One-armed antibody targeting kidney and not forming s-s bond in hinge region

Abstract

One aspect of the present disclosure provides an antigen-binding molecule comprising a dimeric Fc region and an antigen-binding domain, wherein: either or both of a first polypeptide chain comprising one monomer of the Fc region and a second polypeptide chain comprising another monomer of the Fc region do not include an antibody hinge region capable of forming a disulfide bond; and the antigen-binding domain is linked only to the first polypeptide chain, and the antigen-binding domain is not linked to the second polypeptide chain. Antigen binding molecules of this format have high glomerular permeability and can accumulate in the kidney.

Description

A one-armed antibody that targets the kidney and does not form S-S bonds in the hinge region.

[0001] This disclosure relates to antigen-binding molecules capable of targeting the kidney. In one embodiment, this disclosure relates to antigen-binding molecules that have high glomerular permeability and can accumulate in the kidney.

[0002] In the kidney, conventional bivalent IgG antibodies (molecular weight approximately 150 kDa) are blocked by a size barrier (molecules larger than approximately 70 kDa cannot penetrate the glomerulus) and a charge barrier (molecules with low pI, i.e., negative charge, cannot penetrate the glomerulus), and therefore cannot permeate the glomerulus. On the other hand, antibody fragments with smaller molecular weights, such as Fab', Fab, and scFv (molecules approximately 55 kDa, 50 kDa, and 28 kDa, respectively), are known to be able to permeate the glomerulus. For example, Patent Document 1 discloses that a bispecific Fab-scFv fusion protein with a smaller molecular weight than conventional bivalent IgG antibodies is filtered by kidney. However, because these small molecular weight antibody fragments do not contain an Fc region in their structure, it has been difficult to adjust the pharmacological properties and antibody functions of the antibody in vivo, such as extending the blood half-life, exerting antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC) functions via the Fc region, and adding sweeping function (Patent Document 2).

[0003] Despite the usefulness of antibodies containing the Fc region, conventional bivalent IgG antibodies containing a large molecular weight Fc region cannot penetrate the glomerulus due to the aforementioned size barrier and other problems. Antibodies that cannot penetrate the glomerulus cannot access the tissue beyond the glomerulus and are returned to the circulatory system by the efferent arteriole, and therefore cannot accumulate in the kidney tissue itself. Thus, targeting the kidney with conventional bivalent IgG antibodies containing the Fc region has been technically difficult.

[0004] Patent document 3 discloses a technique for tumor-specific accumulation of anti-DOTAM antibodies, using Fc region-containing antibodies in various formats. This document shows that a small amount of Pb-DOTAM that was not trapped in the tumor was detected in the kidney, but renal accumulation of the antibody itself was not detected.

[0005] In Non-Patent Document 1, Rafidi H et al. disclose that one-armed IgG antibodies with an Fc region undergo glomerular filtration, are reuptaken in the proximal tubule, and distributed to the kidney. This document shows that, among one-armed IgG antibodies, antibodies in which the hinge region is replaced with an IgA2 hinge called a "rigid hinge" accumulate in large quantities in the kidney, and concludes that by fixing one-armed IgG antibodies to a rigid conformation, they acquire the property of allowing antibodies with large molecular weights to permeate the glomeruli.

[0006] US 7052872 B1WO 2016 / 098357 A1WO 2022152656 A1

[0007] Mol Cancer Ther. 2021 Oct;20(10):2008-2015

[0008] This invention has been made in view of the above circumstances, and one of its objectives is to clarify the technical characteristics of an antibody that can permeate the glomeruli and / or accumulate in the kidney despite containing an Fc region.

[0009] The inventors, through diligent research, discovered that, contrary to the aforementioned known conditions, monovalent antibodies (Ab_4 in Figure 1) fixed in a rigid conformation (with only one of the Fab heavy chain and Fab light chain linked to each of the two Fc region monomers without a hinge region) had low concentrations in the kidneys. On the other hand, the inventors discovered that one-armed IgG antibodies in which the antigen-binding domain is linked to only one of the Fc regions of the dimer, and which do not contain an antibody hinge region capable of forming a disulfide bond on either or both of the polypeptide chains containing the Fc region (for example, Ab_6, Ab_7, and Ab_8 in Figure 1), had high concentrations in the kidneys. Furthermore, the inventors found that, as long as these molecular shape conditions are met, one-armed IgG antibodies can efficiently permeate the glomeruli and accumulate in the kidneys even without having an IgA2 hinge called a rigid hinge.

[0010] The present invention is based on such findings and specifically includes the embodiments described below as illustrative examples. [A1] An antigen-binding molecule comprising a dimeric Fc region and an antigen-binding domain, wherein either or both of a first polypeptide chain comprising one monomer of the Fc region and a second polypeptide chain comprising the other monomer of the Fc region do not contain an antibody hinge region capable of forming a disulfide bond, and the antigen-binding domain is linked only to the first polypeptide chain and not to the second polypeptide chain. [A2] The antigen-binding molecule according to [A1], wherein only one antigen-binding domain is linked to the first polypeptide chain. [A3] The antigen-binding molecule according to [A1] or [A2], wherein the second polypeptide chain comprises only the Fc region, or, in order from the N-terminus, only the linker region and the Fc region. [A4] An antigen-binding molecule according to any one of [A1] to [A3], wherein the first polypeptide chain comprises a first linker region and the second polypeptide chain comprises a second linker region, and in the antigen-binding molecule, a disulfide bond cannot be formed between the first linker region and the second linker region. [A5] An antigen-binding molecule according to [A4], wherein the C-terminus of the first linker region is linked to the N-terminus of an Fc region contained in the first polypeptide chain. [A6] An antigen-binding molecule according to [A4] or [A5], wherein the C-terminus of the second linker region is linked to the N-terminus of an Fc region contained in the second polypeptide chain. [A7] An antigen-binding molecule according to any one of [A4] to [A6], wherein the amino acid sequence of the first linker region and the amino acid sequence of the second linker region are the same. [A8] An antigen-binding molecule according to any one of [A4] to [A7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG. [A9] The antigen-binding molecule according to any one of [A4] to [A8], wherein the first linker region and the second linker region are modified hinge regions in which one or more Cys amino acids of the hinge region of natural IgG are substituted with amino acids other than Cys.[A10] The antigen-binding molecule according to any one of [A4] to [A7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having an amino acid other than Cys at position 226 (EU index) and position 229 (EU index). [A11] The antigen-binding molecule according to any one of [A4] to [A7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having Ala or Ser at position 226 (EU index) and position 229 (EU index). [A12] The antigen-binding molecule according to [A10] or [A11], wherein the hinge region variant further has different amino acids at positions other than 226 (EU index) and 229 (EU index) compared to the native IgG hinge region of the corresponding isotype. [A13] The antigen-binding molecule according to [A10] to [A12], wherein the hinge region variant of IgG2 is a modified hinge region further having amino acids other than Cys at positions 219 and 220 (EU index). [A14] The sequence of the first linker region and the second linker region is as follows: (1) EPKSCDKTHTAPPAP (Sequence ID: 19); (2) EPKSCDKTHTSPPSP (Sequence ID: 20); (3) EPKSCDKTHTAPPSP (Sequence ID: 21); (4) EPKSCDKTHTSPPAP (Sequence ID: 22); (5) DKTHTAPPAP (Sequence ID: 24); (6) DKTHTSPPSP (Sequence ID: 25); (7) DKTHTAPPSP (Sequence ID: 26); (8) DKTHTSPPAP (Sequence ID: 27); (9) ERKCCVEAPPAP (Sequence ID: 29); (10) ERKCCVESPPSP (Sequence ID: 30); (11) ERKAAVEAPPAP (Sequence ID: 31); (12) ERKSSVEAPPAP (Sequence ID: 32); (13) ERKASVEAPPAP (SEQ ID NO: 33); (14) ERKSAVEAPPAP (SEQ ID NO: 34); (15)An antigen-binding molecule according to any one of [A4] to [A8], which is one of the following: (16) ERKAAVESPPSP (SEQ ID NO: 35); (17) ERKASVESPPSP (SEQ ID NO: 37); (18) ERKSAVESPPSP (SEQ ID NO: 38); (19) ESKYGPPAPPAP (SEQ ID NO: 40); (20) ESKYGPPAPSAP (SEQ ID NO: 41); (21) ESKYGPPSPSSP (SEQ ID NO: 42); (22) ESKYGPPSPPSP (SEQ ID NO: 43); (23) ESKYGPPAPPSP (SEQ ID NO: 45); (24) ESKYGPPSPPAP (SEQ ID NO: 46). [A15] An antigen-binding molecule according to any one of [A4] to [A7], wherein the sequence of the first linker region and the second linker region is not VPPPPP (SEQ ID NO: 49). [A16] The antigen-binding molecule according to [A4], wherein the sequence of one of the first and second linker regions is EPPPPP (SEQ ID NO: 50) and the sequence of the other is GPPPPP (SEQ ID NO: 51). [A17] The antigen-binding molecule according to any one of [A4] to [A7], wherein the sequences of the first and second linker regions are: (1) a glycine polymer; (2) a glycine-serine polymer; (3) a glycine-alanine polymer; (4) an alanine-serine polymer; or (5) a synthetic compound linker. [A18] The antigen-binding molecule according to any one of [A1] to [A3], wherein only one of the first and second polypeptide chains includes a linker region linked to the N-terminus of an antibody CH2 domain. [A19] The antigen-binding molecule according to [A18], wherein the linker region is a natural antibody hinge region or a variant thereof. [A20] The sequence of the linker region is as follows: (1) EPKSCDKTHTCPPCP (Sequence ID: 18); (2) EPKSCDKTHTAPPAP (Sequence ID: 19); (3) EPKSCDKTHTSPPSP (Sequence ID: 20); (4) EPKSCDKTHTAPPSP (Sequence ID: 21); (5) EPKSCDKTHTSPPAP (Sequence ID: 22); (6) DKTHTCPPCP (Sequence ID: 23); (7)(8) DKTHTAPPAP (Sequence ID: 24); (9) DKTHTSPPSP (Sequence ID: 25); (10) DKTHTAPPSP (Sequence ID: 26); (11) DKTHTSPPAP (Sequence ID: 27); (12) ERKCCVECPPCP (Sequence ID: 28); (13) ERKCCVEAPPAP (Sequence ID: 29); (14) ERKCCVESPPSP (Sequence ID: 30); (15) ERKAAVEAPPAP (Sequence ID: 31); (16) ERKSSVEAPPAP (Sequence ID: 32); (17) ERKASVEAPPAP (Sequence ID: 33); (18) ERKSAVEAPPAP (Sequence ID: 34); (19) ERKAAVESPPSP (Sequence ID: 35); (16) ERKSSVESPPSP (Sequence ID: 36); (20) ERKASVESPPSP (Sequence ID: 37); (21) (22) ERKSAVESPPSP (SEQ ID NO: 38); (23) ESKYGPPCPSCP (SEQ ID NO: 39); (24) ESKYGPPAPPAP (SEQ ID NO: 40); (25) ESKYGPPAPSAP (SEQ ID NO: 41); (26) ESKYGPPSPSSP (SEQ ID NO: 42); (27) ESKYGPPSPPSP (SEQ ID NO: 43); (28) ESKYGPPCPPCP (SEQ ID NO: 44); (29) ESKYGPPAPPSP (SEQ ID NO: 45); (30) ESKYGPPSPPAP (SEQ ID NO: 46); (31) EPPPCP (SEQ ID NO: 47); (32) GPPPCP (SEQ ID NO: 48); (33) VPPPPP (SEQ ID NO: 49); (34) EPPPPP (SEQ ID NO: 50); (35) GPPPPP (SEQ ID NO: 51); (36) Glycine polymer; An antigen-binding molecule according to [A18], which is one of the following: (37) a glycine-serine polymer; (38) an alanine-serine polymer; or (39) a synthetic compound linker. [A21] An antigen-binding molecule according to [A1] or [A2], wherein the antigen-binding domain is linked to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker, and the second polypeptide chain comprises only the Fc region.[A22] The antigen-binding molecule according to any one of [A1] to [A21], wherein the molecular weight of the antigen-binding domain is 50 kDa or less. [A23] The antigen-binding molecule according to any one of [A1] to [A22], wherein the antigen-binding domain is Fab, scFv, or sdAb. [A24] The antigen-binding molecule according to any one of [A1] to [A23], wherein the Fc region contained in the first polypeptide chain and the Fc region contained in the second polypeptide chain have different sequences. [A25] The antigen-binding molecule according to any one of [A1] to [A24], wherein the Fc region is the Fc region of natural IgG or a variant thereof. [A26] The antigen-binding molecule according to any one of [A1] to [A24], wherein the Fc region is the Fc region of natural IgG or a variant thereof and does not have a glycosylation sequence.

