more first antigen-binding moieties and one or more second antigen-binding moieties; The polypeptide complex according to any one of [A1] to [A12], wherein one of the second antigen-binding portions and two of the first antigen-binding portions are linked without a linker. [A14] One or more first antigen-binding moieties and one or more second antigen-binding moieties; The polypeptide complex according to any one of [A1] to [A12], wherein one of the second antigen-binding portions and one of the first antigen-binding portions are linked without a linker. [A15] The polypeptide complex according to any one of [A1] to [A14], wherein the second antigen-binding portion comprises Fab or scFv. [A16] The second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [A1] to [A15], wherein the N-terminal amino acid of the heavy chain variable region and / or the light chain variable region of the Fab is linked to the C-terminal amino acid of the first antigen-binding portion without a linker. [A17] Two first antigen-binding moieties and one second antigen-binding moiety; the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [A1] to [A13], [A15] and [A16], wherein the N-terminal amino acids of the heavy chain variable region and the light chain variable region of the Fab are linked to the C-terminal amino acid of each of the first antigen-binding portions without a linker. [A18] Four first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [A1] to [A13], [A15] and [A16], wherein, in each of the Fabs, the N-terminal amino acid of the heavy chain variable region and the light chain variable region is linked to the C-terminal amino acid of each of the first antigen-binding portions without a linker. [A19] One first antigen-binding portion and one second antigen-binding portion, the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [A1] to [A12] and [A14] to [A16], wherein the N-terminal amino acid of the heavy chain variable region of the Fab and the C-terminal amino acid of the first antigen-binding portion are linked without a linker. [A20] A polypeptide comprising one first antigen-binding portion and one second antigen-binding portion, the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [A1] to [A12] and [A14] to [A16], wherein the N-terminal amino acid of the light chain variable region of the Fab and the C-terminal amino acid of the first antigen-binding portion are linked without a linker. [A21] Two first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [A1] to [A12] and [A14] to [A16], wherein, in each of the Fabs, the N-terminal amino acid of the heavy chain variable region thereof and the C-terminal amino acid of each of the first antigen-binding portions are linked without a linker. [A22] Two first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [A1] to [A12] and [A14] to [A16], wherein, in each of the Fabs, the N-terminal amino acid of the light chain variable region thereof and the C-terminal amino acid of each of the first antigen-binding portions are linked without a linker. [A23] The polypeptide complex according to any one of [A1] to [A22], wherein the first antigen-binding portion is a Fab, scFv, VHH, a VH single domain, a VL single domain, or a peptide. [A24] The polypeptide complex according to any one of [A1] to [A23], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. [A25] The first antigen-binding portion is a peptide comprising the amino acid sequence LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44]LP (SEQ ID NO: 40). (where, [X3] is selected from E, S, Q or C; [X6] is selected from E, S or C; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S, C or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and P at position 46 is either present or absent. The polypeptide complex according to any one of [A1] to [A23], [A26] The polypeptide complex of any one of [A1] to [A23], wherein the first antigen-binding portion comprises a peptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. [A27] The polypeptide complex according to any one of [A1] to [A23], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO:4. [A28] The polypeptide complex of any one of [A1] to [A23], wherein the first antigen-binding portion optionally comprises a streptococcal scaffold protein that binds to albumin. [A29] The polypeptide complex of any of [A1] to [A23] and [A28], wherein the first antigen-binding portion comprises an albumin-binding domain derived from protein G of Streptococcus strain G148, and optionally the albumin-binding domain comprises three α-helices. [A30] The polypeptide complex according to any one of [A1] to [A23], [A28], and [A29], wherein the first antigen-binding portion is a non-immunoglobulin-derived affinity protein. [A31] The polypeptide complex according to any one of [A1] to [A30], further comprising an antibody Fc region. [A32] The polypeptide complex of any one of [A1] to [A31], further comprising an antibody Fc region, wherein the second antigen-binding portion is Fab, and the N-terminal amino acid of the antibody Fc region is linked to the C-terminal amino acid of the heavy chain of the Fab. [A33] The polypeptide complex according to [A31] or [A32], wherein the antibody Fc region is a mutant Fc region. [A34] The polypeptide complex according to any one of [A1] to [A33], further comprising a Fab capable of binding to a human transferrin receptor. [A35] A pharmaceutical composition comprising the polypeptide complex according to any one of [A1] to [A34] and a pharmaceutically acceptable carrier. [A36] An isolated nucleic acid encoding the polypeptide complex according to any one of [A1] to [A34]. [A37] A vector comprising the nucleic acid according to [A36]. [A38] A host cell containing the vector described in [A37]. [A39] A method for producing the polypeptide complex according to any one of [A1] to [A34], which comprises a step of culturing the host cell according to [A38]. [A40] A method for detecting and / or targeting an antigen in the central nervous system (CNS), comprising systemically administering to a subject a polypeptide complex according to any one of [A1] to [A34]. [A41] The method according to [A40], wherein the systemic administration is intravenous administration or subcutaneous administration.

[0011] [B1] A polypeptide complex comprising a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, the target antigen is not a plasma protein, the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker; Polypeptide complexes. [B2] The polypeptide complex according to [B1], wherein the non-cleavable linker is a peptide of 4 amino acid residues or less. [B3] The polypeptide complex according to [B1], wherein the non-cleavable linker is a peptide of 3 amino acid residues or less. [B4] The polypeptide complex according to [B1], wherein the non-cleavable linker is a peptide of one amino acid residue. [B5] A polypeptide complex according to any one of [B1] to [B4], which does not bind to a target antigen when bound to a plasma protein. [B6] The polypeptide complex according to any one of [B1] to [B4], which has a lower binding activity to the target antigen in the presence of plasma proteins than in the absence of plasma proteins. [B7] A polypeptide complex according to any one of [B1] to [B4], wherein the binding activity to the target antigen in the presence of a plasma protein at a first concentration is different from the binding activity to the target antigen in the presence of a plasma protein at a second concentration. [B8] The polypeptide complex according to any one of [B1] to [B4], whose binding activity to the target antigen in a human plasma sample is lower than its binding activity to the target antigen in the absence of human plasma proteins. [B9] A polypeptide complex according to any one of [B1] to [B4], which has lower binding activity to the target antigen in a human plasma sample than binding activity to the target antigen in a human cerebrospinal fluid (CSF) sample. [B10] The polypeptide complex according to any one of [B1] to [B4], whose binding activity to a target antigen in the presence of 50 mg / ml of plasma protein is lower than its binding activity to a target antigen in the presence of 0.25 mg / ml of plasma protein. [B11] The polypeptide complex according to any one of [B1] to [B4], whose binding activity to a target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is lower than its binding activity to a target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [B12] A polypeptide complex described in any of [B1] to [B4], wherein the KD value for the target antigen in the presence of 50 mg / ml of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of 0.25 mg / ml of plasma protein. [B13] A polypeptide complex according to any one of [B1] to [B4], wherein the KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [B14] The polypeptide complex according to any one of [B1] to [B13], wherein the plasma protein is albumin. [B15] The polypeptide complex according to [B14], wherein the albumin is human albumin. [B16] Two or more first antigen-binding moieties and one or more second antigen-binding moieties; The polypeptide complex according to any one of [B1] to [B15], wherein one of the second antigen-binding portions and two of the first antigen-binding portions are linked via a non-cleavable linker. [B17] One or more first antigen-binding moieties and one or more second antigen-binding moieties; The polypeptide complex according to any one of [B1] to [B15], wherein one of the second antigen-binding portions and one of the first antigen-binding portions are linked via a non-cleavable linker. [B18] The polypeptide complex according to any one of [B1] to [B17], wherein the second antigen-binding portion comprises Fab or scFv. [B19] Each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [B1] to [B18], wherein the N-terminal amino acid of the heavy chain variable region and / or the light chain variable region of the Fab is linked to the C-terminal amino acid of the first antigen-binding portion via a non-cleavable linker. [B20] Two first antigen-binding moieties and one second antigen-binding moiety; the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [B1] to [B16], [B18] and [B19], wherein the N-terminal amino acids of the heavy chain variable region and the light chain variable region of the Fab are linked to the C-terminal amino acid of each of the first antigen-binding portions via a non-cleavable linker. [B21] Four first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [B1] to [B16], [B18] and [B19], wherein in each of the Fabs, the N-terminal amino acid of the heavy chain variable region and the light chain variable region is linked to the C-terminal amino acid of the first antigen-binding portion via a non-cleavable linker. [B22] One first antigen-binding moiety and one second antigen-binding moiety; the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [B1] to [B15] and [B17] to [B19], wherein the N-terminal amino acid of the heavy chain variable region of the Fab and the C-terminal amino acid of the first antigen-binding portion are linked via a non-cleavable linker. [B23] A polypeptide comprising one first antigen-binding moiety and one second antigen-binding moiety, the second antigen-binding portion comprises a Fab; The polypeptide complex according to any one of [B1] to [B15] and [B17] to [B19], wherein the N-terminal amino acid of the light chain variable region of the Fab and the C-terminal amino acid of the first antigen-binding portion are linked via a non-cleavable linker. [B24] Two first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [B1] to [B15] and [B17] to [B19], wherein, in each of the Fabs, the N-terminal amino acid of the heavy chain variable region thereof and the C-terminal amino acid of each of the first antigen-binding portions are linked via a non-cleavable linker. [B25] Two first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The polypeptide complex according to any one of [B1] to [B15] and [B17] to [B19], wherein, in each of the Fabs, the N-terminal amino acid of the light chain variable region thereof and the C-terminal amino acid of each of the first antigen-binding portions are linked via a non-cleavable linker. [B26] The polypeptide complex according to any one of [B1] to [B25], wherein the first antigen-binding portion is a Fab, scFv, VHH, a VH single domain, a VL single domain, or a peptide. [B27] The polypeptide complex according to any one of [B1] to [B26], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. [B28] A peptide comprising the amino acid sequence of the first antigen-binding portion: LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44]LP (SEQ ID NO: 40). (where, [X3] is selected from E, S, Q or C; [X6] is selected from E, S or C; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S, C or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and P at position 46 is either present or absent. The polypeptide complex according to any one of [B1] to [B26], [B29] The polypeptide complex of any one of [B1] to [B26], wherein the first antigen-binding portion comprises a peptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. [B30] The polypeptide complex according to any one of [B1] to [B26], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO:4. [B31] The polypeptide complex of any one of [B1] to [B26], wherein the first antigen-binding portion optionally comprises a streptococcal scaffold protein that binds to albumin. [B32] The polypeptide complex of any of [B1] to [B26] and [B31], wherein the first antigen-binding portion comprises an albumin-binding domain derived from protein G of Streptococcus strain G148, and optionally the albumin-binding domain comprises three α-helices. [B33] The polypeptide complex according to any one of [B1] to [B26], [B31], and [B32], wherein the first antigen-binding portion is a non-immunoglobulin-derived affinity protein. [B34] The polypeptide complex according to any one of [B1] to [B33], further comprising an antibody Fc region. [B35] The polypeptide complex according to any one of [B1] to [B34], further comprising an antibody Fc region, wherein the second antigen-binding portion is Fab, and the N-terminal amino acid of the antibody Fc region is linked to the C-terminal amino acid of the heavy chain of the Fab. [B36] The polypeptide complex according to [B29] or [B35], wherein the antibody Fc region is a mutant Fc region. [B37] The polypeptide complex according to any one of [B1] to [B36], further comprising a Fab capable of binding to a human transferrin receptor. [B38] A pharmaceutical composition comprising the polypeptide complex according to any one of [B1] to [B37] and a pharmaceutically acceptable carrier. [B39] An isolated nucleic acid encoding the polypeptide complex according to any one of [B1] to [B37]. [B40] A vector comprising the nucleic acid according to [B39]. [B41] A host cell containing the vector according to [B40]. [B42] A method for producing the polypeptide complex according to any one of [B1] to [B37], which comprises a step of culturing the host cell according to [B41]. [B43] A method for detecting and / or targeting an antigen in the central nervous system (CNS), comprising systemically administering to a subject a polypeptide complex according to any one of [B1] to [B37]. [B44] The method according to [B43], wherein the systemic administration is intravenous administration or subcutaneous administration.

[0012] In a non-limiting specific embodiment, the present invention includes the following. [C1] A polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a heavy chain variable region (VH), and a CH1 domain (CH1) of a heavy chain constant region; the second polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a light chain variable region (VL), and a light chain constant region (CL); the VH and VL form a binding moiety for a target antigen; the target antigen is not a plasma protein; Polypeptide complexes. [C2] A polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a heavy chain variable region (VH), and a CH1 domain (CH1) of a heavy chain constant region; the second polypeptide chain comprises, in order from the N-terminus, a light chain variable region (VL) and a light chain constant region (CL); the VH and VL form a binding moiety for a target antigen; the target antigen is not a plasma protein; Polypeptide complexes. [C3] A polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises, in order from the N-terminus, a heavy chain variable region (VH) and a CH1 domain (CH1) of a heavy chain constant region; the second polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a light chain variable region (VL), and a light chain constant region (CL); the VH and VL form a target antigen-binding portion; the target antigen is not a plasma protein; Polypeptide complexes. [C4] The polypeptide complex according to any one of [C1] to [C3], wherein the N-terminal amino acid of the VH and / or VL is linked to the C-terminal amino acid of the plasma protein-binding portion without a linker. [C5] The polypeptide complex according to any one of [C1] to [C3], wherein the N-terminal amino acid of the VH and / or VL is linked to the C-terminal amino acid of the plasma protein-binding portion via a non-cleavable linker. [C6] The polypeptide complex according to [C5], wherein the non-cleavable linker is a peptide of 4 amino acid residues or less. [C7] The polypeptide complex according to [C5], wherein the non-cleavable linker is a peptide of 3 amino acid residues or less. [C8] The polypeptide complex according to [C5], wherein the non-cleavable linker is a peptide of one amino acid residue. [C9] The polypeptide complex according to any one of [C1] to [C8], further comprising an antibody Fc region. [C10] The polypeptide complex according to [C9], wherein the N-terminal amino acid of the antibody Fc region is linked to the C-terminal amino acid of the CH1. [C11] The polypeptide complex according to [C9] or [C10], wherein the antibody Fc region is a mutant Fc region. [C12] The polypeptide complex according to any one of [C1] to [C11], further comprising a Fab capable of binding to a human transferrin receptor. [C13] The polypeptide complex according to any one of [C1] to [C12], which has a lower binding activity to the target antigen in the presence of plasma proteins than in the absence of plasma proteins. [C14] The polypeptide complex according to any one of [C1] to [C12], wherein the binding activity to the target antigen in the presence of a plasma protein at a first concentration is different from the binding activity to the target antigen in the presence of a plasma protein at a second concentration. [C15] The polypeptide complex according to any one of [C1] to [C12], whose binding activity to the target antigen in a human plasma sample is lower than its binding activity to the target antigen in the absence of human plasma proteins. [C16] The polypeptide complex according to any one of [C1] to [C12], wherein the binding activity to the target antigen in a human plasma sample is lower than the binding activity to the target antigen in a human cerebrospinal fluid (CSF) sample. [C17] A polypeptide complex according to any one of [C1] to [C12], whose binding activity to a target antigen in the presence of 50 mg / ml of plasma protein is lower than its binding activity to a target antigen in the presence of 0.25 mg / ml of plasma protein. [C18] The polypeptide complex according to any one of [C1] to [C12], wherein the binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is lower than the binding activity to the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [C19] A polypeptide complex according to any one of [C1] to [C12], wherein the KD value for the target antigen in the presence of 50 mg / ml of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of 0.25 mg / ml of plasma protein. [C20] A polypeptide complex according to any one of [C1] to [C12], wherein the KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 35 times the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. [C21] The polypeptide complex according to any one of [C1] to [C20], wherein the plasma protein-binding portion is a Fab, an scFv, a VHH, a VH single domain, a VL single domain, or a peptide. [C22] The polypeptide complex according to any one of [C1] to [C21], wherein the plasma protein-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. [C23] The plasma protein binding moiety is A peptide comprising the amino acid sequence LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44]LP (SEQ ID NO: 40) (where, [X3] is selected from E, S, Q or C; [X6] is selected from E, S or C; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S, C or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and P at position 46 is either present or absent. The polypeptide complex according to any one of [C1] to [C21], [C24] A polypeptide complex described in any of [C1] to [C21], wherein the plasma protein-binding portion comprises a peptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. [C25] The polypeptide complex according to any one of [C1] to [C21], wherein the plasma protein-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO:4. [C26] The polypeptide complex of any one of [C1] to [C21], wherein the first antigen-binding portion optionally comprises a streptococcal scaffold protein that binds to albumin. [C27] The polypeptide complex of any of [C1] to [C21] and [C26], wherein the first antigen-binding portion comprises an albumin-binding domain derived from protein G of Streptococcus strain G148, and optionally the albumin-binding domain comprises three α-helices. [C28] The polypeptide complex according to any one of [C1] to [C21], [C26], and [C27], wherein the first antigen-binding portion is a non-immunoglobulin-derived affinity protein. [C29] The polypeptide complex according to any one of [C1] to [C28], wherein the plasma protein is albumin. [C30] The polypeptide complex according to [C29], wherein the albumin is human albumin. [C31] A pharmaceutical composition comprising the polypeptide complex according to any one of [C1] to [C30] and a pharmaceutically acceptable carrier. [C32] An isolated nucleic acid encoding the polypeptide complex according to any one of [C1] to [C30]. [C33] A vector comprising the nucleic acid according to [C32]. [C34] A host cell containing the vector according to [C33]. [C35] A method for producing the polypeptide complex according to any one of [C1] to [C30], which comprises a step of culturing the host cell according to [C34].

[0013] In a non-limiting specific embodiment, the present invention includes the following. [D1] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen both in the presence and absence of plasma proteins; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of the plasma protein compared to in the absence of the plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D2] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of a plasma protein at a first concentration and the presence of a plasma protein at a second concentration; (b) selecting a polypeptide complex that has a different binding activity to a target antigen in the presence of a plasma protein at a first concentration and in the presence of a plasma protein at a second concentration; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D3] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen both in a human plasma sample and in the absence of human plasma proteins; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to in the absence of plasma proteins; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D4] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to a target antigen in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample; (b) selecting a polypeptide complex that has a lower binding activity to a target antigen in a human plasma sample compared to a human cerebrospinal fluid (CSF) sample; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D5] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of 50 mg / ml plasma protein and the presence of 0.25 mg / ml plasma protein; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of 50 mg / ml of plasma protein compared to the presence of 0.25 mg / ml of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D6] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (b) selecting polypeptide complexes that have lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D7] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of 50 mg / ml plasma protein and the presence of 0.25 mg / ml plasma protein; (b) selecting polypeptide complexes whose KD value for the target antigen in the presence of 50 mg / ml of plasma protein is 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30-fold or more, or 35-fold or more than the KD value for the target antigen in the presence of 0.25 mg / ml of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D8] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (b) selecting polypeptide complexes that have a KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, wherein the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; method. [D9] The method according to any one of [D1] to [D8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked without a linker. [D10] The method according to any one of [D1] to [D8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker. [D11] The method according to [D10], wherein the non-cleavable linker is a peptide of 4 amino acid residues or less. [D12] The method according to [D10], wherein the non-cleavable linker is a peptide of 3 amino acid residues or less. [D13] The method according to [D10], wherein the non-cleavable linker is a peptide of one amino acid residue. [D14] The method according to any one of [D1] to [D13], wherein the plasma protein is albumin. [D15] The method according to [D14], wherein the albumin is human albumin. [D16] The method according to any one of [D1] to [D15], wherein the target antigen is not a plasma protein. [D17] The method according to any one of [D1] to [D16], further comprising, after step (d), recovering the polypeptide complex from the cells or their culture medium.

[0014] In a non-limiting specific embodiment, the present invention includes the following. [E1] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is lower in the presence of plasma proteins compared to in the absence of plasma proteins; method. [E2] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is different between in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration; method. [E3] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is lower in a human plasma sample compared to that in the absence of plasma proteins; method. [E4] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is lower in a human plasma sample compared to a human cerebrospinal fluid (CSF) sample; method. [E5] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is lower in the presence of 50 mg / ml of plasma protein compared to the presence of 0.25 mg / ml of plasma protein; method. [E6] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the polypeptide complex has a lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; method. [E7] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the KD value of the polypeptide complex for the target antigen in the presence of 50 mg / ml of plasma protein is 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, or 35 times or more than the KD value of the polypeptide complex for the target antigen in the presence of 0.25 mg / ml of plasma protein; method. [E8] A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen; and (b) the KD value of the polypeptide complex for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value of the polypeptide complex for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; method. [E9] The method of any of [E1] to [E8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked without a linker. [E10] The method of any of [E1] to [E8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker. [E11] The method according to [E10], wherein the non-cleavable linker is a peptide of 4 amino acid residues or less. [E12] The method according to [E10], wherein the non-cleavable linker is a peptide of 3 amino acid residues or less. [E13] The method according to [E10], wherein the non-cleavable linker is a peptide of one amino acid residue. [E14] The method according to any one of [E1] to [E13], wherein the plasma protein is albumin. [E15] The method according to [E14], wherein the albumin is human albumin. [E16] The method according to any one of [E1] to [E15], wherein the target antigen is not a plasma protein. [E17] The method according to any one of [E1] to [E16], further comprising the step of recovering the polypeptide complex from the cells or their culture medium.