[0011] [B1] A polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains contains an Fc region, and either or both of the first and second polypeptide chains do not contain an antibody hinge region capable of forming a disulfide bond, and a protein-binding molecule with a molecular weight of 50 kDa or less is linked only to the first polypeptide chain, while the second polypeptide chain is not linked to the protein-binding molecule. [B2] The polypeptide complex according to [B1], wherein the protein-binding molecule is Fab, scFv, sdAb, or a ligand. [B3] The polypeptide complex according to [B1] or [B2], wherein the second polypeptide chain contains only an Fc region, or contains only a linker region and an Fc region in that order from the N-terminus. [B4] The polypeptide complex according to any one of [B1] to [B3], wherein the first polypeptide chain comprises a first linker region and the second polypeptide chain comprises a second linker region, and in the polypeptide complex, a disulfide bond cannot be formed between the first linker region and the second linker region. [B5] The polypeptide complex according to [B4], wherein the C-terminus of the first linker region is linked to the N-terminus of an Fc region contained in the first polypeptide chain. [B6] The polypeptide complex according to [B4] or [B5], wherein the C-terminus of the second linker region is linked to the N-terminus of an Fc region contained in the second polypeptide chain. [B7] The polypeptide complex according to any one of [B4] to [B6], wherein the amino acid sequence of the first linker region and the amino acid sequence of the second linker region are the same. [B8] The polypeptide complex according to any one of [B4] to [B7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG. [B9] The polypeptide complex according to any one of [B4] to [B8], wherein the first linker region and the second linker region are modified hinge regions in which one or more Cys amino acids of the hinge region of natural IgG are substituted with amino acids other than Cys.[B10] The polypeptide complex according to any one of [B4] to [B7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having an amino acid other than Cys at position 226 (EU index) and position 229 (EU index). [B11] The polypeptide complex according to any one of [B4] to [B7], wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having Ala or Ser at position 226 (EU index) and position 229 (EU index). [B12] The polypeptide complex according to [B10] or [B11], wherein the variant of the hinge region further has different amino acids at positions other than 226 (EU index) and 229 (EU index) compared to the native IgG hinge region of the corresponding isotype. [B13] The polypeptide complex according to [B10] to [B12], wherein the variant of the hinge region of IgG2 is a modified hinge region further having amino acids other than Cys at positions 219 and 220 (EU index). [B14] The sequence of the first linker region and the second linker region is as follows: (1) EPKSCDKTHTAPPAP (Sequence ID: 19); (2) EPKSCDKTHTSPPSP (Sequence ID: 20); (3) EPKSCDKTHTAPPSP (Sequence ID: 21); (4) EPKSCDKTHTSPPAP (Sequence ID: 22); (5) DKTHTAPPAP (Sequence ID: 24); (6) DKTHTSPPSP (Sequence ID: 25); (7) DKTHTAPPSP (Sequence ID: 26); (8) DKTHTSPPAP (Sequence ID: 27); (9) ERKCCVEAPPAP (Sequence ID: 29); (10) ERKCCVESPPSP (Sequence ID: 30); (11) ERKAAVEAPPAP (Sequence ID: 31); (12) ERKSSVEAPPAP (Sequence ID: 32); (13) ERKASVEAPPAP (Sequence ID: 33); (14)A polypeptide complex described in any one of [B4] to [B8], which is one of the following: (15) ERKSAVEAPPAP (SEQ ID NO: 34); (16) ERKAAVESPPSP (SEQ ID NO: 35); (17) ERKASVESPPSP (SEQ ID NO: 37); (18) ERKSAVESPPSP (SEQ ID NO: 38); (19) ESKYGPPAPPAP (SEQ ID NO: 40); (20) ESKYGPPAPSAP (SEQ ID NO: 41); (21) ESKYGPPSPSSP (SEQ ID NO: 42); (22) ESKYGPPSPPSP (SEQ ID NO: 43); (23) ESKYGPPAPPSP (SEQ ID NO: 45); (24) ESKYGPPSPPAP (SEQ ID NO: 46). [B15] The polypeptide complex according to any one of [B4] to [B7], wherein the sequences of the first linker region and the second linker region are not VPPPPP (SEQ ID NO: 49). [B16] The polypeptide complex according to [B4], wherein the sequence of one of the first linker region and the second linker region is EPPPPP (SEQ ID NO: 50) and the sequence of the other is GPPPPP (SEQ ID NO: 51). [B17] The polypeptide complex according to any one of [B4] to [B7], wherein the sequences of the first linker region and the second linker region are any of the following: (1) glycine polymer; (2) glycine-serine polymer; (3) glycine-alanine polymer; (4) alanine-serine polymer; (5) synthetic compound linker. [B18] The polypeptide complex according to any one of [B1] to [B3], wherein only one of the first polypeptide chain and the second polypeptide chain includes a linker region linked to the N-terminus of an antibody CH2 domain. [B19] The linker region is a natural antibody hinge region or a variant thereof, according to the polypeptide complex described in [B18]. [B20] The sequences of the linker region are: (1) EPKSCDKTHTCPPCP (SEQ ID NO: 18); (2) EPKSCDKTHTAPPAP (SEQ ID NO: 19); (3) EPKSCDKTHTSPPSP (SEQ ID NO: 20); (4) EPKSCDKTHTAPPSP (SEQ ID NO: 21); (5)(6) EPKSCDKTHTSPPAP (Sequence ID: 22); (7) DKTHTCPPCP (Sequence ID: 23); (8) DKTHTAPPAP (Sequence ID: 24); (9) DKTHTSPPSP (Sequence ID: 25); (10) DKTHTAPPSP (Sequence ID: 26); (11) DKTHTSPPAP (Sequence ID: 27); (12) ERKCCVECPPCP (Sequence ID: 28); (13) ERKCCVEAPPAP (Sequence ID: 29); (14) ERKCCVESPPSP (Sequence ID: 30); (15) ERKAAVEAPPAP (Sequence ID: 31); (16) ERKSSVEAPPAP (Sequence ID: 32); (17) ERKASVEAPPAP (Sequence ID: 33); (18) ERKSAVEAPPAP (Sequence ID: 34); (19) ERKAAVESPPSP (Sequence ID: 35); ERKSSVESPPSP (Sequence ID: 36); (20) ERKASVESPPSP (Sequence ID: 37); (21) ERKSAVESPPSP (Sequence ID: 38); (22) ESKYGPPCPSCP (Sequence ID: 39); (23) ESKYGPPAPPAP (Sequence ID: 40); (24) ESKYGPPAPSAP (Sequence ID: 41); (25) ESKYGPPSPSSP (Sequence ID: 42); (26) ESKYGPPSPPSP (Sequence ID: 43); (27) ESKYGPPCPPCP (Sequence ID: 44); (28) ESKYGPPAPPSP (Sequence ID: 45); (29) ESKYGPPSPPAP (Sequence ID: 46); (30) EPPPCP (Sequence ID: 47); (31) GPPPCP (Sequence ID: 48); (32) VPPPPP (Sequence ID: 49); (33) The polypeptide complex described in [B18] is any of the following: (34) EPPPPP (SEQ ID NO: 50); (35) glycine polymer; (36) glycine-serine polymer; (37) glycine-alanine polymer; (38) alanine-serine polymer; or (39) synthetic compound linker.[B21] The polypeptide complex according to [B1] or [B2], wherein the protein-binding molecule is linked to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker, and the second polypeptide chain contains only an Fc region. [B22] The polypeptide complex according to any one of [B1] to [B21], wherein the Fc region in the first polypeptide chain and the Fc region in the second polypeptide chain have different sequences. [B23] The polypeptide complex according to any one of [B1] to [B22], wherein the Fc region is the Fc region of natural IgG or a variant thereof. [B24] The polypeptide complex according to any one of [B1] to [B23], wherein the Fc region is the Fc region of natural IgG or a variant thereof and does not have a glycosylation sequence.

[0012] [C1] An antigen-binding molecule according to any one of [A1] to [A26], or a polypeptide complex according to any one of [B1] to [B24], conjugated with a cytotoxic substance. [C2] An antigen-binding molecule according to any one of [A1] to [A26], or a polypeptide complex according to any one of [B1] to [B24], conjugated with one or more nucleic acids, peptides (including cyclic peptides), and low molecular weight compounds. [C3] An antigen-binding molecule or polypeptide complex according to [C1] or [C2], wherein the molecular weight of the cytotoxic substance, nucleic acid, peptide (including cyclic peptides), or low molecular weight compound conjugated to the antigen-binding molecule or polypeptide complex is 6 kDa or less. [C4] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], which passes through the renal glomeruli and is taken up by proximal tubular cells, podocytes, distal tubular cells, or collecting duct cells. [C5] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], which accumulates in the kidney. [C6] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], which accumulates in the cells of the proximal tubule. [C7] An isolated nucleic acid encoding an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3]. [C8] A host cell containing the nucleic acid described in [C7]. [C9] A method for producing an antigen-binding molecule or polypeptide complex, comprising the step of culturing the host cell described in [C8] so that the antigen-binding molecule or polypeptide complex is produced. [C10] The method according to [C9], further comprising the step of recovering the antigen-binding molecule or the polypeptide complex from the host cell.

[0013] [D1] A pharmaceutical preparation comprising an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3] and a pharmaceutically acceptable carrier. [D2] A pharmaceutical composition comprising an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3] and a pharmaceutically acceptable carrier. [D3] A pharmaceutical composition for the treatment of kidney disease comprising an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3] and a pharmaceutically acceptable carrier. [D4] A composition for renal imaging comprising a fluorescently labeled or radiolabeled antigen-binding molecule according to any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex according to any one of [B1] to [B24] or [C1] to [C3]. [D5] A pharmaceutical composition for renal delivery comprising an antigen-binding molecule according to any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex according to any one of [B1] to [B24] or [C1] to [C3], and a pharmaceutically acceptable carrier. [D6] A method for treating a kidney disease comprising the step of administering an antigen-binding molecule according to any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex according to any one of [B1] to [B24] or [C1] to [C3]. [D7] A method for kidney imaging, comprising the step of administering a fluorescently labeled or radiolabeled antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3]. [D8] A method for delivering a cytotoxic substance, nucleic acid, peptide (including cyclic peptides), or small molecule compound to the kidney, comprising the step of administering an antigen-binding molecule or polypeptide complex described in any one of [C1] to [C3].[D9] Use of an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], in the manufacture of a pharmaceutical product. [D10] Use of an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], in the manufacture of a pharmaceutical product for the treatment of kidney disease. [D11] Use of an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], in the manufacture of a reagent for kidney imaging. [D12] Use of an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], in the manufacture of a pharmaceutical composition for kidney delivery. [D13] A kit for the treatment of kidney disease comprising an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], and a pharmaceutically acceptable carrier. [D14] A kit for kidney imaging comprising a fluorescently labeled or radiolabeled antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], and a pharmaceutically acceptable carrier. [D15] A kit for renal delivery comprising an antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], and a pharmaceutically acceptable carrier. [D16] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], for use as a pharmaceutical.[D17] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], for use in the treatment of kidney disease. [D18] A fluorescently labeled or radiolabeled antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], for use in kidney imaging. [D19] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], for use as a reagent for kidney delivery. [D20] An antigen-binding molecule described in any one of [A1] to [A26] or [C1] to [C3], or a polypeptide complex described in any one of [B1] to [B24] or [C1] to [C3], for accumulation in the kidney. [D21] An antigen-binding molecule or polypeptide complex described in any one of [C1] to [C3], for accumulation in the kidney of a cytotoxic substance, nucleic acid, peptide (including cyclic peptide), or low molecular weight compound. [D22] An antigen-binding molecule or polypeptide complex described in any one of [C1] to [C3], for accumulation in proximal tubular cells, podocytes, distal tubular cells, or collecting duct cells of a cytotoxic substance, nucleic acid, peptide (including cyclic peptide), or low molecular weight compound.

[0014] [E1] An antigen-binding molecule according to any one of [A1] to [A26], wherein the antigen-binding domain is Fab. [E2] An antigen-binding molecule according to any one of [A1] to [A26], wherein the antigen-binding domain is an antibody fragment in which either or both of the VL or VH chains in Fab are replaced with VHH. [E3] An antigen-binding molecule according to any one of [A1] to [A26], wherein the antigen-binding domain is VHH. [E4] A polypeptide complex according to any one of [B1] to [B24], wherein the protein-binding molecule is Fab. [E5] A polypeptide complex according to any one of [B1] to [B24], wherein the protein-binding molecule is an antibody fragment in which either or both of the VL or VH chains in Fab are replaced with VHH. [E6] A polypeptide complex according to any one of [B1] to [B24], wherein the protein-binding molecule is VHH.

[0015] This figure shows the molecular shapes of the antibodies produced in Example 1-1. The thick lines in the figure indicate disulfide bonds (also called SS bonds). Ab_1 and Ab_5 are two-arm format antibodies containing the hinge region of the IgG class, and Ab_5 does not form disulfide bonds in the hinge region. Ab_2 and Ab_6 are one-arm format antibodies containing the hinge region of IgG1, and Ab_6 does not form disulfide bonds in the hinge region. Ab_3 and Ab_7 are one-arm format antibodies containing the hinge region of IgA2, and Ab_7 does not form disulfide bonds in the hinge region. Ab_4 and Ab_8 are monovalent antibodies that do not contain the hinge region. In Ab_8, one Fab is linked to only one of the two monomeric Fc regions constituting the dimeric Fc region, whereas in Ab_4, either the Fab heavy chain or the Fab light chain constituting one Fab is linked to each of the two monomeric Fc regions constituting the dimeric Fc region. This figure shows the antibody concentration in mouse plasma calculated by ELISA. This figure shows the antibody concentration in mouse kidney calculated by fluorescence intensity. This figure shows that an antibody having only one antigen-binding domain and lacking inter-heavy-chain disulfide bonds in the hinge region can efficiently pass through glomerular filtration. This figure shows the molecular shape of the antibody produced in Example 2-1. The thick lines in the figure indicate disulfide bonds. The x marks in the figure indicate modifications that eliminate the glycosylation sequence. Ab_9 and Ab_10 are one-arm format antibodies containing the hinge region of IgG1, and Ab_10 does not form disulfide bonds in the hinge region. Ab_11 and Ab_12 are one-arm format antibodies containing the hinge region of IgG2, and Ab_12 does not form a disulfide bond in the hinge region. Ab_13 and Ab_14 are one-arm format antibodies containing the hinge region of IgG4, and Ab_14 does not form a disulfide bond in the hinge region. Ab_15 and Ab_16 are modified versions of Ab_9 and Ab_10 in which the glycosylation sequence has been removed. The figure shows the antibody concentration in mouse plasma calculated by electrochemiluminescence ligand binding assay. The figure shows the antibody concentration in mouse kidney calculated by fluorescence intensity.

[0016] The techniques and procedures described or referenced herein are generally well understood, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CCBlackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993) are examples of conventional methodologies commonly used by those skilled in the art.

[0017] The following definitions and detailed explanations are provided to facilitate understanding of the disclosure described herein.