[0015] In a non-limiting specific embodiment, the present invention includes the following. [DE1] The polypeptide complex comprises two or more first antigen-binding moieties and one or more second antigen-binding moieties; A method described in any one of [D1] to [D17] and [E1] to [E17], wherein one of the second antigen-binding portions and two of the first antigen-binding portions are linked. [DE2] The polypeptide conjugate comprises one or more first antigen-binding moieties and one or more second antigen-binding moieties; A method described in any one of [D1] to [D17] and [E1] to [E17], wherein one of the second antigen-binding portions and one of the first antigen-binding portions are linked to each other. [DE3] The method of any of [D1] to [D17] and [E1] to [E17], wherein the second antigen-binding portion comprises a Fab or scFv. [DE4] the second antigen-binding portion comprises a Fab; The method described in any one of [D1] to [D17] and [E1] to [E17], wherein the N-terminal amino acid of the heavy chain variable region and / or light chain variable region of the Fab is linked to the C-terminal amino acid of the first antigen-binding portion. [DE5] The polypeptide conjugate comprises two first antigen-binding moieties and one second antigen-binding moiety; the second antigen-binding portion comprises a Fab; The method according to any one of [D1] to [D17] and [E1] to [E17], wherein the N-terminal amino acids of the heavy chain variable region and light chain variable region of the Fab are linked to the C-terminal amino acid of each of the first antigen-binding portions. [DE6] The polypeptide complex comprises four first antigen-binding moieties and two second antigen-binding moieties; each of the second antigen-binding portions comprises a Fab; The method according to any one of [D1] to [D17] and [E1] to [E17], wherein in each of the Fabs, the N-terminal amino acid of the heavy chain variable region and the light chain variable region is linked to the C-terminal amino acid of the first antigen-binding portion. [DE7] The polypeptide conjugate comprises one first antigen-binding portion and one second antigen-binding portion; the second antigen-binding portion comprises a Fab; The method described in any one of [D1] to [D17] and [E1] to [E17], wherein the N-terminal amino acid of the heavy chain variable region of the Fab is linked to the C-terminal amino acid of the first antigen-binding portion. [DE8] The polypeptide conjugate comprises one first antigen-binding portion and one second antigen-binding portion; the second antigen-binding portion comprises a Fab; The method described in any one of [D1] to [D17] and [E1] to [E17], wherein the N-terminal amino acid of the light chain variable region of the Fab is linked to the C-terminal amino acid of the first antigen-binding portion. [DE9] The polypeptide complex comprises two first antigen-binding portions and two second antigen-binding portions; each of the second antigen-binding portions comprises a Fab; The method of any of [D1] to [D17] and [E1] to [E17], wherein in each of the Fabs, the N-terminal amino acid of the heavy chain variable region is linked to the C-terminal amino acid of each of the first antigen-binding portions. [DE10] The polypeptide complex comprises two first antigen-binding portions and two second antigen-binding portions; each of the second antigen-binding portions comprises a Fab; The method of any of [D1] to [D17] and [E1] to [E17], wherein in each of the Fabs, the N-terminal amino acid of the light chain variable region is linked to the C-terminal amino acid of each of the first antigen-binding portions. [DE11] The method described in any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE10], wherein the first antigen-binding portion is a Fab, scFv, VHH, a VH single domain, a VL single domain, or a peptide. [DE12] A method described in any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE11], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. [DE13] The first antigen-binding portion is a peptide comprising the amino acid sequence LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44]LP (SEQ ID NO: 40). (where, [X3] is selected from E, S, Q or C; [X6] is selected from E, S or C; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S, C or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and P at position 46 is either present or absent. The method according to any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE11], wherein [DE14] The method of any of [D1] to [D17], [E1] to [E17], and [DE1] to [DE11], wherein the first antigen-binding portion comprises a peptide having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4. [DE15] The method described in any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE11], wherein the first antigen-binding portion is a peptide comprising the amino acid sequence set forth in SEQ ID NO:4. [DE16] A polypeptide complex described in any of [D1] to [D17], [E1] to [E17], and [DE1] to [DE11], wherein the first antigen-binding portion optionally comprises a streptococcal scaffold protein that binds to albumin. [DE17] A polypeptide complex according to any of [D1] to [D17], [E1] to [E17], [DE1] to [DE11], and [DE16], wherein the first antigen-binding portion comprises an albumin-binding domain derived from protein G of Streptococcus strain G148, and optionally the albumin-binding domain comprises three α-helices. [DE18] A polypeptide complex described in any of [D1] to [D17], [E1] to [E17], [DE1] to [DE11], [DE16], and [DE17], wherein the first antigen-binding portion is a non-immunoglobulin-derived affinity protein. [DE19] The method of any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE18], wherein the polypeptide complex further comprises an antibody Fc region. [DE20] The method of any of [D1] to [D17], [E1] to [E17], and [DE1] to [DE19], wherein the polypeptide complex further comprises an antibody Fc region, the second antigen-binding portion is a Fab, and the N-terminal amino acid of the antibody Fc region is linked to the C-terminal amino acid of a heavy chain of the Fab. [DE21] The method described in [DE19] or [DE20], wherein the antibody Fc region is a mutated Fc region. [DE22] The method of any one of [D1] to [D17], [E1] to [E17], and [DE1] to [DE21], wherein the polypeptide complex further comprises a Fab capable of binding to the human transferrin receptor.

[0016] In a non-limiting specific embodiment, the present invention includes the following. [F1] A method for screening for a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting a target antigen with a polypeptide complex in both the presence and absence of plasma proteins; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence and absence of plasma proteins; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of the plasma protein compared to in the absence of the plasma protein; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F2] A method for screening for a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting a target antigen with a polypeptide complex in the presence of both a first concentration of plasma protein and a second concentration of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration, respectively; and (c) selecting a polypeptide complex that has a different binding activity to the target antigen in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F3] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting a target antigen with a polypeptide complex both in a human plasma sample and in the absence of human plasma proteins; (b) measuring the binding activity of the polypeptide complex to the target antigen in a human plasma sample and in the absence of human plasma proteins, respectively; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to in the absence of plasma proteins; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F4] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting a target antigen with a polypeptide complex in a human plasma sample and in a human cerebrospinal fluid (CSF) sample, respectively; (b) measuring the binding activity of the polypeptide complex to the target antigen in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to a human cerebrospinal fluid (CSF) sample; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F5] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting the target antigen with the polypeptide complex in the presence of both 50 mg / ml plasma protein and 0.25 mg / ml plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of 50 mg / ml of plasma protein and in the presence of 0.25 mg / ml of plasma protein, respectively; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of 50 mg / ml of plasma protein compared to that in the presence of 0.25 mg / ml of plasma protein; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F6] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting the target antigen with the polypeptide complex in the presence of both about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein, respectively; and (c) selecting polypeptide complexes that have lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F7] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting the target antigen with the polypeptide complex in the presence of both 50 mg / ml plasma protein and 0.25 mg / ml plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of 50 mg / ml of plasma protein and in the presence of 0.25 mg / ml of plasma protein, respectively; and (c) selecting polypeptide complexes whose KD value for the target antigen in the presence of 50 mg / ml of plasma protein is 5-fold or more, 10-fold or more, 15-fold or more, 20-fold or more, 25-fold or more, 30-fold or more, or 35-fold or more than the KD value for the target antigen in the presence of 0.25 mg / ml of plasma protein; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F8] A method for screening a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) contacting the target antigen with the polypeptide complex in the presence of both about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein, respectively; and (c) selecting polypeptide complexes that have a KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen; method. [F9] The method according to any one of [F1] to [F8], further comprising, after step (c), a step of selecting a polypeptide complex that exhibits specific binding activity to a plasma protein. [F10] The method according to any one of [F1] to [F8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked without a linker. [F11] The method according to any one of [F1] to [F8], wherein in the polypeptide complex, the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker. [F12] The method according to [F11], wherein the non-cleavable linker is a peptide of 4 amino acid residues or less. [F13] The method according to [F11], wherein the non-cleavable linker is a peptide of 3 amino acid residues or less. [F14] The method according to [F11], wherein the non-cleavable linker is a peptide of one amino acid residue. [F15] The method according to any one of [F1] to [F14], wherein the plasma protein is albumin. [F16] The method according to [F15], wherein the albumin is human albumin. [F17] The method according to any one of [F1] to [F16], wherein the target antigen is not a plasma protein. [Effects of the Invention]

[0017] The present invention provides a polypeptide complex (plasma protein switch molecule) whose binding activity to a target antigen changes depending on the concentration of a plasma protein. The polypeptide complex of the present invention does not bind to (or has low binding activity with) a target antigen in the presence of a plasma protein (or in the presence of a high concentration of a plasma protein), but can bind to (or has high binding activity with) a target antigen in the absence of a plasma protein (or in the presence of a low concentration of a plasma protein).

[0018] Plasma protein concentrations are high in systemic blood but low in the CNS. Therefore, the polypeptide complexes of the present invention do not exert their medicinal effects in systemic blood where plasma protein concentrations are high, but can exert their medicinal effects in the CNS where plasma protein concentrations are low. Furthermore, even after the polypeptide complexes of the present invention have reached the CNS and become capable of exerting their medicinal effects, their binding to the target antigen decreases again when they return to systemic blood where plasma protein concentrations are high. This is expected to improve medicinal efficacy in the CNS and reduce side effects. In one embodiment, the plasma protein is preferably albumin.

[0019] Specifically, the polypeptide complex of the present invention is expected to have the following effects. The polypeptide complex of the present invention does not bind to the target antigen in non-CNS tissues, but binds to the target antigen only in the CNS and exerts its pharmacological activity. Because the polypeptide complexes of the present invention do not require a linker that can be cleaved by a protease, they can be used to treat diseases in CNS tissues that do not express disease-specific proteases. Furthermore, when using protease-dependent antigen-binding molecules, protease cleavage is irreversible, and if the protease-cleaved molecules are redistributed throughout the body, they may act on normal tissues and cause side effects. On the other hand, the polypeptide complexes of the present invention can achieve high efficacy while reducing systemic side effects ("on-target but non-target tissue effects"). In the polypeptide conjugates of the present invention, the plasma protein-binding moiety and the target antigen-binding moiety are connected without a linker or via an extremely short, non-cleavable linker, which avoids the increased immunogenicity that accompanies the addition of long linkers and makes them suitable for the development of therapeutic antibodies. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 illustrates the concept of controlling binding to target antigens using plasma protein binding moieties. [Figure 2]FIG. 1 illustrates the concept of controlling binding to target antigens using plasma protein binding moieties. [Figure 3] 1 is a graph showing the amount of complex formed between anti-IL-6R antibodies and human soluble IL-6R, and the amount of complex formed between various anti-IL-6R albumin switch molecules and human soluble IL-6R in mouse plasma. [Figure 4] 10 is a graph showing (i) the amount of complex formed between an anti-IL-6R antibody and human soluble IL-6R, and the amount of complex formed between various anti-IL-6R albumin switch molecules and human soluble IL-6R in human or rat plasma, and (ii) the amount of complex formed between an anti-IL-6R antibody and monkey soluble IL-6R, and the amount of complex formed between various anti-IL-6R albumin switch molecules and monkey soluble IL-6R in monkey plasma. [Figure 5] Fig. 10 is a graph showing the amount of complex formed between an anti-IL-6R antibody and human soluble IL-6R, and the amount of complex formed between an anti-IL-6R albumin switch molecule (an antibody conjugated with ABD035 or G418) and human soluble IL-6R in mouse plasma. [Figure 6] These are histograms of antibody binding when IC17, anti-IL6R antibody (H54 / / IC17), or anti-IL6R albumin switch molecule (ABD094(H / L)-H54 / / IC17) was reacted with IL6R-CHO cells at 4°C for 60 minutes. (a) MSA-free medium, (b) 50 mg / mL MSA-containing medium, (c) mouse plasma. The x-axis shows antibody fluorescence intensity, and the y-axis shows cell count. [Figure 7] Fig. 10 is a graph showing the plasma antibody concentration-time profile when sIL6R was administered IV 20 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered IV. [Figure 8] 10 is a graph showing the plasma sIL6R concentration-time profile when sIL6R was administered IV 20 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered IV. [Figure 9]Fig. 10 is a graph showing the plasma antibody concentration-time profile when an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered IV at (a) 0.2 mg / kg or (b) 1 mg / kg, respectively, followed 20 minutes later by IV administration of sIL6R. [Figure 10] 1 is a graph showing the plasma sIL6R concentration-time profile when sIL6R was administered IV 20 minutes after IV administration of an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule at (a) 0.2 mg / kg or (b) 1 mg / kg, respectively. [Figure 11] 1 is a graph showing the plasma total sIL6R concentration 5 minutes after sIL6R administration, when sIL6R was administered IV 20 minutes after anti-IL-6R antibody or anti-IL-6R albumin switch molecule was administered IV at (a) 0.2 mg / kg or (b) 1 mg / kg, respectively. [Figure 12] 1 is a graph showing the plasma free sIL6R concentration 5 minutes after sIL6R administration, when sIL6R was administered IV 20 minutes after an anti-IL-6R antibody or anti-IL-6R albumin switch molecule was administered IV at (a) 0.2 mg / kg or (b) 1 mg / kg, respectively. [Figure 13] 1 is a graph showing the plasma concentration of bound sIL6R 5 minutes after sIL6R administration, when sIL6R was administered IV 20 minutes after anti-IL-6R antibody or anti-IL-6R albumin switch molecule was administered IV at (a) 0.2 mg / kg or (b) 1 mg / kg, respectively. [Figure 14] Fig. 10 is a graph showing the concentration-time profile of total antibody in the CSF when sIL6R was administered to the lateral ventricle 10 minutes after administration of an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule. [Figure 15] 10 is a graph showing the concentration-time profile of total sIL6R in the CSF when sIL6R was administered to the lateral ventricle 10 minutes after administration of an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule. [Figure 16] 10 shows graphs showing the total antibody concentration in the CSF (a) 10 minutes and (b) 90 minutes after sIL6R administration, when sIL6R was administered to the lateral ventricle 10 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered to the lateral ventricle. [Figure 17] 10 shows graphs showing the total sIL6R concentration in the CSF (a) 10 minutes and (b) 90 minutes after sIL6R administration when sIL6R was administered to the lateral ventricle 10 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered to the lateral ventricle. [Figure 18] 10 is a graph showing the concentration of free sIL6R in the CSF 90 minutes after sIL6R administration when sIL6R was administered to the lateral ventricle 10 minutes after administration of an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule. [Figure 19] 10 is a graph showing the concentration of bound sIL6R in CSF (a) 10 minutes and (b) 90 minutes after sIL6R administration when sIL6R was administered to the lateral ventricle 10 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered to the lateral ventricle. [Figure 20] Fig. 11 is a graph showing the amount of complex formed between anti-IL-6R antibodies and human soluble IL-6R in mouse plasma, and the amount of complex formed between various anti-IL-6R albumin switch molecules (with / without linkers) and human soluble IL-6R. [Figure 21] 10 is a graph showing the plasma sIL6R concentration-time profile when sIL6R was administered IV 20 minutes after an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule was administered IV. [Figure 22] 1 is a graph showing the plasma sIL6R concentration-time profile when sIL6R was administered IV 20 minutes after IV administration of an anti-IL-6R antibody or an anti-IL-6R albumin switch molecule. Each point represents the mean ± standard deviation (n=3). [Figure 23]This is a graph showing the amount of complex formed between anti-mouse MOG antibody and mouse soluble MOG in mouse plasma, and the amount of complex formed between various anti-mouse MOG albumin switch molecules (with / without linker) and mouse soluble MOG. [Figure 24] Mouse brain cell suspensions were incubated with IC17, anti-MOG antibody (MOG / / IC17), or anti-MOG albumin switch molecule (ABD094(H / L)-MOG / / IC17) for 60 minutes at 4°C, and antibody fluorescence intensity in the oligodendrocyte fraction was measured by FACS. (a) DPBS without MSA, (b) DPBS with 50 mg / mL MSA, (c) mouse plasma. The x-axis represents antibody fluorescence intensity, and the y-axis represents cell count. [Figure 25] 1 shows a graph depicting the plasma antibody concentration-time profile when anti-MOG antibody or anti-MOG albumin switch molecule was administered IV followed by IV administration of MOG 20 minutes later, where time indicates time after MOG administration. [Figure 26] 1 shows a graph depicting the plasma antigen concentration-time profile when anti-MOG antibody or anti-MOG albumin switch molecule was administered IV followed by MOG 20 minutes later. Time indicates time after MOG administration. [Figure 27] 1 is a graph showing the plasma antibody concentration-time profile following IV administration of anti-MOG antibody or anti-MOG albumin switch molecule. [Figure 28] 1 is a graph showing the brain antibody concentration-time profile following IV administration of anti-MOG antibody or anti-MOG albumin switch molecule. [Figure 29] 1 is a graph showing plasma antigen (sMOG) concentration-time profiles following IV administration of anti-MOG antibodies or anti-MOG albumin switch molecules with various linker lengths. DETAILED DESCRIPTION OF THE INVENTION

[0021] Polypeptide Complex In one aspect, the present invention relates to a polypeptide complex comprising a first antigen-binding portion capable of specifically binding to a plasma protein (also referred to herein as a "plasma protein-binding portion") and a second antigen-binding portion capable of binding to a target antigen (also referred to herein as a "target antigen-binding portion"). In one aspect, the first antigen-binding portion and the second antigen-binding portion are linked without a linker or via an extremely short, non-cleavable linker. In one aspect, the polypeptide complex of the present invention is a fusion polypeptide in which the first antigen-binding portion and the second antigen-binding portion are directly fused. In one aspect, the polypeptide complex of the present invention is a "plasma protein switch molecule" whose binding activity to a target antigen changes depending on the concentration of a plasma protein. Various plasma proteins are available. For example, when albumin is used, the polypeptide complex of the present invention is an "albumin switch molecule" whose binding activity to a target antigen changes depending on the concentration of albumin.

[0022] The polypeptide complex of the present invention has reduced binding activity to a target antigen when the plasma protein-binding moiety is bound to a plasma protein, compared to when the plasma protein-binding moiety is not bound to a plasma protein. In one embodiment, the polypeptide complex of the present invention cannot bind to a target antigen when the plasma protein-binding moiety is bound to a plasma protein. The polypeptide complex of the present invention can regain its binding activity to a target antigen when the plasma protein is released from the plasma protein-binding moiety.

[0023] Antibodies whose binding activity to a target antigen of a therapeutic protein is controlled include antibodies whose binding activity to the target antigen changes depending on the protease (protease-dependent antigen-binding antibodies). Protease-dependent antigen-binding antibodies consist of a peptide that inhibits the antigen-binding site, a protease cleavage site, and a target antigen-binding site. In protease-dependent antigen-binding antibodies, the antigen-binding site acquires binding activity to the target antigen upon cleavage of the protease cleavage site by a protease (e.g., WO2019222282, WO2013192546). As seen in protease-dependent antigen-binding antibodies, when target antigen binding is controlled by masking the antigen-binding site of a therapeutic protein with another peptide, the protease cleavage site has been considered an essential component. However, the polypeptide complexes of the present invention may not contain a protease cleavage site (including a protease-cleavable linker or peptide linker). It was a surprising discovery that the binding of a polypeptide complex to a target antigen can be controlled without using a protease cleavage site.

[0024] In one embodiment, the polypeptide complex of the present invention may comprise a linker (non-cleavable linker) that is not cleaved by proteases or the like between the plasma protein-binding moiety and the target antigen-binding moiety. In one embodiment, the non-cleavable linker in the present disclosure may be a linker that does not contain a sequence that can be cleaved by a protease. In one embodiment, the non-cleavable linker may be a non-cleavable peptide linker. Thus, in the polypeptide complex of the present invention, the plasma protein-binding moiety and the target antigen-binding moiety may be linked via a non-cleavable linker or a non-cleavable peptide linker.