[0018] Definition of Amino Acids In this specification, amino acids are described by a one-letter code, a three-letter code, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V.

[0019] Amino acid modification refers to substitution, deletion, addition, and insertion, or a combination thereof. In this disclosure, amino acid modification may be rephrased as amino acid mutation or amino acid modification. For amino acid modification in the amino acid sequence of antigen-binding molecules, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlapping extension PCR may be used as appropriate. Furthermore, several known methods may also be used as amino acid modification methods for substitution with non-natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is appropriate to use a cell-free translation system (Clover Direct (Protein Express)) that contains tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons.

[0020] In this specification, expressions indicating amino acid modifications may be used as appropriate, with a number representing a specific position, followed by a one-letter or three-letter code for the original and modified amino acid residues, respectively, to the left and right of the number. In one embodiment, the number representing the position indicates the position of the amino acid residue according to EU numbering (also called the EU index). In another embodiment, the number representing the position indicates the position of the amino acid residue according to Kabat numbering (also called the Kabat index). For example, the modification N100bL or Asn100bLeu, used when substituting amino acids contained in the antibody variable region, represents the substitution of Asn with Leu at position 100b (according to Kabat numbering). That is, the number indicates the position of the amino acid residue according to Kabat numbering, the one- or three-letter amino acid code written before the number (to the left of the number) indicates the original amino acid residue, and the one- or three-letter amino acid code written after the number (to the right of the number) indicates the substituted amino acid residue. Similarly, the modifications P238D or Pro238Asp, used when substituting amino acids in the Fc region within the constant region of an antibody, represent the substitution of Pro with Asp at position 238 (according to EU numbering). In other words, the number indicates the position of the amino acid residue according to EU numbering, the one- or three-letter amino acid code before the number (to the left of the number) indicates the amino acid residue before substitution, and the one- or three-letter amino acid code after the number (to the right of the number) indicates the amino acid residue after substitution.

[0021] Polypeptide As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain of two or more amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, “proteins,” “amino acid chains,” “polypeptide chains,” or any other terms used to refer to chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” may be used in place of or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, protein cleavage, or modification with non-natural amino acids. Polypeptides may originate from natural biological sources or may be produced by recombinant technology, but do not necessarily have to be translated from a specified nucleic acid. They may be produced by any method, including chemical synthesis. Polypeptides described herein may have sizes of approximately 3 or more amino acids, 5 or more amino acids, 10 or more amino acids, 20 or more amino acids, 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, 100 or more amino acids, 200 or more amino acids, 500 or more amino acids, 1,000 or more amino acids, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, but they are not necessarily required to have such a structure. Polypeptides having a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but can take on many different stereostructures are referred to as unfolded. When multiple polypeptide chains form a complex through interactions other than amide bonds (peptide bonds) (e.g., non-covalent bonds, disulfide bonds, etc.), the complex may be called a polypeptide complex or polypeptide polymer (e.g., a dimer).

[0022] Antigen-binding molecule As used herein, the term “antigen-binding molecule” refers to any molecule having binding activity to an antigen, and refers to any molecule (e.g., a peptide or protein) containing an antigen-binding domain (also called an antigen-binding site or antigen-binding portion), and in one embodiment, is a peptide or protein having a length of about 5 amino acids or more. In one embodiment, the antigen-binding molecule is a peptide or protein that specifically binds to an antigenic determinant (epitope). Peptides and proteins are not limited to those of biological origin; for example, they may be polypeptides produced from artificially designed sequences. They may be any natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. A scaffold molecule, which includes a known stable three-dimensional structure such as an α / β barrel as a scaffold, and in which part of the molecule becomes the antigen-binding domain, is also one embodiment of an antigen-binding molecule described herein. In one embodiment, the antigen-binding molecule is an antibody, antibody fragment, or antibody derivative. In one embodiment, the antigen-binding molecule of this disclosure includes a dimer Fc region and an antigen-binding domain. In one embodiment, the antigen-binding molecule of the present disclosure is an antigen-binding molecule that specifically binds to an antigen through an antigen-binding domain (antigen-binding site, antigen-binding moiety, or any molecule having antigen-binding activity) fused to a first Fc-region variant, contained in an Fc polypeptide comprising a first Fc-region variant and a second Fc-region variant. In a particular embodiment, the antigen-binding molecule of the present disclosure is an antigen-binding molecule comprising a dimeric Fc region and an antigen-binding domain, but lacking a hinge region that forms an inter-heavy-chain disulfide bond.

[0023] "Binding activity" refers to the total strength of non-covalent interactions between one or more binding sites on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In one embodiment, antigen-binding molecules and antibodies analyzed by the method of this disclosure may be tested for their antigen-binding activity by known methods such as ELISA, Western blotting, biolayer interference, and surface plasmon resonance.

[0024] In this specification, "specific binding" or "specific binding" means that one molecule of a specifically binding molecule binds without showing any significant binding to any molecules other than the one or more molecules it binds to. It is also used when the antigen-binding domain is specific to a particular epitope among several epitopes contained in a given antigen. Furthermore, if the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0025] Antibody In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity. In one embodiment, an antibody comprises a dimeric Fc region and an antigen-binding domain. In a particular embodiment, the antigen-binding molecule of this disclosure is an antibody comprising a dimeric Fc region and an antigen-binding domain, but lacking a hinge region that forms an inter-heavy-chain disulfide bond.

[0026] The term "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0027] The term "immunoglobulin molecule" refers to a protein that has the structure of a natural antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two light chains (L chains) and two heavy chains (H chains) linked by disulfide bonds. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, extending from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant domains. Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, extending from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain, also called a light chain constant domain. The heavy chain of an immunoglobulin may be assigned to one of five types called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further classified into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types called κ and λ, based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0028] In this specification, the "hinge region" refers to the region located between the CH1 and CH2 regions in an antibody. Specifically, in IgG antibodies, the hinge region is the region indicated by amino acid residues at positions 216–230 in the EU numbering system or 226–243 in the Kabat numbering system (https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html), and in this specification, it includes not only the wild type (natural type) but also variants in which amino acid residues have been substituted, added, or deleted from the wild type. In IgA antibodies, the hinge region is the region indicated as amino acid residues at positions 235–240 in J Biol Chem. 1979 Apr 25;254(8):2865-74., Antibodies 2019, 8(4), 57. This specification includes not only the wild type (natural type) but also variants in which amino acid residues are substituted, added, or deleted from the wild type. Hinge region variants in this specification have an amino acid sequence homology of 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, and 30% or less compared to the hinge region of the wild type (natural type).

[0029] In some embodiments, the hinge region as defined herein includes a hinge region variant. For example, a hinge region variant includes one or more amino acid substitutions within the hinge region. In one embodiment, the one or more amino acid substitutions included in the hinge region variant are substitutions to native amino acids. In one embodiment, the one or more amino acid substitutions included in the hinge region variant are substitutions to amino acids other than Cys, with particularly preferred embodiments being substitutions to Ala or Ser. For example, a hinge region variant includes a shorter hinge region than the wild-type (natural-type) hinge region, which includes a portion of the wild-type (natural-type) hinge region. In one embodiment, a short hinge region means a hinge region in which one or more amino acids are deleted from the wild-type (natural-type) hinge region, and includes an amino acid sequence containing one or more, two or more, three or more, four or more, or five or more consecutive amino acids within the hinge region.

[0030] In this specification, the term "constant region" refers to a region within the same class of Ig molecules where there is no significant difference in their amino acid sequence. In mammals, there are five isotypes of constant regions: α, δ, ε, γ, and μ. The difference in isotype and the number of basic units that constitute them represent the difference in classes. Each isotype corresponds to IgA (α), IgD (δ), IgE (ε), IgG (γ), and IgM (μ). The constant regions of α, δ, and γ consist of three domains, each consisting of approximately 340 amino acids, while the constant regions of μ and ε consist of four domains, each consisting of approximately 440 amino acids. In some embodiments, the constant region refers to the α, γ, and δ constant regions of IgG, IgA, and IgD, respectively, and in certain embodiments, it refers to the region containing the CH1, CH2, CH3, and hinge regions (H chain constant region), and / or the region containing the CL region (L chain constant region). In one embodiment of the present disclosure, the constant region is preferably an antibody constant region, more preferably an antibody constant region of type IgG1, IgG2, IgG3, or IgG4, and even more preferably a human antibody constant region of type IgG1, IgG2, IgG3, or IgG4. In another embodiment of the present disclosure, the constant region is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. The amino acid sequences of the human IgG1, human IgG2, human IgG3, and human IgG4 constant regions are known. As for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, several allotype sequences due to genetic polymorphism are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, but any of these may be used in the present disclosure. In one embodiment, the antigen-binding molecule of the present disclosure includes a constant region in which one or more amino acid residues are modified. In one embodiment, engineering techniques to control the association between heavy chains may be applied to the steady-state region.Examples of heavy chain-heavy chain control include methods using Knobs into holes and charge modification (Nat Biotechnol. 1998 Jul;16(7):677-81., WO2006106905).

[0031] Unless otherwise indicated herein, amino acid residues in the light chain constant region are numbered according to Kabat et al., and the numbering of amino acid residues in the heavy chain constant region follows the EU numbering system (also called EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The numbering of amino acid residues at positions not defined in the EU numbering system (EU index) in the heavy chain constant region follows Kabat numbering (also called Kabat index). For the numbering of amino acid residues of IgG and IgA, for IgG, it follows the definition described at https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html, and for IgA, it follows the definition described in J Biol Chem. 1979 Apr 25;254(8):2865-74., Antibodies 2019, 8(4), 57.

[0032] The term “Fc region” (also called “Fc domain” in this specification is used to define the C-terminal region of an immunoglobulin heavy chain, which includes at least a portion of the heavy chain constant region. This term includes the native sequence Fc region and variant Fc regions. In one embodiment, the term “Fc region” includes a fragment essentially consisting of the CH2 and CH3 domains of the heavy chain constant region. The Fc region of an IgG class means, for example, the region from Pro230 (EU numbering) to the C-terminus, but is not limited thereto. The Fc region can preferably be obtained by partially digesting an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a protease such as pepsin, and then re-eluting the fraction adsorbed to a protein A column or protein G column. Such proteases are not particularly limited, as long as they can digest the full-length antibody to restrictively form Fab or F(ab')2 under appropriately set enzyme reaction conditions (e.g., pH). Examples may include pepsin and papain.

[0033] In the present invention, for example, an Fc region derived from natural (wild-type) IgG can be used as the "Fc region" of an antigen-binding molecule. Here, natural IgG means a polypeptide belonging to a class of antibodies that have the same amino acid sequence as naturally occurring IgG and are substantially encoded by the immunoglobulin γ gene. Natural human IgG means, for example, natural human IgG1, natural human IgG2, natural human IgG3, or natural human IgG4. Natural IgG also includes variants that arise spontaneously. Multiple allotype sequences based on genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242 as constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, and any of them can be used in this disclosure. In particular, the sequence of human IgG1 may have DEL or EEM as the amino acid sequence at EU numbering positions 356-358.

[0034] In some embodiments, the dimeric Fc region is a polypeptide chain consisting of a pair of monomeric Fc regions or Fc domains. For example, the dimeric Fc region of an immunoglobulin G (IgG) molecule is a dimer in which each of the two subunits (monomers) contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the dimeric Fc region can stably associate with each other.

[0035] In one embodiment, the dimeric Fc region is an IgG dimeric Fc region. In certain embodiments, the dimeric Fc region is an IgG1 dimeric Fc region, an IgG2 dimeric Fc region, an IgG3 dimeric Fc region, or an IgG4 dimeric Fc region. In further certain embodiments, the dimeric Fc region is a human IgG1 dimeric Fc region, a human IgG2 dimeric Fc region, a human IgG3 dimeric Fc region, or a human IgG4 dimeric Fc region.

[0036] In some embodiments, an antigen-binding molecule comprising the dimeric Fc region of the present disclosure is a one-armed antibody or Fc fusion protein comprising a domain capable of binding to an antigen (antigen-binding domain). Examples of antigens that can be bound by such antibodies and Fc fusion proteins include, but are not limited to, ligands (such as cytokines and chemokines), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins, and immune complexes comprising partially immunoglobulins.

[0037] One-armed antigen-binding molecule: In one aspect, the antigen-binding molecule of the Disclosure may be a “one-armed antigen-binding molecule” (also called a one-arm antigen-binding molecule, one-armed antibody, single-arm antibody, or antibody in one-arm format). In one embodiment, a “one-armed antigen-binding molecule” refers to an antigen-binding molecule that specifically binds to an antigen through an antigen-binding domain fused to one monomer of a dimeric Fc region contained in the antigen-binding molecule. In one embodiment, a “one-armed antigen-binding molecule” refers to an antigen-binding molecule in which the antigen-binding domain is fused to only one monomer of a dimeric Fc region contained in the antigen-binding molecule, and the antigen-binding domain is not fused to the other monomer of the dimeric Fc region. In a particular embodiment, the one-armed antigen-binding molecule of the Disclosure specifically binds to an antigen through a single antigen-binding domain fused to one monomer of a dimeric Fc region contained in the antigen-binding molecule. In one embodiment, a "one-armed antigen-binding molecule" is an antibody (one-armed antibody) lacking one of the two arms (antigen-binding domains, e.g., Fab) that possess antigen-binding activity. In a particular embodiment, a "one-armed antigen-binding molecule" is an IgG-based one-armed antibody (one-armed IgG antibody). The "one-armed" antigen-binding molecules provided herein can exhibit higher glomerular permeability and accumulate in the kidneys compared to conventional two-armed antigen-binding molecules. The amount of the antigen-binding molecule accumulated in the kidney can be calculated, for example, by comparing the blood concentration and the renal concentration of the antigen-binding molecule, and the renal transfer ability of the antigen-binding molecule can be evaluated by the amount of renal accumulation thus calculated. Furthermore, the blood concentration and renal concentration of the antigen-binding molecule can be calculated by methods known to those skilled in the art.

[0038] Two-armed antigen-binding molecules: In contrast, a "two-armed antigen-binding molecule" (also called a two-arm antigen-binding molecule or a two-arm format antibody) refers to an antigen-binding molecule that specifically binds to an antigen through two antigen-binding domains. Unless otherwise specified, one of the two antigen-binding domains is linked (fused) to two monomers in the dimeric Fc region of the "two-armed antigen-binding molecule." In a non-limiting embodiment, a conventional bivalent antibody of the IgG type may be an example of a "two-armed antigen-binding molecule."

[0039] As used herein, an antigen-binding molecule containing at least two antigen-binding moieties (or antigen-binding domains), wherein at least one antigen-binding moiety binds to a first epitope in the antigen molecule and at least another antigen-binding moiety binds to a second epitope in the antigen molecule, is called a multispecific antigen-binding molecule in terms of its reaction specificity.

[0040] When a single antigen-binding molecule binds to two different epitopes through two antigen-binding domains it contains, this antigen-binding molecule is called a "bispecific antigen-binding molecule." When a single antigen-binding molecule binds to three different epitopes through three antigen-binding domains it contains, this antigen-binding molecule is called a "triplespecific antigen-binding molecule."

[0041] In one embodiment, the paratope in the antigen-binding moiety that binds to a first epitope in the antigen molecule has a different structure from the paratope in the antigen-binding moiety that binds to a second epitope that is structurally different from the first epitope. Therefore, an antigen-binding molecule containing at least two antigen-binding moieties (or domains), where at least one of the antigen-binding moieties binds to a first epitope in the antigen molecule and at least another of the antigen-binding moieties binds to a second epitope in the antigen molecule, is called a "multiparatopic antigen-binding molecule" in terms of its structure and specificity.

[0042] When a single antigen-binding molecule binds to two different epitopes through two antigen-binding moieties, it is called a "double-paratope antigen-binding molecule." When a single antigen-binding molecule binds to three different epitopes through three antigen-binding moieties, it is called a "triple-paratope antigen-binding molecule."

[0043] Multivalent, multispecific or multiparatope antigen-binding molecules containing one or more antigen-binding moieties and methods for preparing them are described in non-patent literature such as Conrath et al. (J. Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302), and Kontermann RE ((2011) Bispecific Antibodies (Springer-Verlag)), as well as in patent literature such as WO1996 / 034103 and WO1999 / 023221. The antigen-binding molecules of the present invention can be produced using the multispecific or multiparatope antigen-binding molecules and methods for preparing them described in these documents.

[0044] In one embodiment of fusion, “fused” means that components (e.g., an antigen-binding domain and an Fc region) are linked by covalent bonds (e.g., peptide bonds) either directly or via one or more linkers (e.g., peptide linkers). In another embodiment, “fused” means that a linker (linker region) is linked to another component (e.g., an antigen-binding domain or an Fc region) by covalent bonds.

[0045] In one embodiment, “conjugate” means that the components (e.g., a one-armed antibody and a small molecule compound, an antigen-binding domain and a small molecule compound, an Fc region and a small molecule compound) are linked together by covalent bonds, either directly or via one or more linkers (e.g., synthetic compound linkers), or a complex thus linked. In one aspect, the present disclosure provides an antigen-binding molecule or polypeptide complex (immunoconjugate) of the present disclosure that is conjugated with a bioactive molecule. In one embodiment, the bioactive molecule conjugated to the antigen-binding molecule or polypeptide complex is one or more of cytotoxic substances, nucleic acids, peptides (including cyclic peptides), and small molecule compounds. In one embodiment, the cytotoxic substance is a chemotherapeutic agent or chemotherapy drug, a growth inhibitor, a toxin (e.g., a protein toxin, enzymatically active toxin, or fragment thereof of bacterial, fungal, plant, or animal origin), or a radioisotope. In one embodiment, the molecular weight of the physiologically active molecule is approximately 15 kDa or less, approximately 13 kDa or less, approximately 11 kDa or less, approximately 10 kDa or less, approximately 9 kDa or less, approximately 8 kDa or less, approximately 7 kDa or less, approximately 6 kDa or less, approximately 5 kDa or less, approximately 4 kDa or less, approximately 3 kDa or less, or approximately 2 kDa or less.

[0046] In one aspect of linking, "linked" is a broader concept than "fused" and "conjugated," meaning that the constituent components are connected directly or indirectly by one or more linkers.

[0047] As used herein, the terms “first,” “second,” and “third” relating to components (e.g., polypeptides, e.g., antigen-binding domains or Fc regions) are used for convenience to distinguish between two or more types of each component. The use of these terms is not intended to confer any particular order or orientation of components in an antigen-binding molecule, unless otherwise specified.