[0025] To confer multiple antigen specificities to therapeutic proteins, proteins containing multiple fused antigen-binding moieties are often created. In many of these fusion proteins, the multiple antigen-binding moieties are linked using a linker or peptide linker. Linkers or peptide linkers are used for various reasons, but linkers of a certain length are sometimes used to prevent multiple antigen-binding moieties from interfering with each other or to prevent one antigen-binding moiety from affecting the antigen-binding activity of another antigen-binding moiety. For example, in DVD-Ig, in which the variable region of one antibody is linked to the N-terminus of another antibody variable region, it has been reported that the antigen-binding activity is affected by the linker length. Specifically, it has been shown that the binding affinity of the inner antigen-binding site is reduced when a short linker (5 or 6 amino acid residues) is used compared to when a long linker (12 or 13 amino acid residues) is used (Jakob CG, et., al. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig)). TM ) molecule. MAbs. 2013 May-Jun;5(3):358-63). Thus, when linking multiple antigen-binding moieties, the use of linkers, selection of the binding site, and selection of the type and length of the linker are not simple processes, but rather require trial and error to obtain a therapeutic protein with desired properties. Based on this common technical knowledge, it was surprisingly found that a polypeptide conjugate with desired properties can be obtained by linking a plasma protein-binding moiety and a target antigen-binding moiety without a linker or peptide linker. In particular, it was an unexpected discovery that the binding activity of the target antigen-binding moiety to the target antigen (in the absence of plasma proteins or the presence of low concentrations of plasma proteins) is maintained even without the use of a linker or peptide linker, or even when an extremely short linker or peptide linker is used.

[0026] In one embodiment, the polypeptide complexes of the present invention can include a non-cleavable linker of 4 or fewer amino acid residues, 3 or fewer amino acid residues, 2 or fewer amino acid residues, or 1 amino acid residue between the plasma protein-binding moiety and the target antigen-binding moiety. Preferably, the non-cleavable linker is 3 or fewer amino acid residues. More preferably, the polypeptide complexes of the present invention can include a non-cleavable linker of 1 amino acid residue or no linker or peptide linker between the plasma protein-binding moiety and the target antigen-binding moiety. Most preferably, the polypeptide complexes of the present invention can include no linker or peptide linker between the plasma protein-binding moiety and the target antigen-binding moiety. In other words, in the polypeptide complexes of the present invention, the plasma protein-binding moiety and the target antigen-binding moiety can be linked without a linker or peptide linker. That is, in one aspect, the present invention relates to a polypeptide complex comprising a first antigen-binding portion (plasma protein-binding portion) capable of specifically binding to a plasma protein, and a second antigen-binding portion (target antigen-binding portion) capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a linker consisting of 0 to 4 amino acid residues that is not cleaved by proteases.

[0027] The above phrase "linked via a linker consisting of 0 to 4 amino acid residues" may be interpreted as "an amino acid sequence of 0 to 4 amino acid residues has been inserted." Alternatively, the above phrase "linked via a linker consisting of 0 to 4 amino acid residues" may be interpreted as "linked without a linker, or linked via a linker consisting of 1 to 4 amino acid residues." That is, in one embodiment, the polypeptide complex of the present invention has an amino acid sequence of 4 or fewer amino acid residues, 3 or fewer amino acid residues, 2 or fewer amino acid residues, or 1 amino acid residue inserted between the plasma protein-binding portion and the target antigen-binding portion. Preferably, the inserted amino acid sequence is 3 or fewer amino acid residues. More preferably, the inserted amino acid sequence is 1 amino acid residue, or no amino acid sequence is inserted at all.

[0028] Examples of non-cleavable linkers include, for example, a glycine linker (G)n (n is 1, 2, 3, or 4), a glycine-serine linker (e.g., (GS)n (n is 1 or 2), and (GGGS: SEQ ID NO: 42), etc.), a glycine-alanine linker, an alanine-serine linker, and other linkers known in the art. Examples of flexible linkers consisting of a glycine-serine linker include, but are not limited to, Ser Gly (corresponding to N10 in the Examples of the present application) Gly Ser (GS) (corresponding to N11 in the Examples of the present application) Ser Gly(SG) Gly Gly Ser (GGS) (corresponding to N12 in the Examples of this application) Gly-Ser-Gly (GSG) Ser Gly Gly (SGG) Gly Ser Ser (GSS) Ser-Ser-Gly(SSG) Ser Gly Ser (SGS) Gly Gly Gly Ser (GGGS, SEQ ID NO: 42) (corresponding to N13 in the Examples of the present application) Gly Gly Ser Gly (GGSG, SEQ ID NO: 43) Gly Ser Gly Gly (GSGG, SEQ ID NO: 44) Ser Gly Gly Gly (SGGG, SEQ ID NO: 45) Gly Ser Ser Gly (GSSG, SEQ ID NO: 46) Examples include:

[0029] Preferably, the non-cleavable linker is Gly, Gly Ser (GS), Gly Gly Ser (GGS), or Gly Gly Gly Ser (GGGS, SEQ ID NO: 42). More preferably, the non-cleavable linker is Gly, Gly Ser (GS), or Gly Gly Ser (GGS). Most preferably, the non-cleavable linker is Gly.

[0030] As described above, linkers have been used in conventional fusion polypeptides to prevent multiple antigen-binding moieties from interfering with each other or to prevent one antigen-binding moiety from affecting the antigen-binding activity of another antigen-binding moiety. In the present invention, "without a linker" and "without a linker" refer to the absence of a peptide sequence between the first and second antigen-binding moieties for this purpose. Therefore, even if the C-terminus of the first antigen-binding moiety and / or the N-terminus of the second antigen-binding moiety contain glycine, serine, alanine, etc., which are often found in peptide linkers, resulting in a moiety with a linker-like sequence when the C-terminal amino acid residue of the first antigen-binding moiety is linked to the N-terminal amino acid residue of the second antigen-binding moiety, a polypeptide complex containing such a moiety is included in the category of polypeptide complexes in which the first and second antigen-binding moieties are linked without a linker.

[0031] First antigen-binding portion (plasma protein-binding portion) Examples of the first antigen-binding moiety (plasma protein-binding moiety) of the present invention include a low molecular weight compound having specific binding activity to a plasma protein, a peptide having specific binding activity to a plasma protein, a plasma protein-binding protein domain, or a fragment thereof, and the origin thereof is not particularly limited.

[0032] The plasma proteins of the present invention are not particularly limited, as long as they are present at higher concentrations in brain tissue interstitial fluid or cerebrospinal fluid (CSF) than in plasma. The ratio of the plasma concentration to the brain tissue interstitial fluid or cerebrospinal fluid (CSF) concentration is not particularly limited, but may be, for example, 10-fold or more, preferably 50-fold or more, and more preferably 100-fold or more. Examples of plasma proteins of the present invention include, but are not limited to, albumin, cystinylated albumin, IgG, β-trace (prostaglandin D synthase), transthyretin, transferrin, α1-antitrypsin, apolipoprotein A, γ-trace (cystatin C), orosomucoid, and hemopexin. Furthermore, the species from which the plasma proteins are derived is not limited. Examples of plasma proteins of the present invention include plasma proteins derived from humans, rabbits, mice, rats, monkeys, cows, etc. These plasma proteins can be easily obtained by those skilled in the art using known techniques. Alternatively, they may be purchased from suppliers. In the polypeptide complex of the present invention, a first antigen-binding portion having specific binding activity for a plasma protein derived from human, rabbit, mouse, rat, monkey, cow, or the like can be used.

[0033] Examples of plasma protein-binding moieties of the present invention include small molecules, peptides, protein domains, or fragments thereof. Furthermore, plasma protein-binding moieties may be antibody fragments such as Fab, scFv, domain antibodies (dAbs), and non-immunoglobulin affinity proteins. Examples of domain antibodies include VH single domains, VL single domains, VHHs, and nanobodies. Examples of non-immunoglobulin affinity proteins include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 (adnectins), finomers, Kunitz domains, and O-bodies (Simeon, R. et al., In vitro-engineered non-antibody protein therapeutics. Protein Cell 9, 3-14 (2018)).

[0034] In the present invention, Fab is a full-length antibody from which the Fc region has been removed, and is a set of a polypeptide chain comprising a heavy chain variable region and the CH1 domain (CH1) of the heavy chain constant region, and a polypeptide chain comprising a light chain variable region and the light chain constant region. Fab can be obtained by treating an antibody with papain to generate antibody fragments, or by constructing a gene encoding an antibody fragment, introducing it into an expression vector, and then expressing it in an appropriate host cell (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-666; Bird, RE et al., See TIBTECH (1991) 9, 132-137).

[0035] scFvs can be obtained by linking the heavy and light chain variable regions of an antibody. In these scFvs, the heavy and light chain variable regions are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The peptide linker linking the variable regions can be, for example, any single-chain peptide consisting of 12 to 19 amino acid residues.

[0036] A single-domain antibody is an antibody fragment comprising all or a portion of an antibody heavy-chain variable region or heavy-chain variable domain, or all or a portion of an antibody light-chain variable region or light-chain variable domain. In a specific embodiment, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Pat. No. 6,248,516 B1). The structure of a single-domain antibody is not limited as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity by forming a variable region through pairing of VH and VL, whereas single-domain antibodies can exhibit antigen-binding activity solely through their own domain structure, without pairing with other domains. Single-domain antibodies usually have a relatively low molecular weight and exist in a monomeric form.

[0037] The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, each containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH domain or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds to that antigen to screen a complementary library of VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0038] Examples of single domain antibodies include, but are not limited to, antigen-binding molecules that congenitally lack light chains, such as camelid VHHs and shark VNARs, or antibody fragments comprising all or a portion of the VH domain or all or a portion of the VL domain of an antibody. Examples of single domain antibodies, which are antibody fragments comprising all or a portion of the VH / VL domains of an antibody, include, but are not limited to, single domain antibodies artificially produced starting from a human antibody VH or human antibody VL, such as those described in U.S. Patent No. 6,248,516 B1. In some embodiments of the present invention, a single domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0039] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids and transgenic animals into which genes capable of producing single-domain antibodies have been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which genes capable of producing single-domain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO 2015 / 143414 and U.S. Patent Publication No. US 2011 / 0123527 A1. Humanized single-domain antibodies can also be obtained by substituting human germline sequences or sequences similar thereto for the framework sequences of single-domain antibodies obtained from such animals. Humanized single-domain antibodies (e.g., humanized VHHs) are also an embodiment of the single-domain antibodies of the present invention.

[0040] Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11 (1691-1701)).

[0041] In one embodiment of the present invention, albumin is an example of a plasma protein. In this case, a first antigen-binding moiety capable of binding to albumin (albumin-binding moiety) is used as the first antigen-binding moiety capable of specifically binding to a plasma protein. Examples of such first antigen-binding moieties include low-molecular-weight compounds having specific binding activity to albumin, peptides having specific binding activity to albumin, albumin-binding protein domains, or fragments thereof, and the like, and their origins are not particularly limited. Furthermore, the biological species from which albumin is derived is not particularly limited. Examples of albumin in the present invention include human albumin, rabbit albumin, mouse albumin, rat albumin, monkey albumin, and bovine albumin. These albumins can be easily obtained by those skilled in the art using known techniques. Alternatively, they may be purchased from a supplier. In the polypeptide complexes of the present invention, first antigen-binding moieties (albumin-binding moieties) having specific binding activity to albumin derived from humans, rabbits, mice, rats, monkeys, bovines, etc. can be used.

[0042] In one embodiment, examples of albumin-binding moieties include small molecules, peptides, protein domains, or fragments thereof described in Zorzi A, et., al. Non-covalent albumin-binding ligands for extending the circulating half-life of small biotherapeutics. Medchemcomm. 2019 Jun 6;10(7):1068-1081. The albumin-binding moiety may also be an antibody fragment such as a Fab, scFv, domain antibody (dAb), or non-immunoglobulin-affinity protein. Examples of domain antibodies include a VH single domain, a VL single domain, a VHH, and a nanobody. Examples of non-immunoglobulin affinity proteins include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 (adnectins), finomers, Kunitz domains, and O-bodies (Simeon, R. et., al. In vitro-engineered non-antibody protein therapeutics. Protein Cell 9, 3-14 (2018)).

[0043] Albumin-binding peptide (ABP) In the present invention, peptides having binding activity to albumin (albumin-binding peptides) can also be used as the first antigen-binding moiety. Albumin-binding peptides are peptides of up to 20 amino acids in length that contain two cysteines that bind to albumin and are obtained by phage display. These peptides were obtained by panning against albumin using phages containing a peptide library in which amino acids appear randomly, and selecting clones that bind to albumin. In particular, it has been demonstrated that albumin-binding peptides containing the core sequence "DICLPRWGCLW" (SEQ ID NO: 38) have high binding affinity to albumin derived from humans, rabbits, rats, and mice (Dennis MS, et al., Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J. Biol. Chem. 2002 Sep. 20;277(38):35035-43).

[0044] In one embodiment, the albumin-binding peptide of the present invention is a peptide of 20 amino acid residues or less (e.g., 19, 18, 17, 16, 15, 14, 13, 12, or 11 residues) comprising the amino acid sequence DICLPRWGCLW (SEQ ID NO: 38). Preferably, the albumin-binding peptide of the present invention comprises a peptide having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. Alternatively, the albumin-binding peptide of the present invention is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 39. The amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 39 are identical to the amino acid sequences of SA06 and SA21, which are albumin-binding peptides reported in J Biol Chem. 2002 Sep 20;277(38):35035-43.

[0045] Albumin-binding protein domain or fragment thereof Albumin-binding protein domains are or contain three helical protein domains found in various surface proteins expressed by Gram-positive bacteria. The albumin-binding protein derived from streptococcal scaffold protein G has 214 amino acids and contains three albumin-binding domains (ABD 1-3) that bind to human serum albumin and evade the host immune system. Albumin-binding domain 3, a 46-amino acid sequence, has been demonstrated to bind to human serum albumin. These studies have yielded numerous ABD polypeptides with diverse properties.

[0046] Albumin-binding proteins are also found in other bacteria. For example, naturally occurring albumin-binding proteins include certain surface proteins from Gram-positive bacteria, such as streptococcal M proteins (e.g., M1 / Emm1, M3 / Emm3, M12 / Emml2, EmmL55 / Emm55, Emm49 / EmmL49, and protein H), streptococcal proteins G, MAG, and ZAG, and PPL and PAB from certain strains of Finegoldia magna.

[0047] In one embodiment, an example of an albumin-binding moiety of the present invention includes a streptococcal scaffold protein that optionally binds to albumin. In one embodiment, an albumin-binding moiety of the present invention comprises an albumin-binding domain (ABD) polypeptide that is or is derived from the streptococcal protein G albumin-binding domain. In a further embodiment, the ABD polypeptide is or is derived from the complete streptococcal protein G albumin-binding domain 3, or a functional fragment and / or variant thereof. In a further embodiment, the ABD polypeptide is or is derived from protein G of streptococcal strain G148. In a further embodiment, the albumin-binding domain may comprise three α-helices. In one embodiment, the albumin-binding domain of the present invention is G148-ABD3, ALB8-GA, G148-ABD1, G148-ABD2, ALB1-GA, ALB8-uGA, ALB1B-uGA, L3316-GA1, L3315-GA2, L3316-GA3, L3316-GA4, DG12-GA1, DG12-GA2, ZAG-GA, MAG-GA1, MAG-GA2, PSD-1, or ABDstable, as disclosed in Nilvebrant J., et al., The albumin-binding domain as a scaffold for protein engineering. Comput. Struct. Biotechnol. J. 2013 Sep 1. In a further embodiment, the albumin-binding moiety of the present invention comprises a non-immunoglobulin affinity protein. Examples of non-immunoglobulin affinity proteins include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 (adnectins), finomers, Kunitz domains, and O-bodies (Simeon, R., et al., In vitro-engineered non-antibody protein therapeutics. Protein Cell 9, 3-14 (2018)).

[0048] In a preferred embodiment, the albumin-binding moiety of the present invention comprises albumin-binding domain 3 of protein G of Streptococcus hemolyticus strain G148 (G148-GA3) (SEQ ID NO: 24) or a mutant thereof. G148-GA3 consists of 46 amino acid residues that form a stable three-helix bundle and has been reported to bind to albumin from humans, monkeys, mice, etc. (Johansson, MU et al. (2002) Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Modules J. Biol. Chem. 277(10):8114-8120).

[0049] An example of such a G148-GA3 variant is an albumin-binding domain having the amino acid sequence shown below (MU Johansson, IM Frick, H. Nilsson, et al. Structure, specificity, and mode of interaction for bacterial albumin-binding modules, J. Biol. Chem., 277 (10) (2002), pp. 8114-8120). L[X2][X3]AKE[X7]AI[X10]ELK[X14][X15]GI[X18]SD[X21]Y[X23][X24][X25]INKAKTVEGV[X36]ALK[X40]EIL[X44][X45] (Array number: 69) where: [X2] is selected from A, D, K, L, Q, or S; [X3] is selected from E, K, L, N, Q, or R; [X7] is selected from A, D, E, K, L, or M; [X10] is selected from A, I, K, L, N, Q, or R; [X14] is selected from A, E, K, Q, R, or V; [X15] is selected from A, K, L, N, or Y; [X18] is selected from F, I, L, or Y, or is absent; [X21] is selected from F, I, L, or Y, or is absent; [X23] is selected from F, I, L, K, T, or V; [X24] is selected from D, K, N, S, or T; [X25] is selected from A, K, L, N, or Q; [X36] is selected from E, K, M, N, T, or V; [X40] is selected from A, D, E, N, or Q; [X44] is selected from A, E, K, N, Q, or S, or is absent; and [X45] is selected from A, Q, or S, or is absent.

[0050] Alternatively, an example of such a G148-GA3 variant is an albumin-binding domain having the amino acid sequence shown below (WO2009 / 016043). LAEAK [X6] [X7] A [X9] [X10] EL [X13] KYGVSD [X20] YK [X23] [X24] I [X26] [X27] A [X29] TVEGV [X35] AL [X38] [X39] [X40] ILAALP (Sequence number: 70) where: [X6] is selected from V or E; [X7] is selected from L, E or D; [X9] is selected from N, L or I; [X10] is selected from R or K; [X13] is selected from D or K; [X20] is selected from Y or F; [X23] is selected from N, R or S; [X24] is selected from V, I, L, M, F or Y; [X26] is selected from N, S, E or D; [X27] is selected from R, K or N; [X29] is selected from K or R; [X35] is selected from D, N, Q, E, H, S, R or K; [X38] is selected from K, I or T; [X39] is selected from A, S, T, G, H, L or D, and [X40] is selected from H, E or D.

[0051] Alternatively, an example of such a G148-GA3 mutant is ABD035 (SEQ ID NO: 23) (Jonsson A, et., al. Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng Des Sel. 2008 Aug;21(8):515-27). ABD035 is a G148-GA3 mutant with improved albumin-binding activity, and is known to bind strongly to human, rat, mouse, and monkey albumin, as well as to rabbit and bovine serum albumin (see Table 11 and Figure 6 in Jonsson A, et., al., supra). In a preferred embodiment, the albumin-binding domain of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 23 (ABD035). Alternatively, the albumin-binding domain of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 23 (ABD035).

[0052] A further example of a G148-GA3 variant is an albumin-binding domain having the amino acid sequence shown below (WO2012 / 004384). LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44]LP (Sequence number: 40) where: [X3] is selected from E, S, Q or C; [X6] is selected from E, S or C; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S, C or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and The P at position 46 is either present or absent.

[0053] A preferred example of the G148-GA3 mutant is an albumin-binding domain having the amino acid sequence shown below. LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44] LP (Sequence number: 71) where: [X3] is selected from E, S or Q; [X6] is selected from E or S; [X7] is selected from A or S; [X10] is selected from A, S or R; [X14] is selected from A, S or K; [X26] is selected from D or E; [X39] is selected from D or E; [X40] is selected from A or E; [X43] is selected from A or K; [X44] is selected from A, S or E; L at position 45 is present or absent, and The P at position 46 is either present or absent.

[0054] A more preferred example of the above G148-GA3 mutant is an albumin binding domain having the amino acid sequence shown below. LA[X3]AK[X6][X7]AN[X10]ELD[X14]YGVSDFYKRLI[X26]KAKTVEGVEALK[X39][X40]IL[X43][X44] LP (Sequence number: 72) where: [X3] is selected from E or S; [X6] is E; [X7] is A; [X10] is selected from A or R; [X14] is selected from A, S or K; [X26] is D; [X39] is D; [X40] has A; [X43] is A; [X44] is A; L at position 45 is present or absent, and The P at position 46 is either present or absent.

[0055] A further example of a G148-GA3 variant is an albumin-binding domain having the amino acid sequence shown below (WO2013177398). LKEAKEKAIEELKKAGITSD[X21][X22]FDLINKA[X30][X31]VEGVN[X37]LKD[X41]ILKA (SEQ ID NO: 73) where: [X21] is selected from A, K or Y; [X22] is selected from Y, A, V or S; [X30] is selected from K or D; [X31] is selected from T or A; [X37] is selected from V, Y or A; [X41] is selected from E or Q.

[0056] Alternatively, an albumin-binding domain having the amino acid sequence shown below is also suitable for use in the polypeptide complex of the present invention (WO2014048977). LAEAKEAANAELDSYGVSDFYK [X23] LIDKAKTVEGVEALKDAILAALP (SEQ ID NO: 74) where: [X23] is selected from K, R, N, and S.

[0057] Alternatively, an albumin-binding domain having the amino acid sequence shown below is also suitable for use in the polypeptide complex of the present invention. LA[X3]AKEAANAELD[X14]YGVSDFYKRLIDKAKTVEGVEALKDAILAALP (SEQ ID NO: 41) where: [X3] is E or S, [X14] is A, S, C or K.

[0058] In a most preferred embodiment, the albumin-binding domain of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 4 (ABD094). Alternatively, the albumin-binding domain of the present invention consists of the amino acid sequence set forth in SEQ ID NO: 4 (ABD094). ABD094 is a variant of ABD035. ABD035 was "deimmunized" by modifying it to remove T-cell epitopes, resulting in ABD094, which has reduced immunogenicity while maintaining stability, solubility, and high binding affinity (Frejd F (2012) Half-Life Extension by Binding to Albumin through an Albumin Binding Domain In: Kontermann R, editor. Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives. Weinheim: Wiley-VCH Verlag GmbH & Co.). The amino acid sequence of ABD094 is identical to the amino acid sequence of PP013 disclosed in WO2012004384.

[0059] In one embodiment, the albumin binding domain of the present invention may comprise an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence of ABD035 (SEQ ID NO: 23). Preferably, the albumin binding domain of the present invention may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of ABD035 (SEQ ID NO: 23).

[0060] Alternatively, the albumin binding domain of the present invention may be one in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations (e.g., additions, insertions, deletions, substitutions, etc.) have been added to the amino acid sequence of ABD035 (SEQ ID NO: 23).

[0061] In one embodiment, the albumin binding domain of the present invention may comprise an amino acid sequence having at least 80%, 85%, or 90% sequence identity to the amino acid sequence of ABD094 (SEQ ID NO: 4). Preferably, the albumin binding domain of the present invention may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of ABD094 (SEQ ID NO: 4).

[0062] Alternatively, the albumin binding domain of the present invention may have the amino acid sequence of ABD094 (SEQ ID NO: 4) modified by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (for example, addition, insertion, deletion, substitution, etc.).

[0063] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the entire length of the sequences being compared.

[0064] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, along with user documentation, has been submitted to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0065] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: fraction X / Y × 100. where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program, as described in the immediately preceding paragraph.

[0066] Amino acid addition, deletion, substitution, and / or insertion can be performed by methods known in the art. For example, site-directed mutagenesis (Kunkel et al., Proc. Natl. Acad. Sci. USA 82, 488-492 (1985)) or overlap extension PCR can be performed on a nucleic acid encoding the amino acid sequence. These methods may be performed alone or in combination, as appropriate.

[0067] In general, modifications (e.g., conservative substitutions, deletions, insertions, and / or additions) of one or more (e.g., 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, or 3 or less, or 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more than 10) amino acids in a protein are known not to affect the function of the peptide or even to enhance the function of the original protein. Amino acid residues are classified, for example, into the following groups based on the properties of the side chains contained in their structure: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0068] Substitutions of amino acid residues within each of these groups are called conservative substitutions, while substitutions of amino acid residues between groups are called non-conservative substitutions. Domains in which amino acids contained in an albumin-binding domain having the amino acid sequence of SEQ ID NO: 23 or 4 have been modified with other amino acids classified into groups with the same characteristics are also included in the albumin-binding domains of the present invention. However, the albumin-binding domains of the present invention may also contain non-conservative modifications as long as they are functionally equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 23 or 4.

[0069] Second antigen-binding moiety (target antigen-binding moiety) The second antigen-binding portion (target antigen-binding portion) of the present invention can be a domain of any structure, as long as it binds to the intended target antigen. The target antigen-binding portion may be, for example, an antibody fragment such as Fab, scFv, domain antibody (dAb), or non-immunoglobulin affinity protein. Examples of domain antibodies include VH single domain, VL single domain, VHH, and nanobody. Examples of non-immunoglobulin affinity proteins include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 (adnectins), finomers, Kunitz domains, and O-bodies (Simeon, R., et al., In vitro-engineered non-antibody protein therapeutics. Protein Cell 9, 3-14 (2018)).

[0070] In one embodiment, the second antigen-binding portion may incorporate amino acid deletions, substitutions, insertions, or additions into the amino acid sequence of the antigen-binding portion to enhance the pH-dependent binding ability of the antibody to the antigen. When the second antigen-binding portion comprises an antibody variable region (VH and / or VL), such amino acid mutations may be included within the variable region of the antibody, for example, within one or more HVRs (e.g., CDRs). For example, the mutations may include substituting an amino acid within one or more HVRs (e.g., CDRs) of the antibody variable region with another amino acid. Alternatively, the mutations may include substituting one or more amino acids within at least one HVR (e.g., CDR) of the antibody variable region with histidine. In one embodiment, "enhanced pH-dependent binding ability" means that the variant antigen-binding portion exhibits a higher acidic KD / neutral KD ratio or a higher acidic KD / neutral KD ratio than the original "parent" antigen-binding portion before mutagenesis (i.e., an antigen-binding portion with low pH dependence). In one embodiment, the variant antigen-binding portion has an acidic KD / neutral KD ratio of 2 or greater. Alternatively, the variant antigen-binding portion has an acidic kd / neutral kd ratio of 2 or greater.

[0071] target antigen In one embodiment, the second antigen-binding moiety of the present invention binds to a different target antigen than the first antigen-binding moiety. That is, the second antigen-binding moiety of the present invention binds to an antigen that is not a plasma protein (e.g., albumin). The target antigen of the present invention may be any antigen other than a plasma protein (e.g., albumin), and its structure is not limited as long as it contains an epitope to which the second antigen-binding moiety (target antigen-binding moiety) binds. In other words, the target antigen may be either inorganic or organic. Furthermore, the target antigen of the present invention may be a soluble antigen or a membrane antigen (membrane-bound antigen). Examples of target antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, and activin RIB. ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AQP4, AR , ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6), BRK-2, RPK-1, BMPR-II(BRK-3), BMP,b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-related antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CRTH2 (CD294), CEACAM5, CFTR, cGMP, CINC, botulinum toxin, welchii toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL, CXCL8, CXCL9, CXCL10, CXCL11CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement regulatory factor (decay-accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDA R, EGF, EGFR (ErbB-1), EGFRvIII, EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EphA2, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, F GF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4),Herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-3, IL-3R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-7, IL-7R, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL -15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (IFN)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin Insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1,MAdCAM, MAG, MAP2, MARC, MBP (myelin basic protein), MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MOG (myelin oligodendrocyte glycoprotein), MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NCadherin, NCA 90, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, NMDAR (NMDA-type glutamate receptor), nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OSM receptor, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF-D, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PG J2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, FREE PLAP TfR, TGF, TGF-protease, TGF-factor, TGF-factor Pan Specific TGF-link RI(ALK-5), TGF-link RII, TGF-link RIIb, TGF-link RII I, TGF-factor 1, TGF-factor 2, TGF-factor 3, TGF-factor 4, TGF-factor 5, transcription factor Ck-1 Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF- TNF-protease 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1 Apo-2、DR4)、TNFRSF10B(TRAIL R2 DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3 DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT) TNFRSF1A(TNF RI). CD120a, p55-60, TNFRSF1B(TNF RII CD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2);TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFSF, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor) receptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A,WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XEDAR, XIA P, XPD, HMGB1, IgA, A beta, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-2 0R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, Examples include factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, alpha-synuclein, LPA, S1P, LGI1, CASPR2, glycine receptor, GABAA receptor, GABAB receptor, AMPA receptor, mGluR1, mGluR5, DPPX, D2R, IgLON5, neurexin 3 alpha, amphiphysin, septin 5, synapsin, and others, as well as receptors for hormones and growth factors.

[0072] Particularly preferred target antigens include ADAM10, EGFRvIII, EphA2, CD52, HLA-DR, alpha synuclein, TNF-alpha, IL-6R, C1q, C3, and CD20.

[0073] Tissue albumin concentration Albumin is the most abundant protein in blood, binding to various substances such as bilirubin, ions, fatty acids, and exogenous ligands (drugs) and involved in the transport of substances within the body. Albumin is primarily synthesized in the liver, with 30-40% circulating in the blood and also present in the interstitial fluid of various organs (www.ncbi.nlm.nih.gov / books / NBK459198 / ). However, albumin concentrations are known to be lower in cerebrospinal fluid, skeletal muscle, adipose tissue, skin, ear, and eye than in blood. Transport of substances to the brain is particularly limited, with albumin concentrations being particularly low in cerebrospinal fluid and brain tissue. The albumin concentration in human plasma is approximately 600 μM, while that in human cerebrospinal fluid (CSF) is approximately 3 μM (see Kay AD, et. al., CSF and serum concentrations of albumin and IgG in Alzheimer's disease. Neurobiol Aging. 1987 Jan-Feb;8(1):21-5. and Seyfert S., What determines the CSF concentrations of albumin and plasma-derived IgG? J Neurol Sci. 2004 Apr 15;219(1-2):31-3.). Furthermore, the albumin concentration in mouse plasma has been reported to be approximately 160-fold higher than that in mouse CSF (Liddelow, Shane A et al., Cellular specificity of the blood-CSF barrier for albumin transfer across the choroid plexus epithelium. PloS One. 2014;9(9):e106592).

[0074] Plasma protein concentration-dependent binding activity In one embodiment, the polypeptide complex of the present invention exhibits a stronger binding activity to either a plasma protein or a target antigen than to the other. Plasma protein concentrations are high in systemic blood and low in CNS tissues. In an environment with a high plasma protein concentration, such as in plasma, the polypeptide complex of the present invention exhibits a strong binding activity to plasma proteins and weak or almost no binding activity to the target antigen. On the other hand, in an environment with a low plasma protein concentration, such as in CNS tissues, the polypeptide complex exhibits a strong binding activity to the target antigen and weak or almost no binding activity to plasma proteins.

[0075] In the present invention, "binding activity" refers to the strength of the total noncovalent interactions between one or more binding sites of a polypeptide complex and the binding partner of the polypeptide complex (e.g., a target antigen). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., a polypeptide complex and a target antigen). For example, when the members of a binding pair reflect a monovalent 1:1 interaction, this binding activity is specifically referred to as intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of their binding strengths. The binding activity of molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or "amount of analyte bound per unit amount of ligand" (hereinafter sometimes referred to as "binding amount"). It will be understood by those skilled in the art that, in general, the lower the dissociation constant (KD), the higher the binding activity, and the higher the "amount of analyte bound per unit amount of ligand" or "binding amount," the higher the binding activity. Avidity can be measured by conventional methods known in the art, including those described herein. Specific illustrative exemplary embodiments for measuring avidity are described below.

[0076] In one embodiment, the polypeptide complex of the present invention may be a polypeptide complex whose binding activity to a target antigen in the presence of a plasma protein is lower than its binding activity in the absence of a plasma protein.

[0077] In another embodiment, the polypeptide complex of the present invention may be a polypeptide complex whose binding activity to a target antigen in the presence of a plasma protein at a first concentration is different from its binding activity to a target antigen in the presence of a plasma protein at a second concentration. In one embodiment, the polypeptide complex of the present invention may be a polypeptide complex whose binding activity to a target antigen in the presence of a plasma protein at a first concentration is lower than its binding activity in the presence of a plasma protein at a second concentration lower than the first concentration. In one embodiment, the first concentration can be exemplified by a "high concentration" as described below, and the second concentration can be exemplified by a "low concentration" as described below. In this case, the binding activity to a target antigen in the presence of a plasma protein at a first concentration is lower than the binding activity to a target antigen in the presence of a plasma protein at a second concentration.

[0078] In a specific embodiment, the first concentration and the second concentration can be about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / ml and about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / ml, respectively. More preferably, the first concentration and the second concentration can be 50 mg / ml and 0.25 mg / ml, respectively. That is, the polypeptide complex of the present invention can be a polypeptide complex whose binding activity to a target antigen in the presence of 50 mg / ml of a plasma protein (e.g., albumin) is lower than its binding activity in the presence of 0.25 mg / ml of a plasma protein (e.g., albumin).

[0079] In another embodiment, the polypeptide complex of the present invention may be a polypeptide complex whose binding activity to a target antigen in a human plasma sample is lower than its binding activity to the target antigen in the absence of human plasma proteins.

[0080] In a further embodiment, the polypeptide complex of the present invention may be a polypeptide complex whose binding activity to a target antigen in a human plasma sample is lower than its binding activity to a target antigen in a human cerebrospinal fluid (CSF) sample.

[0081] In some embodiments, the binding activity of the polypeptide complex of the present invention to a target antigen can be expressed as a dissociation constant (KD) value. Alternatively, when the target antigen is a membrane-type molecule, the apparent dissociation constant (apparent KD) can be used. The dissociation constant (KD) and apparent dissociation constant (apparent KD) can be measured by methods known to those skilled in the art, such as Biacore (GE Healthcare), Scatchard plot, or flow cytometer. In the present invention, when measuring the binding activity of a polypeptide complex to a target antigen at different plasma protein concentrations, it is preferable to keep all conditions other than the plasma protein concentration the same.

[0082] In one embodiment, the binding activity (KD) of the polypeptide complex to the target antigen is most preferably calculated from the amount of complex formed between the polypeptide complex and the target antigen at a specific plasma protein concentration. A method for calculating such binding activity (KD) is shown below using the plasma protein as an example, where the plasma protein is albumin.

[0083] First, the polypeptide complex of the present invention is added to a solution or sample (e.g., a plasma sample or a cerebrospinal fluid (CSF) sample) containing a certain concentration of albumin, followed by incubation at room temperature. Next, target antigens, the concentrations of which are gradually adjusted, are added to the solution or sample, followed by incubation at room temperature. The polypeptide complex of the present invention and target antigen may be added to the solution or sample simultaneously, or the polypeptide complex may be added first. Furthermore, if the target antigen is endogenously present in a sample (e.g., a plasma sample or a CSF sample) in an amount sufficient for the sensitivity of the measurement system, addition of the target antigen is not necessary. Next, to detect the complex between the polypeptide complex and the target antigen, a labeled antibody (secondary antibody) that specifically binds to the polypeptide complex is added to the solution or sample, followed by incubation at room temperature and then overnight at 4°C. For example, if the polypeptide complex contains an Fc region (delta GK) lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain, an anti-delta GK antibody (an antibody that specifically binds to an Fc region lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain) can be used as the secondary antibody. For example, AF647 can be used as the label. The labeled secondary antibody may be diluted with an appropriate buffer, such as PBS 0.05% Tween 20, before addition.

[0084] The complex between the polypeptide complex and the target antigen can be measured using a Gyrolab xP. Measurements using the Gyrolab xP use a Gyrolab Bioaffy 200 (P0004180, Gyros Protein Technologies) or similar device as the measurement disk. First, a biotin-labeled anti-target antigen antibody ("immobilized antibody") is added to the measurement disk and immobilized in the reaction layer within the disk. If necessary, the immobilized antibody may be diluted with an appropriate buffer (e.g., PBS 0.05% Tween 20) before addition. Preferably, the immobilized antibody binds to the same target antigen as the polypeptide complex, but to a different epitope on the target antigen molecule. When the incubated sample (a mixed solution containing the polypeptide complex, target antigen, and labeled secondary antibody) is added, the "polypeptide complex-target antigen-labeled secondary antibody" complex is captured by the immobilized antibody. The amount of the captured complex is detected by the label signal, e.g., the fluorescent signal of AF647. The AF647 fluorescent signal is analyzed using a Gyro Evaluator (Gyros Protein Technologies). The target antigen concentration that gives a fluorescent signal half the intensity of 100% binding is calculated from the concentration of the target antigen added and the resulting AF647 fluorescent signal, and this can be used as the KD value of the polypeptide complex for the target antigen. When measuring the binding activity of the polypeptide complex for the target antigen at different albumin concentrations, it is preferable to keep all conditions other than the albumin concentration the same.

[0085] If the target antigen is not a soluble antigen (e.g., a membrane antigen), it may be difficult to measure the amount of the complex between the polypeptide complex and the target antigen using the Gyrolab xP. In such cases, the next preferred method for measuring KD may be ELISA. The method for calculating KD using ELISA is shown below, using the plasma protein albumin as an example.

[0086] First, a stepwise concentration of the polypeptide complex of the present invention is added to a solution or sample (e.g., a plasma sample or a cerebrospinal fluid (CSF) sample) containing a certain concentration of albumin, followed by incubation at room temperature. The incubated solution is then added to an ELISA plate on which a target antigen has been immobilized or to an ELISA plate coated with cells expressing the target antigen, followed by incubation at room temperature. After washing away any polypeptide complex of the present invention that has not bound to the target antigen, a labeled antibody (secondary antibody) that specifically binds to the polypeptide complex is added to the sample and incubated at room temperature to detect the complex between the polypeptide complex and the target antigen. For example, if the polypeptide complex contains an Fc region lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain (delta GK), an anti-delta GK antibody (an antibody that specifically binds to an Fc region lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain) can be used as the secondary antibody. Examples of labels that can be used include horseradish peroxidase. The labeled secondary antibody may be diluted with an appropriate buffer, for example, PBS 0.05% Tween 20, and then added. When an unlabeled secondary antibody is used, an antibody that recognizes the secondary antibody may be added for detection. The target antigen concentration that gives a signal that is half the binding intensity of 100% can then be calculated from the detected label signal and used as the KD value of the polypeptide complex for the target antigen. When measuring the binding activity of the polypeptide complex for the target antigen at different albumin concentrations, it is preferable to keep all conditions other than the albumin concentration the same.

[0087] In another embodiment, the binding activity of a polypeptide complex to a target antigen in mouse plasma or cerebrospinal fluid (CSF) can also be calculated from (1) the concentration of the total polypeptide complex, (2) the concentration of free target antigen (target antigen not bound by the polypeptide complex), and (3) the concentration of bound target antigen (target antigen bound by the polypeptide complex) in mouse plasma or CSF after administration of the polypeptide complex to the mouse. Herein, the binding activity (KD) calculated in this manner is also referred to as in vivo affinity (KD).

[0088] To measure the target antigen concentration in mouse plasma, an appropriate amount of the polypeptide complex of the present invention is administered to a mouse (e.g., a 6- to 8-week-old male C57BL / 6J mouse) via the tail vein. Then, at an appropriate time, for example, 10 or 20 minutes after administration of the polypeptide complex, an appropriate amount of the target antigen is administered. Note that if the target antigen is endogenously present, administration of the target antigen is not necessary. Then, at an appropriate time, for example, 5 minutes after administration of the target antigen, blood is collected and centrifuged (12,000 rpm, 4°C, 5 minutes) to obtain plasma.

[0089] To measure the target antigen concentration in mouse cerebrospinal fluid (CSF), an appropriate amount of the polypeptide complex of the present invention is administered into the lateral ventricle of the brain of a mouse (e.g., a 6- to 8-week-old male C57BL / 6J mouse). Then, at an appropriate time, for example, 10 or 20 minutes after administration of the polypeptide complex, an appropriate amount of the target antigen is administered. Note that if the target antigen is endogenously present, administration of the target antigen is not necessary. Then, at an appropriate time, for example, 10 or 90 minutes after administration of the target antigen, CSF is collected.

[0090] The total polypeptide complex concentration in mouse plasma or CSF can be measured by enzyme-linked immunosorbent assay (ELISA). For example, first, a capture antibody (an antibody that binds to the polypeptide complex) is added to a plate. Next, a blocking solution is added to the plate, followed by a plasma sample or CSF sample (which may be diluted as necessary). Then, a biotin-labeled anti-polypeptide complex antibody (detection antibody) and a streptavidin-labeled secondary detection antibody are added. Finally, a substrate (e.g., tetramethylbenzidine) is added, and the absorbance at 650 nm is measured using an absorption spectrophotometer. The polypeptide complex concentration is calculated based on the absorbance.

[0091] The free target antigen concentration in mouse plasma or CSF can be measured using a fully automated ELISA system (Gyrolab xP workstation). For example, a standard curve solution of the target antigen is first prepared, followed by a solution containing a labeled (e.g., biotin-labeled) anti-target antigen antibody (the "capture molecule solution"). Furthermore, a solution containing a labeled (e.g., Alexa Fluor® 647) anti-target antigen antibody (the "detector molecule solution") is prepared. The capture molecule solution, plasma sample or CSF sample (optionally diluted), and detector molecule solution are placed on a PCR plate in this order, and the PCR plate is then sealed with a plate seal. Measurements can be performed by placing the PCR plate and Bioaffy 200 disk on the Gyrolab xP workstation. Using the data processed by Gyros Evaluater 3.6.2.30 software and the standard curve concentrations, the plasma free sIL6R concentration is calculated by logistic regression using XLfit 5.5.0.5 software.