[0048] Antigen-binding domain In this specification, “antigen-binding domain” refers, in one embodiment, to a portion of an antibody that includes a region that specifically binds to and is complementary to a part or all of an antigen. When the molecular weight of the antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. This specific portion is called an epitope. The antigen-binding domain may be provided, for example, by the variable domains (also called antibody variable regions) of one or more antibodies. In one embodiment, the antigen-binding domain includes both the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH). Examples of such antigen-binding domains include “single chain Fv (scFv)”, “single chain antibody”, “Fv”, “single chain Fv2 (scFv2)”, “Fab”, and “F(ab')2”. In another embodiment, the antigen-binding domain includes a single-domain antibody (sdAb), such as VHH, VL, VH, minibody, or any antibody fragment. Any antibody fragment may include an antibody fragment in which either or both of the VL or VH chains in Fab are replaced with VHH. In another embodiment, the antigen-binding domain includes a non-antibody protein or a fragment thereof that binds to a specific antigen. In one embodiment, the antigen-binding domain can orient the entity to which it is attached (e.g., the antigen-binding molecule of this disclosure) to a target site. In one embodiment, the molecular weight of the antigen-binding domain contained in the antigen-binding molecule of this disclosure is about 70 kDa or less, for example, about 50 kDa or less.

[0049] In certain embodiments, the antigen-binding domain of the Disclosure specifically binds to all or part of a partial peptide of an antigen. As used herein, the term “antigen” refers to all or a specific site of a polypeptide macromolecule to which the antigen-binding domain binds (e.g., a three-dimensional structure consisting of a continuous sequence of amino acids or separate regions of discontinuous amino acids) to which the antigen-binding domain binds, forming an antigen-binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surface of viruses, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in a free state in serum, and / or in the extracellular matrix (ECM). Examples of antigens to which the antigen-binding domain of the Disclosure can bind include, but are not limited to, ligands (e.g., cytokines and chemokines), receptors, cancer antigens, viral antigens, MHC antigens, differentiation antigens, immunoglobulins, and immunocomplexes partially composed of immunoglobulins. Unless otherwise specified, the protein referred to as an antigen in this specification may be any natural form of a protein derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human antigen, a cynomolgus monkey antigen, or a mouse antigen, particularly a human antigen. In certain embodiments, the antigen-binding domain is cross-reactive to (i.e., specifically binds to) human and cynomolgus monkey antigens. In other embodiments, the antigen is an antigen of a pathogenic entity, such as a pathogenic microorganism including viruses, bacteria, mycobacteria, fungi, protozoa, etc.

[0050] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0051] HVR or CDR As used herein, the terms “hypervariable region” or “HVR” refer to each region of the variable domain of an antibody that is hypervariable in sequence (“complementarity determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariable loop”) and / or contains an antigen contact residue (“antigen contact”). Hypervariable regions (HVRs) are also referred to as “complementarity determining regions” (CDRs), and these terms are used herein interchangeably with respect to the portion of the variable region that forms the antigen-binding region. Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).Examples of HVRs described herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c) including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0052] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 are also referred to as "H-CDR1," "H-CDR2," "H-CDR3," "L-CDR1," "L-CDR2," and "L-CDR3," respectively.

[0053] Framework "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0054] Human Consensus Framework The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected groups of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0055] Antibody Fragments An "antibody fragment" refers to a molecule other than the complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), VL, VH, and single-domain antibodies. For reviews of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003) and Konning D and Kolmar H, Microb Cell Fact. 2018 Feb 26;17(1):32. For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); also see WO93 / 16185; and U.S. Patents Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments with extended in vivo half-lives containing salvage receptor-binding epitope residues, see U.S. Patent No. 5,869,046. A diabody is an antibody fragment containing two antigen-binding domains, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody.In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1). Antibody fragments can be produced by a variety of methods, including, but are not limited to, proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0056] In this specification, a "Fab molecule" (also called F(ab) or Fab') is composed of an immunoglobulin light chain (Fab light chain) consisting of a VL (variable light chain region) and a CL (constant light chain region), and a portion of an immunoglobulin heavy chain consisting of a VH (variable heavy chain region) and a CH1 (γ1 region in the constant heavy chain region) (Fab heavy chain), and may have a structure in which the portion of the heavy chain and the light chain are linked by a disulfide bond at the C-terminal region.

[0057] A "crossover" Fab molecule (also called a "Crossfab") means a Fab molecule in which either the variable or constant regions of the Fab heavy chain and Fab light chain are exchanged. That is, a crossover Fab molecule includes a peptide chain composed of VL and CH1 and a peptide chain composed of VH and CL. To clarify, in a crossover Fab molecule in which the variable regions of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing CH1 is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing VH is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0058] "Conventional" Fab In contrast, "conventional" Fab molecules refer to Fab molecules in their natural form, namely, Fab molecules that include a Fab heavy chain (VH-CH1) composed of VH and CH1 and a Fab light chain (VL-CL) composed of VL and CL.

[0059] F(ab')2 and Fab' "F(ab')2" or "Fab" refers to antibody fragments produced by treating immunoglobulin molecules with proteases such as pepsin and papain, and by digesting the immunoglobulin molecule near the disulfide bond located between the hinge regions of each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bond located between the hinge regions of each of the two H chains, producing two homologous antibody fragments in which the L chain, containing VL (variable L chain region) and CL (constant L chain region), is linked by a disulfide bond at the C-terminal region to an H chain fragment containing VH (variable H chain region) and CHγ1 (γ1 region in the constant H chain region). These two homologous antibody fragments are each called Fab'.

[0060] "F(ab')2" consists of two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that a disulfide bond is formed between the two heavy chains. The F(ab')2 disclosed herein can be suitably produced as follows: A full-length monoclonal antibody containing the desired antigen-binding domain is partially digested with a protease such as pepsin, and the Fc fragment is removed by adsorption onto a protein A column. The protease is not particularly limited as long as it can selectively cleave the entire antibody to yield F(ab')2 under appropriate setting enzymatic reaction conditions such as pH. For example, such proteases include pepsin and ficin.

[0061] Variable Fragment (Fv) In this specification, the term "variable fragment (Fv)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and the antibody's heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that homogeneous and active antibodies could be prepared from the periplasmic fraction of E. coli by inserting an antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in E. coli (Science (1988) 240 (4855), 1038-1041). In Fv prepared from the periplasmic fraction, VH and VL associate in a manner that binds to the antigen.

[0062] scFv, Monoclonal Antibodies, and sc(Fv)2 In this specification, the terms “scFv,” “monoclonal antibody,” and “sc(Fv)2” all refer to antibody fragments of a single polypeptide chain that include variable regions derived from the heavy and light chains but do not include a constant region. Generally, monoclonal antibodies further include a polypeptide linker between the VH and VL domains, which enables the formation of a desired structure that is expected to allow antigen binding. Monoclonal antibodies are discussed in detail by Pluckthun in “The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, 269-315 (1994).” See also International Publication WO1988 / 001649, U.S. Patents 4,946,778 and 5,260,203. In certain embodiments, monoclonal antibodies may be bispecific and / or humanized.

[0063] scFv is a single-chain low molecular weight antibody in which the VH and VL that form Fv are linked together by a peptide linker (Proc. Natl. Acad. Sci. USA (1988) 85 (16), 5879-5883). The peptide linker allows the VH and VL to be kept in close proximity. sc(Fv)2 is a single-chain antibody in which four variable regions, two VL and two VH, are linked by a linker such as a peptide linker to form a single chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VH and two VL may originate from different monoclonal antibodies. Such sc(Fv)2 can preferably be described as a bispecific sc(Fv)2 that recognizes two epitopes present in a single antigen, such as the one disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be produced by methods known to those skilled in the art. For example, sc(Fv)2 can be produced by linking scFv with a linker such as a peptide linker.

[0064] In this specification, sc(Fv)2 includes two VH units and two VL units arranged in the order VH, VL, VH, VL ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) starting from the N-terminus of a single-stranded polypeptide. The order of the two VH units and two VL units is not limited to the above configuration and may be arranged in any order. Examples of configurations are listed below. [VL]-Linker-[VH]-Linker-[VH]-Linker-[VL] [VH]-Linker-[VL]-Linker-[VL]-Linker-[VH] [VH]-Linker-[VH]-Linker-[VL] [VL]-Linker-[VL]-Linker-[VH]-Linker-[VL]-Linker-[VH] [VL]-Linker-[VH]-Linker-[VL]-Linker-[VH]

[0065] The molecular morphology of sc(Fv)2 is described in detail in WO2006 / 132352. Those skilled in the art can prepare the desired sc(Fv)2 according to these descriptions.

[0066] Linkers used for linking antibody variable regions include any peptide linkers that can be introduced by genetic engineering, synthetic compound linkers, and linkers disclosed, for example, in Protein Engineering, 9 (3), 299-305, 1996. However, peptide linkers are preferred in this disclosure. Typically, three linkers are required to link four antibody variable regions. The linkers used may be of the same type or different types. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. The length is preferably 5 amino acids or more (but not particularly limited, the upper limit is usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. If sc(Fv)2 contains three peptide linkers, their lengths may all be the same or different.