[0092] The concentration of bound target antigen in mouse plasma or CSF can be measured using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (LC / ESI-MS / MS). For example, a calibration curve solution for the target antigen is first prepared. The calibration curve solution and plasma sample are then added to magnetic beads bearing an antibody (secondary antibody) that specifically binds to the polypeptide complex, shaken, and the magnetic beads are washed. If the polypeptide complex contains an Fc region lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain (delta GK), an anti-delta GK antibody (an antibody that specifically binds to an Fc region lacking two amino acids (glycine and lysine) at the C-terminus of the heavy chain) can be used as the secondary antibody. The magnetic beads are then suspended in ammonium bicarbonate containing urea, dithiothreitol, and lysozyme (hen egg white) and shaken. Iodoacetamide is then added and shaken. Sequencing-grade modified trypsin is then added to the ammonium bicarbonate, and the sample is shaken. The reaction was stopped by adding trifluoroacetic acid. The enzyme-digested sample thus prepared was used for LC / ESI-MS / MS analysis. A Xevo TQ-S triple quadrupole instrument (Waters) connected to an I-class plus UPLC (Waters) was used for LC / ESI-MS / MS analysis, and the human IL-6R-specific peptide HVVQLR (SEQ ID NO: 75) was detected by selective reaction monitoring (SRM). The concentration of bound sIL6R in plasma was calculated using the analysis software Masslynx Ver. 4.1 (Waters).

[0093] Using the total polypeptide complex concentration, free target antigen concentration, and bound target antigen concentration in plasma or CSF measured above, the in vivo affinity (KD) can be calculated according to the following formula: TIFF2026502811000002.tif11170where in vivo KD is the in vivo affinity for the target antigen, Ab 全 is the total polypeptide complex concentration, Ag遊離 where IC is the free target antigen concentration and IC is the bound target antigen concentration. For the in vivo affinity (KD) in plasma, if multiple polypeptide complex doses are available, the larger of the calculated in vivo affinities (KD) can be used. For the in vivo affinity (KD) in CSF, if multiple evaluation timings are available, the smaller of the calculated in vivo affinities (KD) can be used. The in vivo affinity (KD) ratio between plasma and CSF is then calculated by dividing the in vivo affinity (KD) in CSF by the in vivo affinity (KD) in plasma. A larger in vivo affinity (KD) ratio indicates a greater difference in binding activity between plasma and CSF, suggesting that such a polypeptide complex is more suitable as a plasma protein switch molecule.

[0094] In one aspect, when the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for a target antigen in the presence of plasma proteins is compared with the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for a target antigen in the absence of plasma proteins, the latter value is smaller than the former value.

[0095] Alternatively, in another aspect, when the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in the presence of a high or higher concentration of plasma protein is compared with the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in the presence of a low or lower concentration of plasma protein, the latter value is smaller than the former value.

[0096] Alternatively, in another aspect, when the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in a human plasma sample is compared with the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in the absence of human plasma proteins, the latter value is smaller than the former value.

[0097] Alternatively, in another aspect, when the KD value, apparent KD value, or in vivo KD value of a polypeptide complex for a target antigen in a human plasma sample is compared with the KD value, apparent KD value, or in vivo KD value of a polypeptide complex for a target antigen in a human cerebrospinal fluid (CSF) sample, the latter value is smaller than the former value.

[0098] Alternatively, in another embodiment, when the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein (e.g., albumin) is compared with the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein (e.g., albumin), the latter value is smaller than the former value. Preferably, when the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for a target antigen in the presence of 50 mg / mL of plasma protein (e.g., albumin) is compared with the KD value, apparent KD value, or in vivo KD value of the polypeptide complex for a target antigen in the presence of 0.25 mg / mL of plasma protein (e.g., albumin), the latter value is smaller than the former value.

[0099] In one embodiment, the smaller KD value, apparent KD value, or in vivo KD value is, for example, 9×10 -7 M or less, 8×10 -7Below M, 7×10 -7 Below M, 6×10 -7 Below M, 5×10 -7 Below M, 4×10 -7 Below M, 3×10 -7 Below M, 2×10 -7 Below M, 1×10 -7 Below M, 9×10 -8 Below M, 8×10 -8 Below M, 7×10 -8 Below M, 6×10 -8 Below M, 5×10 -8 Below M, 4×10 -8 Below M, 3×10 -8 Below M, 2×10 -8 Below M, 1×10 -8 Below M, 9×10 -9 Below M, 8×10 -9 Below M, 7×10 -9 Below M, 6×10 -9 Below M, 5×10 -9 Below M, 4×10 -9 Below M, 3×10 -9 Below M, 2×10 -9 Below M, 1×10 -9 Below M, 9×10 -10 Below M, 8×10 -10 Below M, 7×10 -10 Below M, 6×10 -10 Below M, 5×10 -10 Below M, 4×10 -10 Below M, 3×10 -10 Below M, 2×10 -10 Below M, 1×10 -10 Below M, 9×10 -11 Below M, 8×10 -11 Below M, 7×10 -11 Below M, 6×10 -11 Below M, 5×10 -11 Below M, 4×10 -11 Below M, 3×10 -11 Below M, 2×10 -11 Below M, または1×10 -11 M and below are the same.

[0100] In one embodiment, the larger KD value, apparent KD value, or in vivo KD value is, for example, 1×10 -9 M or more, 2×10 -9 M or more, 3×10 -9 M or more, 4×10 -9 M or more, 5×10 -9 M or more, 6×10 -9 M or more, 7×10 -9 M or more, 8×10 -9 M or more, 9×10 -9 M or more, 1×10 -8 M or more, 2×10 -8 M or more, 3×10 -8 M or more, 4×10 -8 M or more, 5×10 -8 M or more, 6×10 -8 M or more, 7×10 -8 M or more, 8×10 -8 M or more, 9×10 -8 M or more, 1×10 -7 M or more, 2×10 -7 M or more, 3×10 -7 M or more, 4×10 -7 M or more, 5×10 -7 M or more, 6×10 -7 M or more, 7×10 -7 M or more, 8×10 -7 M or more, 9×10 -7 M or more, 1×10 -6 M or more, 2×10 -6 M or more, 3×10 -6 M or more, 4×10 -6 M or more, 5×10 -6 M or more, 6×10 -6 M or more, 7×10 -6 M or more, 8×10 -6 M or more, or 9 x 10 -6 It can be M or more.

[0101] In one embodiment, the KD value, apparent KD value, or in vivo KD value in the absence or presence of low concentrations of plasma proteins is compared to the KD value, apparent KD value, or in vivo KD value in the presence of plasma proteins or high concentrations of plasma proteins. The ratio of KD values ​​can be, for example, 2x or more, 3x or more, 4x or more, 5x or more, 6x or more, 7x or more, 8x or more, 9x or more, 10x or more, 11x or more, 12x or more, 13x or more, 14x or more, 15x or more, 16x or more, 17x or more, 18x or more, 19x or more, 20x or more, 21x or more, 22x or more, 23x or more, 24x or more, 25x or more, 26x or more, 27x or more, 28x or more, 29x or more, 30x or more, 31x or more, 32x or more, 33x or more, 34x or more, 35x or more, 36x or more, 37x or more, 38x or more, 39x or more, 40x or more, 50x or more, 100x or more, 200x or more, 300x or more, 500x or more, 1x10 3 More than twice, 2×10 3 More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 It can be more than double.

[0102] In another embodiment, the KD value, apparent KD value, or in vivo KD value of the polypeptide complex of the present invention for a target antigen is, for example, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 11-fold or more, 12-fold or more, 13-fold or more, 14-fold or more, 15-fold or more, 16-fold or more, 17-fold or more, 18-fold or more, 19-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 or more, in the presence of plasma protein or in the presence of a high concentration of plasma protein, compared to that of a control polypeptide complex having the same structure as the polypeptide complex of the present invention except that it does not have the first antigen-binding portion. 3 More than twice, 2×10 3 More than double, 3×10 3More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 It can be more than twice as large.

[0103] Alternatively, the kd (dissociation rate constant) can be used as the value of antigen-binding activity when the target antigen is a soluble molecule, and the apparent kd (apparent dissociation rate constant) can be used when the target antigen is a membrane-type molecule. The kd (dissociation rate constant) and apparent kd (apparent dissociation rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), a flow cytometer, etc.

[0104] Alternatively, the k a (binding rate constant) can be used as the value of antigen-binding activity when the target antigen is a soluble molecule, and the apparent k a (apparent binding rate constant) can be used when the target antigen is a membrane-type molecule. The k a (binding rate constant) and apparent k a (apparent binding rate constant) can be measured by methods known to those skilled in the art, for example, using Biacore (GE Healthcare), a flow cytometer, etc.

[0105] In another embodiment, the binding activity of the polypeptide complex of the present invention may be expressed, for example, as the amount of target antigen bound to the polypeptide complex. For example, in a surface plasmon resonance assay, the amount of bound polypeptide complex immobilized on a sensor chip and the amount of bound target antigen further bound thereto are each measured in resonance units (RU). The antigen-binding activity may be expressed using the amount of bound target antigen as an index, or alternatively, the value obtained by dividing the amount of bound target antigen by the amount of bound polypeptide complex (i.e., the amount of bound target antigen per unit amount of polypeptide complex) as an index.

[0106] In some embodiments, the amount of target antigen binding in the presence of plasma proteins is greater than the amount of target antigen binding in the absence of plasma proteins.

[0107] Alternatively, in another aspect, when the amount of binding of the target antigen in the presence of a high or higher concentration of plasma protein is compared to the amount of binding of the target antigen in the presence of a low or lower concentration of plasma protein, the latter value is greater than the former value.

[0108] In another embodiment, when the amount of bound target antigen in a human plasma sample is compared with the amount of bound target antigen in the absence of human plasma proteins, the latter value is greater than the former value.

[0109] Alternatively, in another embodiment, when the amount of binding of the target antigen in a human plasma sample is compared with the amount of binding of the target antigen in a human cerebrospinal fluid (CSF) sample, the latter value is greater than the former value.

[0110] In another embodiment, when the amount of target antigen binding in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein (e.g., albumin) is compared with the amount of target antigen binding in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein (e.g., albumin), the latter value is greater than the former value. Preferably, when the amount of target antigen binding in the presence of 50 mg / mL of plasma protein (e.g., albumin) is compared with the amount of target antigen binding in the presence of 0.25 mg / mL of plasma protein (e.g., albumin), the latter value is greater than the former value.

[0111] In one embodiment, the binding activity of the polypeptide complex of the present invention to a target antigen may be expressed as the amount of complex between the polypeptide complex and the target antigen formed at a specific concentration of plasma protein. For example, in measurements using a Gyrolab xP, the fluorescent signal value of the complex between the polypeptide complex of the present invention and the target antigen can be calculated by setting the fluorescent signal obtained from a complex between a control polypeptide complex having the same structure as the polypeptide complex of the present invention except that it does not contain the first antigen-binding portion at 100. In this case, the fluorescent signal value of the complex between the polypeptide complex of the present invention and the target antigen can be, for example, 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, or 95% or less in the presence of plasma protein or a high concentration of plasma protein, compared to the fluorescent signal obtained from a complex between a control polypeptide complex having the same structure as the polypeptide complex of the present invention and the target antigen except that it does not contain the first antigen-binding portion. The amount of captured complex is detected by the signal of the label, for example, the fluorescent signal of AF647. The AF647 fluorescent signal is analyzed using a Gyro Evaluator (Gyros Protein Technologies). The antigen concentration that gives a fluorescent signal half the 100% binding intensity is calculated from the concentration of the target antigen added and the obtained AF647 fluorescent signal, and this can be used as the KD value for the antibody to be evaluated against the bound antigen. When measuring the binding activity of a polypeptide complex to a target antigen at different concentrations of plasma protein, it is preferable to keep all conditions other than the plasma protein concentration the same.

[0112] Any concentration of plasma protein (eg, albumin) can be selected as long as a difference in the binding activity of the polypeptide complex is detected. In certain embodiments, high or higher concentrations include, for example, 100 μM or higher, 150 μM or higher, 200 μM or higher, 250 μM or higher, 300 μM or higher, 350 μM or higher, 400 μM or higher, 450 μM or higher, 500 μM or higher, 550 μM or higher, 600 μM or higher, 650 μM or higher, 700 μM or higher, 750 μM or higher, 800 μM or higher, 850 μM or higher, 900 μM or higher, 950 μM or higher, 1 mM or higher, 2 mM or higher, 3 mM or higher, 4 mM or higher, 5 mM or higher, 6 mM or higher, 7 mM or higher, 8 Examples of concentrations include concentrations of 1 mM or higher, 9 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher.

[0113] In one embodiment, the high or higher concentration can include, for example, the plasma protein concentration in the plasma of humans, rabbits, mice, rats, monkeys, cows, and the like.

[0114] In one embodiment, the high or higher concentration herein can be selected as a sufficient amount such that the polypeptide complex exhibits maximum binding activity to the target antigen. In another embodiment, the high or higher concentration herein can be selected as a plasma protein concentration such that the polypeptide complex exhibits a "larger KD value, apparent KD value, or in vivo KD value for the target antigen" and / or a "smaller binding amount of the target antigen" described herein.

[0115] In certain embodiments, low or lower concentrations include, for example, 100 μM or lower, 90 μM or lower, 80 μM or lower, 70 μM or lower, 60 μM or lower, 50 μM or lower, 40 μM or lower, 30 μM or lower, 20 μM or lower, 10 μM or lower, 9 μM or lower, 8 μM or lower, 7 μM or lower, 6 μM or lower, 5 μM or lower, 4 μM or lower, 3 μM or lower, 2 μM or lower, 1 μM or lower, 900 nM or lower, 800 nM or lower, 700 nM or lower, 600 nM or lower, 500 Examples of concentrations include nM or lower, 400 nM or lower, 300 nM or lower, 200 nM or lower, 100 nM or lower, 30 nM or lower, 10 nM or lower, 3 nM or lower, 1 nM or lower, 300 pM or lower, 100 pM or lower, 30 pM or lower, 10 pM or lower, 3 pM or lower, 1 pM or lower, and the like.

[0116] In one embodiment, the low or lower concentration can include plasma protein concentration in central nervous system (CNS) tissue, such as cerebrospinal fluid (CSF), of, for example, humans, rabbits, mice, rats, monkeys, cows, etc.

[0117] In one embodiment, the low concentration herein can be selected as a sufficient amount such that the polypeptide complex exhibits minimal binding activity to the target antigen. A concentration of substantially zero (in the absence of plasma proteins) can also be selected as an embodiment of a low concentration. In another embodiment, a plasma protein concentration that exhibits the "smaller KD value, apparent KD value, or in vivo KD value for the target antigen" and / or the "larger binding amount of the target antigen" described herein can be selected as the low or lower concentration herein.

[0118] In another embodiment, examples of ratios of high concentration to low concentration include 3x or more, 10x or more, 30x or more, 50x or more, 100x or more, 150x or more, 160x or more, 170x or more, 180x or more, 190x or more, 200x or more, 250x or more, 300x or more, 350x or more, 400x or more, 450x or more, 500x or more, 1x10 3 Double or more, 3 x 10 3 Double or more, 1 x 10 4 A value of double or more can be selected.

[0119] Conditions other than the plasma protein concentration when measuring binding activity to a target antigen can be appropriately selected by those skilled in the art and are not particularly limited. For example, measurements can be performed under conditions of HEPES buffer and 37°C. Measurements can be performed using, for example, Biacore (GE Healthcare). When the target antigen is a soluble molecule, the binding activity to soluble molecules can be evaluated by passing the target antigen as an analyte through a chip on which the polypeptide complex is immobilized. When the target antigen is a membrane-type molecule, the binding activity to membrane-type molecules can be evaluated by passing the polypeptide complex as an analyte through a chip on which the target antigen is immobilized.

[0120] To obtain a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of plasma protein, the techniques described below can be applied as appropriate. For example, to confirm that the binding activity of a polypeptide complex to a target antigen in the presence of plasma protein is lower than that in the absence of plasma protein, the binding activity of the polypeptide complex to a target antigen in the absence and presence of plasma protein is compared. In another non-limiting embodiment, to confirm that the binding activity of a polypeptide complex to a target antigen in the presence of a high concentration of plasma protein is lower than that in the presence of a low concentration of plasma protein, the binding activity of the polypeptide complex to a target antigen in the presence of a low concentration of plasma protein is compared.

[0121] In one embodiment, the binding activity of a polypeptide complex can be measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed using a polypeptide complex of interest and its target antigen. For example, the solution binding affinity of a polypeptide complex for a target antigen is measured by measuring the binding affinity of the polypeptide complex to a minimum concentration ( 125Measurements are performed by equilibrating the Fab with I)-labeled antigen and then capturing the bound target antigen using a plate coated with an antibody against the polypeptide complex (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-polypeptide complex antibody in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-target antigen is mixed with serial dilutions of the polypeptide complex of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The polypeptide complex of interest is then incubated overnight, although this incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillation agent (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each polypeptide complex that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0122] In one embodiment, the binding activity of a polypeptide complex is measured using a ligand capture method based on surface plasmon resonance analysis, such as with a BIACORE™ T200 or BIACORE™ 4000 (GE Healthcare, Uppsala, Sweden). The instrument is operated using BIACORE™ Control Software. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. Binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween (registered trademark)-20) as the measurement buffer, at a flow rate of 10 to 30 μL / min, and at a measurement temperature of preferably 25°C or 37°C. When measuring by having a ligand capture molecule capture an antibody as a ligand, the antibody is injected to capture a desired amount, and then a serial dilution of the target antigen (analyte) prepared using the measurement buffer is injected. When measuring by having a ligand capture molecule capture a target antigen as a ligand, the target antigen is injected to capture a desired amount, and then a serial dilution of the antibody (analyte) prepared using the measurement buffer is injected.

[0123] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetic parameter calculations are performed by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model, and the binding rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. Another parameter of binding activity, the "amount of analyte bound per unit amount of ligand," can also be calculated by dividing the amount of analyte bound at a specific concentration (resonance units: RU) by the amount of ligand captured (RU), can also be calculated.

[0124] When the target antigen is a membrane-type molecule, methods for measuring the binding activity of a polypeptide complex containing an antigen-binding portion for the target antigen to target antigen-expressing cells include, for example, the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, assessment can be performed using ELISA or fluorescence-activated cell sorting (FACS) with target antigen-expressing cells as the antigen. The following provides examples of methods for measuring the binding activity of a polypeptide complex that binds to IL-6R to IL-6R-expressing cells. Polypeptide complexes that bind to target antigens other than IL-6R can also be appropriately measured using the following examples.

[0125] In the ELISA format, the binding activity of a polypeptide complex containing an antigen-binding portion for IL-6R to IL-6R-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, the test polypeptide complex is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the test polypeptide complex bound to the cells is detected using an enzyme-labeled antibody that recognizes the test polypeptide complex. Alternatively, in FACS, a dilution series of the test polypeptide complex is prepared, and the antibody binding titer to IL-6R-expressing cells is determined, allowing the binding activity of the test polypeptide complex to IL-6R-expressing cells to be compared.

[0126] The binding of the polypeptide complex to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected by a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE FACSCalibur™ (both are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0127] For example, the following method is an example of a suitable method for measuring the binding activity of a test polypeptide complex containing an antigen-binding portion for IL-6R to a target antigen. First, cells expressing IL-6R are reacted with the test polypeptide complex and stained with an FITC-labeled secondary antibody that recognizes the test polypeptide complex. The test polypeptide complex is diluted with a suitable buffer to prepare a polypeptide complex of the desired concentration. For example, the polypeptide complex can be used at a concentration between 10 μg / ml and 10 ng / ml. Next, fluorescence intensity and cell count are measured using a FACSCalibur (BD). The fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD) reflects the amount of antibody bound to the cells. In other words, by measuring the geometric mean value, the binding activity of the test polypeptide complex, represented by the amount of bound test polypeptide complex, can be obtained.

[0128] Structure of the polypeptide complex Polypeptide complexes of the invention can comprise one or more first antigen-binding moieties (plasma protein-binding moieties) and one or more second antigen-binding moieties (target antigen-binding moieties). In one embodiment, polypeptide complexes of the invention can comprise two or more (e.g., two, three, or four) first antigen-binding moieties (plasma protein-binding moieties) and one or more (e.g., one or two) second antigen-binding moieties (target antigen-binding moieties). In another embodiment, polypeptide complexes of the invention can comprise one or two first antigen-binding moieties (plasma protein-binding moieties) for each second antigen-binding moiety (target antigen-binding moiety) capable of binding to a target antigen.

[0129] Specifically, the polypeptide complex of the present invention includes: a polypeptide complex comprising one first antigen-binding moiety (plasma protein-binding moiety) and one second antigen-binding moiety (target antigen-binding moiety); a polypeptide complex comprising two first antigen-binding moieties (plasma protein-binding moieties) and one second antigen-binding moiety (target antigen-binding moiety); a polypeptide complex comprising one first antigen-binding moiety (plasma protein-binding moiety) and two second antigen-binding moieties (target antigen-binding moieties); a polypeptide complex comprising two first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties); a polypeptide complex comprising three first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties); or a polypeptide complex comprising four first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties); Examples include, but are not limited to, the following.

[0130] When two second antigen-binding portions (target antigen-binding portions) are included, each second antigen-binding portion may bind to the same target antigen or to a different target antigen. Preferably, the second antigen-binding portion binds to a protein that is not a plasma protein.

[0131] In one embodiment, the polypeptide complex of the present invention can comprise one first antigen-binding moiety (plasma protein-binding moiety) and one second antigen-binding moiety (target antigen-binding moiety). In one embodiment, the second antigen-binding moiety (target antigen-binding moiety) can comprise a set of polypeptide chains comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which amino acid residues located in the heavy chain variable region or the light chain variable region are linked to amino acid residues of the first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. For example, the polypeptide complex of the present invention can be a polypeptide complex in which the N-terminal amino acid residue of either the heavy chain variable region or the light chain variable region is linked to the C-terminal amino acid residue of the first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. The non-cleavable linker preferably consists of 0 to 3 amino acid residues, and more preferably 0 to 1. A "non-cleavable linker consisting of 0 amino acid residues" means that it does not contain any linker at all.

[0132] In one embodiment, such a polypeptide complex may be a "polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, in order from the N-terminus, a plasma protein binding moiety, a heavy chain variable region (VH), and a CH1 domain (CH1) of a heavy chain constant region; and the second polypeptide chain comprises, in order from the N-terminus, a light chain variable region (VL) and a light chain constant region (CL)." Alternatively, the polypeptide complex may be a "polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, in order from the N-terminus, a heavy chain variable region (VH) and a CH1 domain (CH1) of a heavy chain constant region; and the second polypeptide chain comprises, in order from the N-terminus, a plasma protein binding moiety, a light chain variable region (VL), and a light chain constant region (CL)." The first polypeptide chain and the second polypeptide chain may be linked by a disulfide bond. In one embodiment, the polypeptide complex of the present invention may comprise one or two sets of a first polypeptide chain and a second polypeptide chain. When the polypeptide complex comprises two sets of first and second polypeptide chains, the first polypeptide chains of one set can be linked to the first polypeptide chains of the other set by disulfide bonds.

[0133] The present inventors have demonstrated that a polypeptide complex in which the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues (no first antigen-binding moiety (plasma protein-binding moiety) is linked to the heavy chain variable region) can more effectively inhibit binding to a target antigen in the presence of plasma proteins than a polypeptide complex in which the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of the first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues (no first antigen-binding moiety (plasma protein-binding moiety) is linked to the light chain variable region). Therefore, the former polypeptide complex can be cited as a preferred example of the polypeptide complex of the present invention.

[0134] In one embodiment, the polypeptide complex of the present invention can comprise two first antigen-binding moieties (plasma protein-binding moieties) and one second antigen-binding moiety (target antigen-binding moiety). In one embodiment, the second antigen-binding moiety (target antigen-binding moiety) can comprise a set of polypeptide chains comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which amino acid residues located in the heavy chain variable region are linked to amino acid residues of one first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and amino acid residues located in the light chain variable region are linked to amino acid residues of the other first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. For example, the polypeptide complex of the present invention can be a polypeptide complex in which the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of one first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the other first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues.

[0135] The present inventors have demonstrated that a polypeptide complex in which two first antigen-binding moieties (plasma protein-binding moieties) are linked to one second antigen-binding moiety (target antigen-binding moiety) can more effectively inhibit target antigen binding in the presence of plasma proteins than a polypeptide complex in which one first antigen-binding moiety (plasma protein-binding moiety) is linked to one second antigen-binding moiety (target antigen-binding moiety). Thus, in the present invention, a polypeptide complex in which two first antigen-binding moieties (plasma protein-binding moieties) are linked to one second antigen-binding moiety (target antigen-binding moiety) can be cited as a preferred example.

[0136] In one embodiment, such a polypeptide complex may be a "polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises, in order from the N-terminus, a plasma protein binding moiety, a heavy chain variable region (VH), and a CH1 domain (CH1) of a heavy chain constant region, and the second polypeptide chain comprises, in order from the N-terminus, a plasma protein binding moiety, a light chain variable region (VL), and a light chain constant region (CL)."

[0137] In the above-described embodiment, the heavy chain variable region (VH), the CH1 domain (CH1) of the heavy chain constant region, the light chain variable region (VL), and the light chain constant region (CL) form the Fab of the antibody. The heavy chain variable region (VH) and the light chain variable region (VL) form a binding moiety for a target antigen. In one embodiment, the C-terminal amino acid residue of the plasma protein-binding moiety is linked to the N-terminal amino acid residue of the heavy chain variable region (VH) and / or the light chain variable region (VL) via a non-cleavable linker consisting of 0 to 4 amino acid residues. Such a polypeptide complex may comprise an antibody Fc region. In this case, the C-terminal amino acid residue of the CH1 domain (CH1) of the heavy chain constant region is linked to the N-terminal amino acid residue of the antibody Fc region.

[0138] In one embodiment, the polypeptide complex of the present invention can comprise one first antigen-binding portion (plasma protein-binding portion) and two second antigen-binding portions (target antigen-binding portions). In one embodiment, the second antigen-binding portion (target antigen-binding portion) can comprise a set of polypeptide chains comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which amino acid residues located in the heavy chain variable region or light chain variable region of either of the two second antigen-binding portions (target antigen-binding portions) are linked to amino acid residues of the first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues. For example, the polypeptide complex of the present invention can be a polypeptide complex in which, in either one of the two second antigen-binding portions (target antigen-binding portions), the N-terminal amino acid residue of either the heavy chain variable region or the light chain variable region is linked to the C-terminal amino acid residue of the first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues. In one aspect, the polypeptide complex of the present invention is preferably a polypeptide complex in which the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues.

[0139] In one embodiment, the polypeptide complex of the present invention can comprise two first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties). In one embodiment, the second antigen-binding moiety (target antigen-binding moiety) can comprise a set of polypeptide chains comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which, in one second antigen-binding moiety (target antigen-binding moiety), amino acid residues located in the heavy chain variable region or light chain variable region are linked to amino acid residues in one first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and in the other second antigen-binding moiety (target antigen-binding moiety), amino acid residues located in the heavy chain variable region or light chain variable region are linked to amino acid residues in the other first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. Alternatively, the polypeptide complexes of the present invention can be polypeptide complexes in which, in one second antigen-binding portion (target antigen-binding portion), amino acid residues located in the heavy chain variable region are linked to amino acid residues in one first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker of 0 to 4 amino acid residues, and amino acid residues located in the light chain variable region are linked to amino acid residues in the other first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker of 0 to 4 amino acid residues, and in the other second antigen-binding portion (target antigen-binding portion), the first antigen-binding portion (plasma protein-binding portion) is not linked. In a specific embodiment, the polypeptide complex of the present invention can be a polypeptide complex in which, in one of the two second antigen-binding portions (target antigen-binding portions), the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of one of the first antigen-binding portions (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and in the other of the two second antigen-binding portions (target antigen-binding portions), the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of the other first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues. In a specific embodiment, the polypeptide complexes of the present invention can be polypeptide complexes in which, in one of the two second antigen-binding moieties (target antigen-binding moieties), the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of one of the first antigen-binding moieties (plasma protein-binding moieties) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and in the other of the two second antigen-binding moieties (target antigen-binding moieties), the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the other first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. In a specific embodiment, the polypeptide complexes of the present invention can be polypeptide complexes in which, in one of the two second antigen-binding portions (target antigen-binding portions), the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of one of the first antigen-binding portions (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and in the other of the two second antigen-binding portions (target antigen-binding portions), the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the other first antigen-binding portion (plasma protein-binding portion) via a non-cleavable linker consisting of 0 to 4 amino acid residues.Alternatively, the polypeptide complex of the present invention can be a polypeptide complex in which, in one of the two second antigen-binding moieties (target antigen-binding moieties), the N-terminal amino acid residue of the heavy chain variable region is linked to the C-terminal amino acid residue of one of the first antigen-binding moieties (plasma protein-binding moieties) via a non-cleavable linker consisting of 0 to 4 amino acid residues, and the N-terminal amino acid residue of the light chain variable region is linked to the C-terminal amino acid residue of the other first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues; and, in the other of the two second antigen-binding moieties (target antigen-binding moieties), the first antigen-binding moiety (plasma protein-binding moiety) is not linked to the N-terminal amino acid residue of either the heavy chain variable region or the light chain variable region.

[0140] In one embodiment, the polypeptide complex of the present invention can comprise three first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties). In one embodiment, the second antigen-binding moieties (target antigen-binding moieties) can comprise a polypeptide chain comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and one set of polypeptide chains comprising a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which, in one of the second antigen-binding moieties (target antigen-binding moieties), amino acid residues located in the heavy chain variable region and light chain variable region are linked to amino acid residues located in two of the three first antigen-binding moieties (plasma protein-binding moieties) without a linker, and in the other of the second antigen-binding moieties (target antigen-binding moieties), amino acid residues located in the heavy chain variable region or light chain variable region are linked to amino acid residues in the remaining one of the three first antigen-binding moieties (plasma protein-binding moieties) without a linker. For example, the polypeptide complexes of the present invention can be polypeptide complexes in which, in either one of the two second antigen-binding moieties (target antigen-binding moieties), the N-terminal amino acid residues of both the heavy chain variable region and the light chain variable region are linked to the C-terminal amino acid residue of two of the three first antigen-binding moieties (plasma protein-binding moieties) via a non-cleavable linker consisting of 0 to 4 amino acid residues; and, in the other of the two second antigen-binding moieties (target antigen-binding moieties), the N-terminal amino acid residue of either the heavy chain variable region or the light chain variable region is linked to the C-terminal amino acid residue of the remaining one of the three first antigen-binding moieties (plasma protein-binding moieties) via a non-cleavable linker consisting of 0 to 4 amino acid residues.

[0141] In one embodiment, the polypeptide complex of the present invention can comprise four first antigen-binding moieties (plasma protein-binding moieties) and two second antigen-binding moieties (target antigen-binding moieties). In one embodiment, each of the second antigen-binding moieties (target antigen-binding moieties) can comprise a polypeptide chain comprising a heavy chain variable region and a CH1 domain (CH1) of a heavy chain constant region, and one set of polypeptide chains comprising a light chain variable region and a light chain constant region, i.e., Fab. In this case, the polypeptide complex of the present invention can be a polypeptide complex in which amino acid residues located in the heavy chain variable region and the light chain variable region of both second antigen-binding moieties (target antigen-binding moieties) are linked to amino acid residues located in each of the first antigen-binding moieties (plasma protein-binding moieties) via a non-cleavable linker consisting of 0 to 4 amino acid residues. For example, the polypeptide complex of the present invention can be a polypeptide complex in which the N-terminal amino acid residues of both the heavy and light chain variable regions of two second antigen-binding moieties (target antigen-binding moieties) are linked to the C-terminal amino acid residue of each first antigen-binding moiety (plasma protein-binding moiety) via a non-cleavable linker consisting of 0 to 4 amino acid residues. In this example, the heavy and light chain variable regions of each of the two second antigen-binding moieties are linked to a single first antigen-binding moiety via a non-cleavable linker consisting of 0 to 4 amino acid residues. Alternatively, the heavy and light chain variable regions of each of the two second antigen-binding moieties are linked to a single first antigen-binding moiety without a linker.

[0142] In the above polypeptide complex, the non-cleavable linker preferably consists of 0 to 3 amino acid residues, more preferably 0 to 1. Note that a "non-cleavable linker consisting of 0 amino acid residues" means that no linker is included at all.

[0143] In one embodiment, the polypeptide complexes of the invention can further comprise an antibody Fc region or a mutated Fc region, or a fragment thereof.

[0144] In the present invention, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0145] As used herein, the term "variant Fc region" refers to an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide, compared to the native-sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology thereto, and more preferably at least about 95%, 96%, 97%, 98%, or 99% homology thereto.

[0146] In another embodiment, the Fc region may be a mutant Fc region created by adding amino acid modifications to a native sequence Fc region, and is preferably an Fc region variant of a native IgG (IgG1, IgG2, IgG3, or IgG4) antibody. In a specific embodiment, the mutant Fc region has enhanced or decreased binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa, compared to a native sequence Fc region.

[0147] In one embodiment, the mutant Fc region has increased FcRn-binding activity in the acidic and neutral pH ranges, particularly in the acidic pH range. In one embodiment, the mutant Fc region of the present invention comprises, as indicated by EU numbering, Ala at position 434; any one of Glu, Arg, Ser, and Lys at position 438; and any one of Glu, Asp, and Gln at position 440; and more preferably, Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440. Preferably, the mutant Fc region further comprises, as indicated by EU numbering, Ile or Leu at position 428 and / or any one of Ile, Leu, Val, Thr, and Phe at position 436. More preferably, the mutant Fc region comprises, as indicated by EU numbering, Leu at position 428 and / or Val or Thr at position 436.

[0148] In one embodiment, the variant Fc region is represented by the EU numbering system: (a)N434A / Q438R / S440E; (b) N434A / Q438R / S440D; (c)N434A / Q438K / S440E; (d)N434A / Q438K / S440D; (e)N434A / Y436T / Q438R / S440E; (f)N434A / Y436T / Q438R / S440D; (g)N434A / Y436T / Q438K / S440E; (h)N434A / Y436T / Q438K / S440D; (i)N434A / Y436V / Q438R / S440E; (j)N434A / Y436V / Q438R / S440D; (k)N434A / Y436V / Q438K / S440E; (l)N434A / Y436V / Q438K / S440D; (m)N434A / R435H / F436T / Q438R / S440E; (n)N434A / R435H / F436T / Q438R / S440D; (o)N434A / R435H / F436T / Q438K / S440E; (p)N434A / R435H / F436T / Q438K / S440D; (q)N434A / R435H / F436V / Q438R / S440E; (r)N434A / R435H / F436V / Q438R / S440D; (s)N434A / R435H / F436V / Q438K / S440E; (t)N434A / R435H / F436V / Q438K / S440D; (u)M428L / N434A / Q438R / S440E; (v)M428L / N434A / Q438R / S440D; (w)M428L / N434A / Q438K / S440E; (x)M428L / N434A / Q438K / S440D; (y)M428L / N434A / Y436T / Q438R / S440E; (z)M428L / N434A / Y436T / Q438R / S440D; (aa)M428L / N434A / Y436T / Q438K / S440E; (ab)M428L / N434A / Y436T / Q438K / S440D; (ac)M428L / N434A / Y436V / Q438R / S440E; (ad)M428L / N434A / Y436V / Q438R / S440D; (ae)M428L / N434A / Y436V / Q438K / S440E; (af)M428L / N434A / Y436V / Q438K / S440D; (ag)L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (ah)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E The amino acid sequence may comprise a combination of amino acid substitutions selected from the group consisting of:

[0149] In a preferred embodiment, the variant Fc region has the following structure, as represented by EU numbering: (a)N434A / Q438R / S440E; (b) N434A / Y436T / Q438R / S440E; (c)N434A / Y436V / Q438R / S440E; (d)M428L / N434A / Q438R / S440E; (e)M428L / N434A / Y436T / Q438R / S440E; (f)M428L / N434A / Y436V / Q438R / S440E; (g) L235R / G236R / S239K / M428L / N434A / Y436T / Q438R / S440E; and (h)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E The amino acid sequence may comprise a combination of substituted amino acids selected from the group consisting of:

[0150] In one embodiment, the mutant Fc region preferably has increased FcRn-binding activity under acidic pH conditions compared to that of a native-sequence Fc region. The increase in FcRn-binding activity of the mutant Fc region in a certain pH range may correspond to an increase in measured FcRn-binding activity compared to that measured for the native-sequence Fc region. In such cases, the difference in binding activity, KD(native-sequence Fc region) / KD(mutant Fc region), may be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 70-fold, 80-fold, 100-fold, 500-fold, or 1000-fold. While such an increase can occur in the acidic and / or neutral pH ranges, an increase in the acidic pH range may be preferred.

[0151] In one embodiment, when the polypeptide complex of the present invention comprises two second antigen-binding portions (target antigen-binding portions), each of which is a Fab, the two second antigen-binding portions (target antigen-binding portions) can form a full-length antibody together with the antibody Fc. Thus, in one embodiment, the polypeptide complex of the present invention can be a polypeptide complex comprising a first antigen-binding portion (plasma protein-binding portion) and an antibody capable of binding to a target antigen.

[0152] In the polypeptide complex of the present invention, the plasma protein is preferably albumin, and the albumin is preferably human albumin. That is, the first antigen-binding portion of the present invention is preferably an albumin-binding portion capable of binding to albumin, and more preferably a human albumin-binding portion capable of binding to human albumin.

[0153] When the polypeptide complex of the present invention comprises an antibody as a part thereof, the antibody may be a chimeric antibody, a humanized antibody, a human antibody, or a mouse antibody. It may also be a monoclonal antibody or a polyclonal antibody. It may also be a multispecific antibody (e.g., a bispecific antibody). In one embodiment, the antibody may be an antibody fragment such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. The antibody may also be an antibody derived from a human, mouse, rat, hamster, rabbit, monkey, or the like. In another embodiment, the antibody is a full-length antibody such as a complete IgG1 antibody, a complete IgG4 antibody, or any other antibody class or isotype defined herein.

[0154] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region or a variant Fc region as defined herein.

[0155] The "class" of an antibody refers to the type of constant domain or constant region possessed by the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0156] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for making monoclonal antibodies, are described herein.

[0157] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0158] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. It can also refer to an antibody comprising an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0159] Methods for producing antibodies with desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0160] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques. Note that the term "monoclonal antibodies" as used herein also includes "humanized antibodies" and "chimeric antibodies."

[0161] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells are screened by a conventional screening method to select hybridomas that produce anti-IL-6R antibodies.

[0162] The IL-6R protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be prepared by chemical synthesis based on the amino acid sequence of human IL-6R, or by incorporating the partial IL-6R gene into an expression vector and expressing it. Alternatively, the partial peptide can be produced by degrading the IL-6R protein with a protease. The region and size of the partial IL-6R peptide are not limited to any particular embodiment. The number of amino acids forming the peptide used as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, more preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0163] Furthermore, fusion proteins in which a desired partial polypeptide or peptide of the IL-6R protein is fused with a different polypeptide can be used as a sensitizing antigen. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitizing antigens. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for preparing IL-6R to be used as a sensitizing antigen and immunization methods using IL-6R are also specifically described in International Publication Nos. WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0164] There are no particular limitations on the mammal to be immunized with the sensitizing antigen. It is preferable to select a mammal in consideration of its compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, and hamsters, rabbits, and monkeys are preferably used.

[0165] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. Commonly used immunization methods include intraperitoneal or subcutaneous administration of the sensitizing antigen to a mammal. Specifically, the sensitizing antigen is diluted to an appropriate dilution ratio with PBS (phosphate-buffered saline), saline, or the like. If desired, a conventional adjuvant, such as Freund's complete adjuvant, is mixed with the sensitizing antigen and the mixture is emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier may also be used when immunizing with the sensitizing antigen. In particular, when a low-molecular-weight partial peptide is used as the sensitizing antigen, it may be desirable to conjugate the sensitizing antigen peptide to a carrier protein such as albumin or keyhole limpet hemocyanin for immunization.

[0166] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed to express a gene encoding an antigen protein in an animal is administered to the immunized animal, resulting in the expression of a sensitizing antigen, thereby stimulating the immune system. Compared to conventional immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages: -It can provide immune stimulation while maintaining the structure of membrane proteins such as IL-6R. - There is no need to purify the immunogen.

[0167] To prepare monoclonal antibodies by DNA immunization, DNA expressing the IL-6R protein is first administered to the animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The resulting DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be used as the expression vector. Commonly used methods can be used to administer vectors to living organisms. For example, DNA immunization can be performed by introducing gold particles coated with the expression vector into the cells of the animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0168] A mammal is immunized as described above, and an increase in the serum titer of IL-6R-binding antibodies is confirmed. Subsequently, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0169] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. The selection marker confers a trait on the cells that allows (or prevents) them from surviving under specific culture conditions. Known selection markers include hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA and die in HAT selective medium. However, when fused with normal cells, they can continue DNA synthesis using the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0170] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells incorporate these pyrimidine analogs into their DNA and die. On the other hand, cells lacking these enzymes cannot incorporate these pyrimidine analogs and can therefore survive in selective media. Another selectable marker, called G418 resistance, is conferred by the neomycin resistance gene and confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs). Various myeloma cell lines suitable for cell fusion are known.