[0067] Peptide Linker In some embodiments, the peptide linker is a GS linker as described herein, for example, (GS)2, (GGGGS: SEQ ID NO: 53)2. The above preferred peptide linkers can be easily selected from a variety of lengths, preferably including 1 amino acid (Gly, etc.) to 300 amino acids, 2 amino acids to 200 amino acids, or 3 amino acids to 100 amino acids including 4 amino acids to 100 amino acids, 5 amino acids to 100 amino acids, 5 amino acids to 50 amino acids, 5 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, or 5 amino acids to 20 amino acids. Examples of peptide linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GGGGS: SEQ ID NO: 53)n, (GSGGS: SEQ ID NO: 80)n, and (GGGS: SEQ ID NO: 56)n, where n is at least an integer of 1) (see Chen X et al., Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69), glycine-alanine polymers, alanine-serine polymers, and other mobile linkers known in the conventional art.Examples of constituent peptide linkers include: Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 53) (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 53))n (SEQ ID NO: 63) Gly Gly Gly Gly Ala (GGGGA, SEQ ID NO: 54) Gly Gly Gly Gly Glu (GGGGE, SEQ ID NO: 55) Gly Gly Gly Ser (GGGS, SEQ ID NO: 56) (Gly Gly Gly Ser (GGGS, SEQ ID NO: 56))n (SEQ ID NO: 66) Gly Gly Gly Ala (GGGA, SEQ ID NO: 57) Gly Gly Gly Glu (GGGE, SEQ ID NO: 58) Gln Gln Gln Gly (QQQG, SEQ ID NO: 59) Gln Gln Gln Gln Gly (QQQQG, SEQ ID NO: 60) Ser Ser Ser Gly (SSSG, SEQ ID NO: 61) Ser Ser Ser Ser Gly (SSSSG, SEQ ID NO: 62), (Glu Ala Ala Ala Lys)n ((EAAAK)n, SEQ ID NO: 107), Ala (Glu Ala Ala Ala Lys)n, Ala (A(EAAAK)nA, SEQ ID NO: 108) (where n is an integer greater than or equal to 1) may be included, but are not limited to these. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0068] GS Linker Among the movable linkers mentioned above, glycine polymers and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and readily function as neutral tethers between components.Examples of movable linkers (GS linkers) made of glycine-serine polymers include: Ser Gly Ser (GS) Ser Gly (SG) Gly Gly Ser (GGS) 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, Sequence ID: 56) Gly Gly Ser Gly (GGSG, Sequence ID: 73) Gly Ser Gly Gly (GSGG, Sequence ID: 74) Ser Gly Gly Gly (SGGG, Sequence ID: 75) Gly Ser Ser Gly (GSSG, Sequence ID: 76) Gly Gly Gly Gly Ser (GGGGS, Sequence ID: 53) Gly Gly Gly Ser Gly (GGGSG, Sequence ID: 77) Gly Gly Ser Gly Gly (GGSGG, Sequence ID: 78) Gly Ser Gly Gly Gly (GSGGG, Sequence ID: 79) Gly Ser Gly Gly Ser (GSGGS, Sequence ID: 80) Ser Gly Gly Gly Gly (SGGGG, Sequence ID: 81) Gly Ser Ser Gly Gly (GSSGG, Sequence ID: 82) Gly Ser Gly Ser Gly (GSGSG, Sequence ID: 83) Ser Gly Gly Ser Gly (SGGSG, Sequence ID: 84) Gly Ser Ser Ser Gly (GSSSG, Sequence ID: 85) Gly Gly Gly Gly Gly Ser (GGGGGS, Sequence ID: 86) Ser Gly Gly Gly Gly Gly (SGGGGG, Sequence ID: 87) Gly Gly Gly Gly Gly Gly Ser (GGGGGGS, Sequence ID: 88) Ser Gly Gly Gly Gly Gly Gly (SGGGGGG, Sequence ID: 89) (Gly Gly Gly Gly Ser (GGGGS, Sequence ID: 53))n (Ser Gly Gly Gly This may include, but is not limited to, Gly(SGGGG, sequence number: 81))n(sequence number: 98) (where n is an integer greater than or equal to 1).However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0069] Synthetic compound linkers (chemical crosslinking agents) are commonly used for crosslinking peptides. Examples include N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinking agents are commercially available.

[0070] In this specification, the term "single-domain antibody" is not limited by its structure, as long as the domain can exert antigen-binding activity on its own. While general antibodies, such as IgG antibodies, exhibit antigen-binding activity when their variable regions are formed by the pairing of VH and VL domains, it is known that the domain structure of a single-domain antibody can exert antigen-binding activity on its own without pairing with another domain. Typically, single-domain antibodies have a relatively low molecular weight and exist in monomeric form.

[0071] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules such as VHH from camelid animals and VNAR from sharks, which inherently lack light chains, and antibody fragments containing all or part of an antibody VH domain or all or part of an antibody VL domain. Examples of single-domain antibodies that are antibody fragments containing all or part of an antibody VH domain or antibody VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from human antibody VH or human antibody VL, as described in U.S. Patent No. 6,248,516B1, etc. In some aspects of the present invention, a single-domain antibody has three types of CDRs (CDR1, CDR2, and CDR3).

[0072] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or by immunization of 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 possessing genes capable of producing single-domain antibodies. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals possessing genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in International Publication No. WO2015 / 143414 and U.S. Patent Publication No. US2011 / 0123527 A1. The framework sequence of the single-domain antibody obtained from such animals may be converted to a human germline sequence or a similar sequence in order to obtain a humanized single-domain antibody. A humanized single-domain antibody (e.g., humanized VHH) is also one embodiment of the single-domain antibody of the present invention.

[0073] Alternatively, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by ELISA or panning, etc. 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); and Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764: 8 (1307-1319)), antibody libraries obtained by immunization of various animals (e.g., Journal of Applied Microbiology 2014 117: 2 (528-536)), and synthetic antibody libraries prepared 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); and AIDS 2016 30: 11 (1691-1701) is one example.

[0074] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable region in which a portion of the heavy chain and / or light chain variable region originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain variable region originates from a different source or species.

[0075] Humanized Antibody A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains 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 contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization. The "humanized antibody variable region" refers to the variable region of a humanized antibody.

[0076] Human Antibodies: "Human antibodies" are antibodies produced by humans or human cells, or antibodies that possess an amino acid sequence corresponding to the amino acid sequence of antibodies derived from the human antibody repertoire or other non-human sources using human antibody coding sequences. This definition of human antibodies explicitly excludes humanized antibodies that contain non-human antigen-binding residues. "Human antibody variable region" refers to the variable region of a human antibody.

[0077] When pyroglutamylated antibodies are expressed in cells, it is known that the antibodies undergo post-translational modification. Examples of post-translational modification include cleavage of the lysine at the C-terminus of the heavy chain by carboxypeptidase; modification to pyroglutamate by pyroglutamylation of glutamine or glutamate at the N-terminus of the heavy and light chains; glycosylation; oxidation; deamide; and glycation. Such post-translational modifications are known to occur in a variety of antibodies (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447).

[0078] The antigen-binding molecules or polypeptide complexes of this disclosure also include antibodies that have undergone post-translational modifications. Examples of antigen-binding molecules or polypeptide complexes of this disclosure that have undergone post-translational modifications include antibodies that have undergone pyroglutamylation of the N-terminus of the heavy chain variable region and / or deletion of lysine at the C-terminus of the heavy chain. It is known in the art that such post-translational modifications, such as pyroglutamylation of the N-terminus and deletion of lysine at the C-terminus, have no effect on antibody activity (Analytical Biochemistry, 2006, Vol. 348, p. 24-39).

[0079] Furthermore, the antigen-binding molecule or polypeptide complex of this disclosure may be conjugated with a carrier polymer such as PEG or an organic compound such as an anticancer agent. Alternatively, a glycosylation sequence may be suitably inserted into the antigen-binding molecule or polypeptide complex so that the glycosylation produces a desired effect.

[0080] Specificity "Specific" means that a molecule that specifically binds to one or more binding partners does not show any significant binding to molecules other than those partners. Furthermore, "specific" is also used when an antigen-binding domain is specific to a particular epitope among several epitopes contained in an antigen. When an antigen-binding molecule specifically binds to an antigen, it is also stated that "the antigen-binding molecule has / shows specificity to / for the antigen." If the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, the antigen-binding molecule containing the antigen-binding domain can bind to various antigens that have those epitopes.

[0081] Affinity / Avidity "Affinity" refers to the strength of the sum of non-covalent interactions between one binding site of a molecule (e.g., an antigen-binding molecule or antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antigen-binding molecule and an antigen, or an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation rate constant and the association rate constant (k, respectively). off and kon It can be expressed by the dissociation constant (KD), which is the ratio of ). Therefore, as long as the ratio of the rate constants remains the same, equivalent affinity may include different rate constants. Affinity can be measured by established methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0082] The structure of the antigen-binding domain of an antibody that binds to an epitope is called a paratope. Paratopes stably bind to epitopes through hydrogen bonds, electrostatic forces, van der Waals forces, or hydrophobic bonds acting between the epitope and the paratope. This binding force between the epitope and the paratope is called "affinity" (see also above). The total binding force when multiple antigen-binding domains bind to multiple antigens is called "avidity." Affinity can work synergistically, for example, when an antibody containing multiple antigen-binding domains (i.e., a polyvalent antibody or multivalent antibody) binds to multiple epitopes, and avidity may be higher than affinity.