[0171] For example, myeloma cells including the following cells can be suitably used. P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1(Current Topics in Microbiology and Immunology(1978)81, 1-7), NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519), MPC-11(Cell(1976)8 (3), 405-415), SP2 / 0(Nature(1978)276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133).

[0172] Cell fusion between the immune cells and myeloma cells is basically carried out using known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0173] More specifically, the cell fusion can be carried out in a standard nutrient medium in the presence of a cell fusion promoter. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an auxiliary agent such as dimethyl sulfoxide can be added to further enhance the fusion efficiency.

[0174] The ratio of immune cells to myeloma cells can be set at will. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. The culture medium used for the cell fusion may be, for example, a medium suitable for growing the myeloma cell line, such as RPMI1640 culture medium, MEM culture medium, or other conventional culture medium used for culturing this type of cell. Furthermore, serum supplements such as fetal calf serum (FCS) may be added.

[0175] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium. A PEG solution (e.g., average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is then added, typically at a concentration of 30% to 60% (w / v). The desired fused cells (hybridomas) are formed by gentle mixing. The appropriate culture medium listed above is then gradually added to the cells, and the mixture is centrifuged repeatedly to remove the supernatant. This allows the removal of cell fusion agents and other substances that are undesirable for hybridoma growth.

[0176] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT culture medium can be continued for a period of time sufficient to allow cells other than the desired hybridoma (unfused cells) to die. This period of time is usually several days to several weeks. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0177] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, HGPRT-deficient or TK-deficient cells can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. By continuing culture in the HAT medium for a sufficient period of time, cells other than the desired hybridoma (non-fused cells) can be killed. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody are then screened and single-cell cloned by the conventional limiting dilution method.

[0178] Screening and monocloning of the desired antibody can be suitably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened by, for example, fluorescence-activated cell sorting (FACS). FACS is a system that measures antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

[0179] To screen for hybridomas producing monoclonal antibodies by FACS, cells expressing IL-6R are first prepared. The preferred cells for screening are mammalian cells overexpressing IL-6R. As a control, non-transformed mammalian cells can be used as host cells to selectively detect the binding activity of antibodies to cell surface IL-6R. Specifically, hybridomas producing anti-IL-6R monoclonal antibodies can be isolated by selecting hybridomas producing antibodies that bind to cells overexpressing IL-6R but not to host cells.

[0180] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies bound to the immobilized cells are detected. When the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody capable of binding to the antigen can be selected by the above screening and cloned by limiting dilution or other methods.

[0181] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium and stored for a long period in liquid nitrogen.

[0182] The hybridomas can be cultured according to conventional methods, and the desired monoclonal antibodies can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be prepared from the ascites fluid. The former method is suitable for obtaining highly pure antibodies.

[0183] Antibodies encoded by antibody genes cloned from antibody-producing cells such as the above-mentioned hybridomas can also be suitably used. The cloned antibody gene is inserted into an appropriate vector, which is then introduced into a host, thereby expressing the antibody encoded by the gene. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0184] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is prepared from hybridoma cells that produce the antibody. To do this, total RNA is first extracted from the hybridoma. Methods for extracting mRNA from cells include, for example, the following. Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0185] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or similar. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be prepared from hybridomas. cDNA encoding antibody variable regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, the SMART RACE cDNA Amplification Kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002; Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, which will be described later, can be introduced at both ends of the cDNA.

[0186] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria. After colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Whether or not the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0187] A convenient way to isolate genes encoding variable regions is to use the 5'-RACE method, which uses primers to amplify the variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template to construct a 5'-RACE cDNA library. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0188] The antibody genes are amplified by PCR using the 5'-RACE cDNA library as a template. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. The nucleotide sequences of these primers vary depending on the immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0189] Specifically, for example, to isolate a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains are used. To amplify IgG variable region genes, a primer that anneals to a portion of the constant region close to the variable region is generally used as the 3' primer. Meanwhile, the primer included in the 5' RACE cDNA library construction kit is used as the 5' primer.

[0190] The PCR product thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibody can be selected using the binding activity of the reconstituted immunoglobulin to IL-6R as an indicator. For example, when the aim is to isolate an antibody against IL-6R, it is more preferable that the antibody binds specifically to IL-6R. Antibodies that bind to IL-6R can be screened, for example, by the following steps: (1) contacting an antibody isolated from a hybridoma, the antibody comprising a variable region encoded by the cDNA, with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0191] Methods for detecting the binding of an antibody to IL-6R-expressing cells are known. Specifically, the binding of an antibody to IL-6R-expressing cells can be detected by techniques such as the above-mentioned FACS. To evaluate the binding activity of an antibody, a fixed sample of IL-6R-expressing cells can be used as appropriate.

[0192] Panning using phage vectors is also a suitable method for screening antibodies based on binding activity. Screening methods using phage vectors are advantageous when isolating antibody genes from a library of heavy and light chain subclasses of a polyclonal antibody-expressing cell population. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form a single-chain Fv (scFv). Inserting a gene encoding an scFv into a phage vector can produce a phage that expresses the scFv on its surface. DNA encoding an scFv with the desired binding activity can be isolated by contacting the phage with the desired antigen and then recovering the phage that binds to the antigen. Repeating this procedure as necessary allows the enrichment of scFv with the desired binding activity.

[0193] After isolating cDNA encoding the variable region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and cleave nucleotide sequences that occur at low frequency in the nucleotide sequence constituting the antibody gene. Furthermore, to insert a single copy of the digested fragment in the correct orientation, it is preferable to insert a restriction site for the enzyme that generates a cohesive end into the vector. An antibody expression vector is constructed by digesting cDNA encoding the variable region of the anti-IL-6R antibody as described above and inserting it into an appropriate expression vector. In this case, a chimeric antibody is obtained by fusing the gene encoding the antibody constant region (C region) with the gene encoding the variable region in frame. Here, the term "chimeric antibody" refers to an antibody in which the origin of the constant region is different from the origin of the variable region. Therefore, in addition to heterogeneous chimeric antibodies such as mouse / human, human / human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the variable region gene into an expression vector already containing the constant region. Specifically, for example, a recognition sequence for a restriction enzyme that excises the variable region gene can be appropriately placed at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region. Two genes digested with the same combination of restriction enzymes are fused in frame to construct a chimeric antibody expression vector.

[0194] To produce an anti-IL-6R monoclonal antibody, the antibody gene is inserted into an expression vector so that its expression is controlled by an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. An appropriate signal sequence may also be added to the amino terminus so that the expressed antibody is secreted extracellularly. The expressed polypeptide is cleaved at the carboxyl terminal of the sequence, and the resulting polypeptide can be secreted extracellularly as a mature polypeptide. Next, recombinant cells expressing DNA encoding the anti-IL-6R antibody can be obtained by transforming an appropriate host cell with this expression vector.

[0195] DNAs encoding the antibody heavy chain (H chain) and light chain (L chain) are inserted separately into separate expression vectors, and the antibody genes are expressed. An antibody molecule having both an H chain and an L chain can be expressed by simultaneously transforming the same host cell with vectors into which the H chain and L chain have been inserted. Alternatively, host cells can be transformed by inserting DNAs encoding the H chain and L chain into a single expression vector (see International Publication WO 1994 / 011523).

[0196] Many combinations of host cells and expression vectors are known for producing antibodies by introducing isolated antibody genes into a suitable host. All of these expression systems are applicable to isolating the antigen-binding moiety of the present invention. Eukaryotic cells suitable for use as host cells include animal cells, plant cells, and fungal cells. Specific examples of animal cells include the following: (1) Mammalian cells: CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma (Sp2 / 0, NS0, etc.), BHK (baby hamster kidney cell line), Hela, Vero, HEK293 (human embryonic kidney cell line containing common adenovirus (Ad) 5 DNA), PER.C6 cells (human embryonic retinal cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), etc. (Current Protocols in Protein Science (May 2001, Unit 5.9, Table 5.9.1)). (2) Amphibian cells: Xenopus oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0197] Furthermore, antibody gene expression systems using plant cells derived from the genus Nicotiana, such as Nicotiana tabacum, are known. Callus cultured cells can be appropriately used for transformation of plant cells.

[0198] Furthermore, the following fungal cells can be used: Yeast: Saccharomyces cerevisiae and other species of the genus Saccharomyces, and Pichia pastoris and other species of the genus Pichia Filamentous fungi: Aspergillus genus, such as Aspergillus niger

[0199] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when bacterial cells are used, bacterial cells such as Escherichia coli and Bacillus subtilis can be appropriately used. An expression vector containing the antibody gene of interest is introduced into these cells by transformation. The transformed cells are cultured in vitro, and the desired antibody can be prepared from the culture of the transformed cells.

[0200] In addition to the host cells described above, transgenic animals can also be used to produce recombinant antibodies. That is, the antibody of interest can be obtained from an animal into which a gene encoding the antibody has been introduced. For example, an antibody gene can be constructed as a fusion gene by inserting it in frame into a gene encoding a protein specifically produced in milk. Examples of proteins secreted into milk include goat beta-casein. A DNA fragment containing a fusion gene with an antibody gene inserted therein is injected into a goat embryo, and the injected embryo is then introduced into a female goat. The desired antibody can be obtained from the milk produced by the transgenic goat (or its offspring) that receives the embryo, as a fusion protein with a milk protein. Furthermore, hormones can be administered to the transgenic goat as appropriate to increase the amount of milk containing the desired antibody produced by the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0201] When the polypeptide complexes described herein are administered to humans, antigen-binding portions of the polypeptide complexes may be appropriately derived from recombinant antibodies that have been artificially modified to reduce heterologous antigenicity to humans, for example. Such recombinant antibodies include, for example, humanized antibodies. These modified antibodies are appropriately produced using known methods.

[0202] The variable region of an antibody used to prepare the antigen-binding portion of the polypeptide complex described herein is typically composed of three complementarity-determining regions (CDRs) sandwiched between four framework regions (FRs). CDRs are the regions that essentially determine the binding specificity of an antibody. The amino acid sequences of CDRs are highly diverse. However, the amino acid sequences that make up FRs often show high identity even among antibodies with different binding specificities. Therefore, the binding specificity of one antibody can generally be transferred to another antibody by CDR grafting.

[0203] Humanized antibodies are also called reshaped human antibodies. Specifically, humanized antibodies in which CDRs from non-human animals, such as mouse antibodies, are grafted onto human antibodies are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting the CDRs of mouse antibodies onto human FRs. In overlap extension PCR, a nucleotide sequence encoding the CDRs of the mouse antibody to be grafted is added to a primer for synthesizing the FRs of a human antibody. Primers are prepared for each of the four FRs. In general, when grafting mouse CDRs onto human FRs, selecting human FRs that are highly identical to the mouse FRs is considered advantageous in terms of maintaining CDR function. In other words, it is generally preferable to use human FRs whose amino acid sequences are highly identical to the amino acid sequences of the FRs adjacent to the mouse CDR to be grafted.

[0204] The nucleotide sequences to be linked are designed to be connected in frame with each other. Human FRs are synthesized individually using each primer. As a result, products are obtained in which mouse CDR-encoding DNAs are added to each FR-encoding DNA. The nucleotide sequences encoding the mouse CDRs of each product are designed to overlap with each other. Subsequently, the overlapping CDR portions of the products synthesized using the human antibody gene as a template are annealed to perform complementary strand synthesis. This reaction links the human FRs via the mouse CDR sequences.

[0205] The full-length variable region gene, in which three CDRs and four FRs are ultimately linked, is amplified using primers that anneal to the 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A humanized antibody expression vector can be constructed by inserting the DNA obtained as described above and DNA encoding a human antibody constant region into an expression vector so that they are linked in frame. After introducing the recombinant vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, resulting in the production of the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400 and International Publication WO 1996 / 002576).

[0206] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, it is possible to suitably select FRs of a human antibody that form a favorable antigen-binding site when linked via the CDRs. If necessary, amino acid residues in the FRs can be substituted so that the CDRs of the reshaped human antibody form a suitable antigen-binding site. For example, amino acid sequence mutations can be introduced into the FRs by applying the PCR method used to graft mouse CDRs onto human FRs. More specifically, partial nucleotide sequence mutations can be introduced into primers annealing to the FRs. Nucleotide sequence mutations are introduced into the FRs synthesized using such primers. By measuring and evaluating the antigen-binding activity of mutant antibodies with amino acid substitutions using the above method, mutant FR sequences with desired properties can be selected (Cancer Res., (1993) 53, 851-856).

[0207] Alternatively, transgenic animals carrying the full repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735) can be immunized by DNA immunization to obtain the desired human antibodies.

[0208] Furthermore, techniques for preparing human antibodies by panning using a human antibody library are also known. For example, the variable regions of human antibodies are expressed on the surface of phages as single-chain antibodies (scFv) using phage display. Phages expressing scFvs that bind to antigens can be selected. The DNA sequence encoding the variable regions of human antibodies that bind to antigens can be determined by analyzing the genes of the selected phages. An expression vector can be prepared by determining the DNA sequence of the scFv that binds to the antigen, fusing the variable region sequence in frame with the sequence of the constant region of a desired human antibody, and then inserting the resulting vector into an appropriate expression vector. The human antibody can be produced by introducing the expression vector into a suitable expression cell such as those listed above and expressing the gene encoding the human antibody. These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0209] In addition to the above techniques, B cell cloning techniques (identification, cloning, and isolation of each antibody-encoding sequence; use in constructing expression vectors for producing each antibody (particularly IgG1, IgG2, IgG3, or IgG4), etc.) as described in Bernasconi et al. (Science (2002) 298, 2199-2202) or International Publication WO2008 / 081008 can also be used appropriately to isolate antibody genes.

[0210] Furthermore, in one embodiment, the polypeptide complex of the present invention can further comprise a substance capable of binding to the human transferrin receptor. The binding activity of the polypeptide complex of the present invention to a target antigen is suppressed in systemic blood where plasma protein concentrations are high, and it exhibits binding activity to a target antigen in the CNS or other areas where plasma protein concentrations are low. On the other hand, the polypeptide complex of the present invention must pass through the blood-brain barrier (BBB) ​​to access the CNS. The BBB tightly regulates the influx of molecules, such as proteins and small molecules, into brain tissue. Therefore, it is preferable to use the polypeptide complex of the present invention in combination with a technique that promotes passage through the BBB.

[0211] Techniques for promoting passage through the BBB include techniques that target receptors expressed in the BBB, such as transferrin receptors, insulin receptors, enkephalin receptors, and glutathione receptors. Incorporation of substances that target these receptors into the polypeptide complexes of the present invention is expected to promote uptake of the polypeptide complexes of the present invention into the CNS. Known molecules that target receptors expressed in the BBB include LRP-1 affinity peptides, anti-insulin receptor antibodies, glutathione-PEG-liposomes, melanotransferrin or melanotransferrin peptides, and anti-transferrin receptor antibodies. In one embodiment, the polypeptide complexes of the present invention can contain a substance that binds to the transferrin receptor. In another embodiment, the substance capable of binding to the human transferrin receptor can be an antibody capable of binding to the human transferrin receptor. In another embodiment, the antibody can be a minibody (low-molecular-weight antibody) such as scFv, Fv, Fab, or VHH. Examples of such antibodies and low-molecular-weight antibodies include the blood-brain barrier shuttles disclosed in WO2014033074A1 and WO2015101588A1. These are anti-transferrin receptor antibodies that can promote drug passage across the BBB by transcytosis mediated by the transferrin receptor on cerebrovascular endothelial cells. The polypeptide complexes of the present invention can contain a substance capable of binding to the human transferrin receptor, which is well known to those skilled in the art.

[0212] In one embodiment, the polypeptide complex of the present invention may have a first antigen-binding portion comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 23, 24, 38-41, and 69-74.

[0213] Furthermore, in one embodiment, the polypeptide complex of the present invention may have an antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the polypeptide complex of the present invention may have an antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 5 or 6. In another embodiment, the polypeptide complex of the present invention may have an antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2. In yet another embodiment, the polypeptide complex of the present invention may have an antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 7 or 8.

[0214] Furthermore, in one embodiment, the polypeptide complexes of the present invention may comprise a fusion polypeptide of a first antigen-binding portion and an antibody heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 13, 19, 25, 27, 36, 57, and 63 to 65, or an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 12. In another embodiment, the polypeptide complexes of the present invention may comprise a fusion polypeptide of a first antigen-binding portion and an antibody light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 18, 20, 26, 28, 37, 58, and 66 to 68, or an antibody light chain comprising the amino acid sequence of SEQ ID NO: 14 or 17.

[0215] In another embodiment, the polypeptide complex of the present invention may have an antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 47. In another embodiment, the polypeptide complex of the present invention may have an antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 6. In another embodiment, the polypeptide complex of the present invention may have an antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 48. In another embodiment, the polypeptide complex of the present invention may have an antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0216] Furthermore, in one embodiment, the polypeptide complex of the present invention may comprise a fusion polypeptide of a first antigen-binding portion and an antibody heavy chain, comprising the amino acid sequence set forth in SEQ ID NO: 51, 53, 55, 76, or 78. In another embodiment, the polypeptide complex of the present invention may comprise a fusion polypeptide of a first antigen-binding portion and an antibody light chain, comprising the amino acid sequence set forth in SEQ ID NO: 52, 54, 56, 77, or 79.

[0217] Pharmaceutical Composition In one aspect, the present invention relates to a pharmaceutical composition comprising the polypeptide complex described herein. The polypeptide complex contained in the pharmaceutical composition of the present invention exhibits (or exhibits stronger) binding activity to a target antigen in the absence of plasma proteins or at low plasma protein concentrations, and exhibits no (or exhibits weaker) binding activity to a target antigen in the presence of plasma proteins or at high plasma protein concentrations. Therefore, the pharmaceutical composition of the present invention is expected to have a strong therapeutic effect against diseases of the central nervous system and other conditions with low plasma protein concentrations, while avoiding side effects caused by cytotoxicity or neutralizing effects on tissues with high plasma protein concentrations.

[0218] The pharmaceutical compositions of the present invention may contain a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0219] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions containing water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate amount within the indicated range is obtained.

[0220] An "effective amount" of a pharmaceutical composition or formulation refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0221] Sterile compositions for injection can be formulated according to common pharmaceutical practice using a vehicle such as distilled water for injection. Aqueous solutions for injection include, for example, physiological saline, and isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, and sodium chloride). Suitable solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (Polysorbate 80™, HCO-50, etc.), can be used in combination.

[0222] Oils include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used as a solubilizer. It is also possible to add buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and / or antioxidants. The prepared injection solution is filled into appropriate ampoules.

[0223] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, the composition may be administered in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, the composition may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0224] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing the polypeptide complex of the present invention can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be set, for example, in the range of 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0225] Nucleic acids, vectors, and host cells The present invention relates to isolated nucleic acids or polynucleotides encoding the polypeptide complexes described herein. A nucleic acid or polynucleotide of the present invention refers to one or more nucleic acid molecules encoding the polypeptides that make up the polypeptide complexes of the present invention. The nucleic acids or polynucleotides of the present invention include one or more nucleic acid molecules encoding at least a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, and include nucleic acid molecules on a single vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0226] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and those with unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by: P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0227] The nucleic acids of the present invention can be used, for example, to produce the polypeptide complex of the present invention. When producing the polypeptide complex of the present invention, typically, a nucleic acid encoding the polypeptide complex of the present invention is inserted into an appropriate expression vector, the vector is introduced into appropriate cells, the transformed cells are cultured, and the expressed polypeptide complex is isolated and purified. The polypeptide complex can also be expressed as a fusion protein with other proteins, for purposes such as facilitating purification. For example, using Escherichia coli as a host, methods such as preparing a fusion protein with maltose-binding protein (vector pMAL series available from New England BioLabs, USA), preparing a fusion protein with glutathione S-transferase (GST) (vector pGEX series available from Amersham Pharmacia Biotech), or preparing a fusion protein by adding a histidine tag (pET series from Novagen) can be used.

[0228] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0229] In one embodiment of the present invention, the nucleic acid of the present invention may be inserted into a vector. For example, when Escherichia coli is used as a host, the vector can be amplified in large quantities in Escherichia coli (e.g., JM109, DH5 alpha, HB101, XL1 Blue) and other strains to prepare large quantities of the vector. Examples of vectors include, but are not limited to, vectors having an "ori" for amplification in Escherichia coli and a selection gene for transformed Escherichia coli (e.g., a drug resistance gene that can be detected by a drug (ampicillin, tetracycline, kanamycin, chloramphenicol, etc.)). Examples of such vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Furthermore, for the purpose of cDNA subcloning and excision, examples of vectors include, in addition to the above vectors, pGEM-T, pDIRECT, pT7, etc. When using a vector for the purpose of producing a polypeptide complex, an expression vector is particularly useful. For example, when expression in E. coli is desired, in addition to the above-mentioned characteristics that allow the vector to be amplified in E. coli, when the host is E. coli such as JM109, DH5 alpha, HB101, or XL1-Blue, the expression vector must have a promoter that enables efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter. Examples of such vectors include pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET.

[0230] The vector may also contain a signal sequence for secreting the polypeptide. In the case of production in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4379) can be used as the signal sequence for polypeptide secretion. The vector can be introduced into the host cell using, for example, the calcium chloride method or electroporation. Furthermore, examples of vectors that can be expressed in plants include vectors such as pMH1, pMH2, and pCAMBIA.

[0231] In addition to E. coli, examples of vectors for producing polypeptide complexes include mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, and pCDM8), insect cell-derived expression vectors (e.g., the "Bac-to-BAC baculovirus expression system" (Gibco BRL), and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., the "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608 and pKTH50).

[0232] When the objective is expression in animal cells such as CHO cells, COS cells, or NIH3T3 cells, it is essential that the vector contain a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), the MMLV-LTR promoter, the EF1 alpha promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), or the CMV promoter. It is even more preferable if the vector contains a gene for selecting for transformation of cells (e.g., a drug resistance gene that can be detected by a drug (neomycin, G418, etc.)). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0233] Host cells are not particularly limited as long as they are suitable for expressing recombinant proteins. In addition to the aforementioned Escherichia coli, for example, yeast, various animal and plant cells, insect cells, etc. can be used. Various methods known to those skilled in the art can be used to introduce vectors into host cells. For example, introduction into Escherichia coli can be achieved using a calcium ion-based introduction method (Mandel, M., Higa, A. (1970) Journal of Molecular Biology, 53, 158-162; Hanahan, D. (1983) Journal of Molecular Biology, 166, 557-580). The modified polypeptide complex expressed in the host cells can be purified and recovered from the host cells, their cell cultures, or culture supernatants by methods known to those skilled in the art. When the polypeptide complex is expressed as a fusion protein with the aforementioned maltose-binding protein, affinity purification can be easily performed.

[0234] Suitable host cells for cloning or expressing vectors encoding the polypeptide complexes of the present invention include prokaryotic or eukaryotic cells as described herein. For example, polypeptide complexes may be produced in bacteria, particularly if glycosylation and Fc effector function are not required. For expression of polypeptide complexes and fragments thereof in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of fragments in E. coli). After expression, the polypeptide complexes may be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0235] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptide complexes with partial or fully human glycosylation patterns, may also be used as suitable cloning or expression hosts for vectors encoding polypeptide complexes. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0236] Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated polypeptide complexes. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0237] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing polypeptide complexes in transgenic plants).

[0238] Vertebrate cells can also be used as hosts, for example, mammalian cell lines that have been adapted to grow in suspension are useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. In one embodiment, for a review of specific mammalian host cell lines suitable for producing antibodies that comprise polypeptide complexes, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0239] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included in the term "host cell" herein.

[0240] The polypeptide complexes of the present invention do not bind (or have low binding activity) to target antigens in the presence of plasma proteins (or in the presence of high concentrations of plasma proteins), but can bind (or have high binding activity) to target antigens in the absence of plasma proteins (or in the presence of low concentrations of plasma proteins). Therefore, the polypeptide complexes of the present invention can be used to detect and / or target antigen proteins in the absence of plasma proteins or in the presence of low concentrations of plasma proteins, such as the central nervous system (CNS). That is, in one aspect, the present invention relates to a composition for detecting and / or targeting an antigen in the central nervous system (CNS) of a subject, comprising a polypeptide complex of the present invention. The present invention also relates to a method for detecting and / or targeting an antigen in the central nervous system (CNS), comprising administering a polypeptide complex of the present invention to a subject. Furthermore, the present invention relates to a polypeptide complex for use in detecting and / or targeting an antigen in the central nervous system (CNS) of a subject. Furthermore, the present invention relates to the use of a polypeptide complex in the manufacture of a composition for detecting and / or targeting an antigen in the central nervous system (CNS) of a subject.

[0241] In one embodiment, "targeting an antigen" refers to inhibiting or promoting the function and / or activity of the antigen by binding of the polypeptide complex of the present invention to the antigen, or inhibiting or promoting the binding of a ligand to a receptor. "Targeting an antigen in the central nervous system (CNS) of a subject" includes the polypeptide complex of the present invention passing through the blood-brain barrier (BBB) ​​of the subject to reach the CNS and binding to an antigen present in the CNS, thereby exerting the above-mentioned effect.

[0242] The route of administration of the polypeptide complex of the present invention is not particularly limited, but parenteral administration is preferred. For example, the polypeptide complex of the present invention can be administered in the form of an injection, intranasal administration, pulmonary administration, or subcutaneous administration. Alternatively, the polypeptide complex of the present invention can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0243] Method for producing polypeptide complexes In one aspect, the invention provides a method for producing a polypeptide complex of the invention, comprising culturing a host cell containing nucleic acid encoding the polypeptide complex under conditions suitable for expression of the polypeptide complex, and optionally recovering the polypeptide complex from the host cell or its culture medium.

[0244] When producing the polypeptide complex of the present invention, typically, a nucleic acid encoding the polypeptide complex of the present invention is inserted into an appropriate expression vector, the vector is introduced into appropriate cells, the transformed cells are cultured, and the expressed polypeptide complex is isolated and purified.

[0245] Expression vectors and host cells known to those skilled in the art can be used, but in one embodiment, those described herein may be used. Regarding culture, when animal cells are used as hosts, culture media such as DMEM, MEM, RPMI1640, and IMDM can be used, and these can be suitably used in combination with serum supplements such as FBS or fetal calf serum (FCS). Cells can also be cultured in a serum-free medium.

[0246] The polypeptide complex produced by culturing host cells containing a nucleic acid encoding the polypeptide complex of the present invention under conditions suitable for its expression can be isolated from the host cells or from the outside of the host cells (culture medium, milk, etc.) and purified as a substantially pure and homogeneous polypeptide complex. Isolation and purification of the polypeptide complex can be performed using isolation / purification methods commonly used for polypeptide purification. Examples of suitable methods include, but are not limited to, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, but is not limited to, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. These chromatographies can be performed using liquid chromatography, such as HPLC and FPLC. Columns used for affinity chromatography include, but are not limited to, Protein A and Protein G columns. Protein A columns include, but are not limited to, Hyper D, POROS, and Sepharose FF (Pharmacia).

[0247] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) the binding activity to the target antigen is lower in the presence of plasma proteins compared to in the absence of plasma proteins; Regarding the method.

[0248] Such polypeptide complexes can be obtained, for example, by measuring the binding activity of the polypeptide complex to a target antigen both in the presence and absence of plasma proteins, and selecting polypeptide complexes that have lower binding activity to a target antigen in the presence of plasma proteins compared to in the absence of plasma proteins.

[0249] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to the target antigen both in the presence and absence of plasma proteins; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of the plasma protein compared to in the absence of the plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex both in the presence and absence of plasma proteins.

[0250] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) the binding activity to the target antigen is different between in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration; Regarding the method. Here, "first concentration" and "second concentration" are as defined herein.

[0251] Such polypeptide complexes can be obtained, for example, by measuring the binding activity of the polypeptide complex to a target antigen in both the presence of a plasma protein at a first concentration and the presence of a plasma protein at a second concentration, and selecting polypeptide complexes whose binding activity to the target antigen differs between the presence of the plasma protein at the first concentration and the presence of the plasma protein at the second concentration.

[0252] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of a plasma protein at a first concentration and the presence of a plasma protein at a second concentration; (b) selecting a polypeptide complex that has a different binding activity to a target antigen in the presence of a plasma protein at a first concentration and in the presence of a plasma protein at a second concentration; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex both in the presence of the plasma protein at the first concentration and in the presence of the plasma protein at the second concentration.

[0253] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) lower binding activity to the target antigen in human plasma samples compared to the absence of plasma proteins; Regarding the method.

[0254] Such a polypeptide complex can be obtained, for example, by measuring the binding activity of the polypeptide complex to a target antigen both in a human plasma sample and in the absence of human plasma proteins, and selecting a polypeptide complex that has lower binding activity to a target antigen in a human plasma sample compared to in the absence of plasma proteins.

[0255] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to the target antigen both in a human plasma sample and in the absence of human plasma proteins; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to in the absence of plasma proteins; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex both in a human plasma sample and in the absence of human plasma proteins.

[0256] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) lower binding activity to the target antigen in human plasma samples compared to human cerebrospinal fluid (CSF) samples; Regarding the method.

[0257] Such a polypeptide complex can be obtained, for example, by measuring the binding activity of the polypeptide complex to a target antigen in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample, and selecting a polypeptide complex that has lower binding activity to the target antigen in the human plasma sample compared to the human cerebrospinal fluid (CSF) sample.

[0258] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to a target antigen in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample; (b) selecting a polypeptide complex that has a lower binding activity to a target antigen in a human plasma sample compared to a human cerebrospinal fluid (CSF) sample; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample.

[0259] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein; Regarding the method. Preferably, the polypeptide complex has lower binding activity to the target antigen in the presence of 50 mg / mL of plasma protein compared to the presence of 0.25 mg / mL of plasma protein.

[0260] Such polypeptide complexes can be obtained, for example, by measuring the binding activity of the polypeptide complex to a target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein, and selecting polypeptide complexes whose binding activity to a target antigen is lower in the presence of 50 mg / mL of plasma protein compared to that in the presence of 0.25 mg / mL of plasma protein. Preferably, such a polypeptide complex can be obtained by, for example, measuring the binding activity of the polypeptide complex to a target antigen in the presence of both 50 mg / mL and 0.25 mg / mL of plasma protein, and selecting a polypeptide complex whose binding activity to a target antigen is lower in the presence of 50 mg / mL of plasma protein than in the presence of 0.25 mg / mL of plasma protein.

[0261] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (b) selecting polypeptide complexes that have lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first and second antigen-binding moieties are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may further comprise, prior to step (a), contacting the target antigen with the polypeptide complex in the presence of both about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex both in the presence of 50 mg / mL of plasma protein and in the presence of 0.25 mg / mL of plasma protein.

[0262] In one aspect, the present invention provides a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising the steps of: culturing a cell containing a polynucleotide encoding the polypeptide complex; wherein the polypeptide complex is (a) comprising a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues; and (b) the KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; Regarding the method.

[0263] The binding activity of such polypeptide complexes to a target antigen is measured in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein, and the binding activity of the polypeptide complexes to a target antigen is measured in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein. The KD value for the target antigen in the presence of plasma protein at about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, is 5 times or more, 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more, or 35 times or more.

[0264] Therefore, in one aspect, the present invention relates to a method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, the method comprising the steps of: (a) measuring the binding activity of the polypeptide complex to the target antigen in both the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (b) selecting polypeptide complexes that have a KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c). The polypeptide complex comprises a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, and the first and second antigen-binding moieties are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. The method of the present invention may further comprise, prior to step (a), contacting the target antigen with the polypeptide complex in the presence of both about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL of plasma protein and about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL of plasma protein. The method of the present invention may include, prior to step (a), a step of contacting the target antigen with the polypeptide complex both in the presence of 50 mg / ml of plasma protein and in the presence of 0.25 mg / ml of plasma protein.

[0265] In the above-mentioned method, the non-cleavable linker preferably consists of 0 to 3 amino acid residues, more preferably 0 to 1. Note that a "non-cleavable linker consisting of 0 amino acid residues" means that it does not contain a linker at all, i.e., the first antigen-binding portion and the second antigen-binding portion are linked without a linker.

[0266] In the production method of the present invention, the plasma protein is preferably albumin, and the albumin is preferably human albumin. Furthermore, the target antigen is preferably a protein that is not a plasma protein. The specific binding activity of the polypeptide complex to a plasma protein and the binding activity to the target antigen can be measured by methods known to those skilled in the art, and in one embodiment, can be measured by the method described herein. Furthermore, the polynucleotide encoding the polypeptide complex can be obtained by determining the nucleotide sequence by methods known to those skilled in the art.

[0267] Method for screening polypeptide complexes The present invention also relates to a method for screening for a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein. The polypeptide complex of the present invention comprises a first antigen-binding portion capable of specifically binding to a plasma protein and a second antigen-binding portion capable of binding to a target antigen, wherein the first antigen-binding portion and the second antigen-binding portion are linked via a non-cleavable linker consisting of 0 to 4 amino acid residues. Here, the non-cleavable linker preferably consists of 0 to 3 amino acid residues, and more preferably 0 to 1 amino acid residue. Note that a "non-cleavable linker consisting of 0 amino acid residues" means that no linker is included, i.e., the first antigen-binding portion and the second antigen-binding portion are linked without a linker.

[0268] In one embodiment, the screening method of the present invention comprises: (a) contacting a target antigen with a polypeptide complex in both the presence and absence of plasma proteins; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence and absence of plasma proteins; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of the plasma protein compared to in the absence of the plasma protein; Includes.

[0269] In another embodiment, the screening method of the present invention comprises: (a) contacting a target antigen with a polypeptide complex in the presence of both a first concentration of plasma protein and a second concentration of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration, respectively; and (c) selecting a polypeptide complex that has a different binding activity to the target antigen in the presence of the plasma protein at a first concentration and in the presence of the plasma protein at a second concentration; Includes. Here, "first concentration" and "second concentration" are as defined herein.

[0270] In another embodiment, the screening method of the present invention comprises: (a) contacting a target antigen with a polypeptide complex both in a human plasma sample and in the absence of human plasma proteins; (b) measuring the binding activity of the polypeptide complex to the target antigen in a human plasma sample and in the absence of human plasma proteins, respectively; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to in the absence of plasma proteins; Includes.

[0271] In another embodiment, the screening method of the present invention comprises: (a) contacting a target antigen with a polypeptide complex in a human plasma sample and in a human cerebrospinal fluid (CSF) sample, respectively; (b) measuring the binding activity of the polypeptide complex to the target antigen in each of a human plasma sample and a human cerebrospinal fluid (CSF) sample; and (c) selecting a polypeptide complex that has a lower binding activity to the target antigen in a human plasma sample compared to a human cerebrospinal fluid (CSF) sample; Includes.

[0272] In another embodiment, the screening method of the present invention comprises: (a) contacting the target antigen with the polypeptide complex both in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein, respectively; and (c) selecting polypeptide complexes that have lower binding activity to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein compared to about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; Includes.

[0273] In another embodiment, the screening method of the present invention comprises: (a) contacting the target antigen with the polypeptide complex both in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; (b) measuring the binding activity of the polypeptide complex to the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein and in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein, respectively; and (c) selecting polypeptide complexes that have a KD value for the target antigen in the presence of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg / mL, preferably 50 mg / mL, of plasma protein that is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold greater than the KD value for the target antigen in the presence of about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / mL, preferably 0.25 mg / mL, of plasma protein; Includes.

[0274] The screening method of the present invention comprises a step of contacting a target antigen with a polypeptide complex. Contact can be carried out, for example, by adding the target antigen and polypeptide complex to a sample containing a specific concentration of plasma protein (e.g., a sample containing plasma protein at a concentration described herein), a sample not containing plasma protein, a plasma sample derived from various animals such as humans, mice, rats, hamsters, rabbits, and monkeys, or a cerebrospinal fluid (CSF) sample. Alternatively, the target antigen and the polypeptide complex may be added to a sample containing either the target antigen or the polypeptide complex, while the other may be added to the sample.

[0275] In the screening method of the present invention, the plasma protein is preferably albumin, and the albumin is preferably human albumin, and the target antigen is preferably a protein that is not a plasma protein.

[0276] The screening method of the present invention can further comprise, after step (c), the step (d) of selecting a polypeptide complex that exhibits specific binding activity to a plasma protein.

[0277] The specific binding activity to plasma proteins and the binding activity to target antigens can be measured by methods well known to those skilled in the art, including, but not limited to, Gyrolab xP (Gyros Protein Technologies), ELISA, Biacore (GE healthcare), Scatchard plot, flow cytometer, etc.

[0278] In the screening method of the present invention, each of steps (a) to (d) may be repeated two or more times, and the cycle of steps (a) to (c) and the cycle of steps (a) to (d) may also be repeated two or more times.

[0279] The first antigen-binding portion capable of specifically binding to a plasma protein and the second antigen-binding portion capable of binding to a target antigen that constitute the polypeptide complex to be screened by the screening method of the present invention are not particularly limited. They may have natural sequences or may have amino acid sequence substitutions.

[0280] All prior art documents cited in this specification are hereby incorporated by reference. [Example]

[0281] Example 1: Concept of antigen binding control using plasma protein binding moieties Antigen-binding molecules, including antibodies, are known to have a low brain penetration rate of 0.1% or less (St-Amour I, et. al., Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier. J Cereb Blood Flow Metab. 2013 Dec;33(12):1983-92). Therefore, antigen-binding molecules administered to a subject are present at high concentrations in plasma and at low concentrations in the central nervous system (CNS), including the brain. To achieve the concentration required for therapeutic efficacy in the CNS, a large amount of antigen-binding molecules must be administered to the subject. As a result, the concentration of antigen-binding molecules present in the plasma and throughout the body becomes excessive, leading to binding to target antigens in tissues other than the CNS and resulting in side effects. For example, the TNF inhibitor infliximab is used to treat neuro-Behcet's disease, but because it suppresses the immune response throughout the body, it can lead to a relapse of tuberculosis and the discontinuation of administration (Horiguchi, N., et. al., A 10-year follow-up of infliximab monotherapy for refractory uveitis in Behcet's syndrome. Sci Rep 10, 22227 (2020)).

[0282] If we could create a molecule that binds to a target antigen in the CNS but has reduced binding to the target antigen in tissues other than the CNS (non-CNS), we cou...

Claims

1. 1. A polypeptide complex comprising a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, the target antigen is not a plasma protein; the first antigen-binding portion and the second antigen-binding portion are linked without a linker; Polypeptide complexes.

2. 1. A polypeptide complex comprising a first antigen-binding moiety capable of specifically binding to a plasma protein and a second antigen-binding moiety capable of binding to a target antigen, the target antigen is not a plasma protein; the first antigen-binding moiety and the second antigen-binding moiety are linked via a non-cleavable linker; Polypeptide complexes.

3. The polypeptide complex of claim 2, wherein the non-cleavable linker is a peptide of four amino acid residues or less.

4. 4. The polypeptide complex of claim 1, which does not bind to the target antigen when bound to the plasma protein.

5. The polypeptide complex according to any one of claims 1 to 3, wherein the binding activity to the target antigen in the presence of the plasma protein is lower than the binding activity to the target antigen in the absence of the plasma protein.

6. 6. The polypeptide complex of claim 1, wherein the plasma protein is albumin, preferably human albumin.

7. 7. The polypeptide complex of claim 1, wherein the second antigen-binding portion comprises a Fab or scFv.

8. A polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a heavy chain variable region (VH), and a CH1 domain (CH1) of a heavy chain constant region; the second polypeptide chain comprises, in order from the N-terminus, a plasma protein binding portion, a light chain variable region (VL), and a light chain constant region (CL); the VH and the VL form a binding moiety for a target antigen; the target antigen is not a plasma protein; Polypeptide complexes.

9. The polypeptide complex of claim 8, wherein the N-terminal amino acid of the VH and / or the VL and the C-terminal amino acid of the plasma protein-binding portion are linked without a linker.

10. 9. The polypeptide complex of claim 8, wherein the N-terminal amino acid of the VH and / or the VL and the C-terminal amino acid of the plasma protein binding moiety are linked via a non-cleavable linker.

11. A pharmaceutical composition comprising the polypeptide complex of any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. 11. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 1 to 10, for targeting an antigen in the central nervous system (CNS) and for systemic administration to a subject.

13. 13. The pharmaceutical composition of claim 12, wherein the systemic administration is intravenous or subcutaneous.

14. 1. A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: (a) measuring the binding activity of the polypeptide complex to the target antigen both in the presence and absence of the plasma protein; (b) selecting a polypeptide complex that has a lower binding activity to the target antigen in the presence of the plasma protein compared to in the absence of the plasma protein; (c) obtaining a polynucleotide encoding the polypeptide complex selected in (b); and (d) culturing cells containing the polynucleotide obtained in (c); Including, the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to the plasma protein and a second antigen-binding portion capable of binding to the target antigen; method.

15. 1. A method for producing a polypeptide complex whose binding activity to a target antigen changes depending on the concentration of a plasma protein, comprising: culturing a cell containing a polynucleotide encoding the polypeptide complex; (a) the polypeptide complex comprises a first antigen-binding portion capable of specifically binding to the plasma protein and a second antigen-binding portion capable of binding to the target antigen; and (b) the binding activity of the polypeptide complex to the target antigen is lower in the presence of the plasma protein than in the absence of the plasma protein; method.

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