[0083] Method for Determining Affinity In certain embodiments, the antigen-binding molecule or antibody provided herein has an affinity for the antigen of ≤1 μM, ≤120 nM, ≤100 nM, ≤80 nM, ≤70 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 It has a dissociation constant (KD) of M. In a particular embodiment, the KD value of the antibody / antigen-binding molecule with respect to the antigen falls within the range of 1–40, 1–50, 1–70, 1–80, 30–50, 30–70, 30–80, 40–70, 40–80, or 60–80 nM.

[0084] In one aspect, KD is measured by a radiolabeled antigen binding assay (RIA). In one aspect, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab for an antigen is determined in the presence of a serial increasing amount of unlabeled antigen at the minimum concentration of ( 125 I) equilibrating the Fab with the radiolabeled antigen and then capturing the bound antigen with a plate coated with an anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To construct the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of the capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM of 125 I]-antigen is mixed with serial dilutions of the Fab of interest (in the same manner as the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997), for example). Then, the Fab of interest is incubated overnight, although this incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is achieved. Thereafter, the mixture is transferred to the capture plate for incubation at room temperature (e.g., 1 hour). Then the solution is removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of the maximum binding is selected for use in the competitive binding assay.

[0085] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. The association rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting association and dissociation sensorgrams using a simple one-to-one Langmuir couple model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on It is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). The on-velocity was 10 by the surface plasmon resonance assay described above. 6 M -1 s -1If it exceeds this, the ON rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C in the presence of gradually increasing concentrations of antigen using a spectrometer (e.g., a stop-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0086] By following the above method for measuring the affinity of antigen-binding molecules or antibodies, those skilled in the art can perform affinity measurements of other antigen-binding molecules or antibodies to various antigens.

[0087] Polynucleotides (Nucleic Acids) As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides such as methylated nucleotides and analogs thereof. Non-nucleotide components may be interposed in the sequence of nucleotides. Polynucleotides may include modifications made after synthesis, such as conjugation to labels. Other types of modifications include, for example, "caps," substitutions of one or more native nucleotides with analogs, internucleotide modifications, such as those involving uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramic acids, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those involving pendant portions such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those involving intercalating agents (e.g., acridine, psoralen, etc.), those involving chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those involving alkylating agents, and those involving modified linkages (e.g., alpha-anomeric nucleic acids, etc.) or unmodified forms of polynucleotides. Furthermore, any hydroxyl group normally present in sugars can be replaced by, for example, a phosphonate group, a phosphate group, protected by a standard protecting group, activated to prepare further linkage to further nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH groups can be substituted with phosphorylation or with amines or organic cap groups of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides may also include analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azid-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methylriboside. One or more phosphodiester bonds may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the 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 optionally an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl substituted or unsubstituted alkyl (1–20C). Not all linkages in a polynucleotide need to be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0088] Isolated nucleic acids: An "isolated nucleic acid molecule" refers to a nucleic acid molecule that has been separated from the components of its original environment. Isolated nucleic acid molecules also include nucleic acid molecules that would normally be found in the cell containing that nucleic acid molecule, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original chromosomal location.

[0089] Vectors As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells using viruses or electroporation. However, vector introduction is not limited to in vitro methods. For example, vectors can also be introduced directly into a target using in vivo methods.

[0090] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0091] Recombination Methods and Configurations: For example, antigen-binding molecules and polypeptide complexes can be produced using recombination methods and configurations, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antigen-binding molecule or polypeptide complex described herein is provided. Such nucleic acid may encode an amino acid sequence containing the VL of an antibody and / or an amino acid sequence containing the VH (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing the VL of an antibody and an amino acid sequence containing the VH of an antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing the VL of an antibody and a second vector containing nucleic acid encoding an amino acid sequence containing the VH of an antibody (e.g., transformed). In one embodiment, the host cells are eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method for producing an antigen-binding molecule or polypeptide complex of the present disclosure is provided, comprising culturing host cells containing a nucleic acid encoding the antigen-binding molecule or polypeptide complex under conditions suitable for the expression of the antigen-binding molecule or polypeptide complex, and optionally recovering the antigen-binding molecule or polypeptide complex from the host cells (or host cell culture medium).

[0092] For the recombinant production of antigen-binding molecules or polypeptide complexes described herein, nucleic acids encoding the antigen-binding molecules or polypeptide complexes are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of antibodies).

[0093] Suitable host cells for cloning or expressing vectors encoding antigen-binding molecules or polypeptide complexes include prokaryotic or eukaryotic cells as described herein. For example, antigen-binding molecules or polypeptide complexes may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For bacterial expression of antibody fragments and polypeptides, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli).) After expression, antigen-binding molecules or polypeptide complexes may be isolated from bacterial cell paste into soluble fractions and further purified.

[0094] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been "humanized" to produce antibodies with partial or complete human glycosylation patterns, are suitable cloning or expression hosts for antibody-coding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0095] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

[0096] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0097] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68). (Described in 1982); MRC5 cells; and FS4 cells, etc. Other useful mammalian host cell lines include DHFR - This includes Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0098] Recombinant production of antigen-binding molecules or polypeptide complexes described herein can be carried out in the same manner as described above by using host cells (e.g., transformed therewith) containing one or more vectors containing nucleic acids encoding an amino acid sequence comprising all or part of the antigen-binding molecule or polypeptide complex.

[0099] And / or, in this specification, the meaning of the term "and / or" includes any combination in which "and" and "or" are appropriately combined. Specifically, for example, "A, B, and / or C" includes the following variations: (a) A, (b) B, (c) C, (d) A and B, (e) A and C, (f) B and C, and (g) A, B, and C.

[0100] Detailed Description of Various Embodiments In one aspect of the antigen-binding molecule, the Disclosure provides an antigen-binding molecule comprising a dimeric Fc region and an antigen-binding domain, wherein either or both of a first polypeptide chain comprising one monomer of the Fc region and a second polypeptide chain comprising the other monomer of the Fc region do not contain an antibody hinge region capable of forming a disulfide bond, and the antigen-binding domain is linked only to the first polypeptide chain and not to the second polypeptide chain (a one-armed antigen-binding molecule without an antibody hinge region capable of forming a disulfide bond). In one embodiment of the antigen-binding molecule of the Disclosure, the first polypeptide chain and the second polypeptide chain associate at the CH3 region, for example, by a non-covalent interaction between their CH3 regions. In one embodiment of the antigen-binding molecule of the Disclosure, the Fc region is the Fc region of natural IgG or a variant thereof. In one embodiment of the antigen-binding molecule of the Disclosure, the Fc region is the Fc region of natural IgG or a variant thereof and does not have a glycosylation sequence. In one embodiment, the antigen-binding molecule of the Disclosure is a monovalent antigen-binding molecule (a monovalent one-armed antigen-binding molecule) in which only one antigen-binding domain is linked to a first polypeptide chain. In another embodiment, the antigen-binding molecule of the Disclosure is a bivalent antigen-binding molecule (a bivalent one-armed antigen-binding molecule) in which two antigen-binding domains fused (linked) to each other by a peptide bond, a covalent bond, or a known linker are linked to a first polypeptide chain. In some embodiments of the monovalent or bivalent one-armed antigen-binding molecules of this disclosure, the molecular weight of one or two antigen-binding domains linked to the first polypeptide chain, including the linker that fuses (links) the two antigen-binding domains together, if present, is about 100 kDa or less, for example, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, or 30 kDa or less, preferably about 70 kDa or less, and particularly preferably about 50 kDa or less. In some embodiments, the molecular weight of the antigen-binding domain is about 70 kDa or less. In one embodiment, the molecular weight of the antigen-binding domain is about 50 kDa or less.In a particularly preferred embodiment, one antigen-binding domain is Fab (e.g., ≤50 kDa) or VHH (e.g., ≤15 kDa). In a particularly preferred embodiment, the combination of two antigen-binding domains is Fab / Fab (e.g., ≤100 kDa), a combination of two Fabs; Fab / VHH (e.g., ≤65 kDa), a combination of one Fab and one VHH; or VHH / VHH (e.g., ≤30 kDa), a combination of two VHHs.

[0101] In some embodiments, the antigen-binding domain is Fab, scFv, VHH, VL, VH, or a single-domain antibody (sdAb), or a ligand. In one embodiment, the antigen-binding domain is Fab, scFv, or sdAb. In a particular preferred embodiment, the antigen-binding domain is Fab. In another particular preferred embodiment, the antigen-binding domain is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH. In yet another particular preferred embodiment, the antigen-binding domain is VHH.

[0102] In the antigen-binding molecule of this disclosure, the antigen-binding domain may be directly linked to the first polypeptide chain or indirectly linked via a linker region. In one embodiment, the antigen-binding domain may be directly linked to the first polypeptide chain. In another embodiment, the antigen-binding domain may be linked to the first polypeptide chain by a covalent bond (peptide bond) using a peptide linker. In a particular embodiment, the antigen-binding domain may be linked to the first polypeptide chain by a covalent bond (e.g., a peptide bond) using a synthetic compound linker.

[0103] In one embodiment, the antigen-binding molecule of the present disclosure is a "one-armed antigen-binding molecule" comprising an Fc polypeptide containing a first Fc region variant and a second Fc region variant, and an antigen-binding domain, which specifically binds to an antigen through an antigen-binding domain (antigen-binding site, antigen-binding moiety, or any molecule having binding activity to an antigen) fused to the first Fc region variant. In certain examples of such embodiments, the second Fc region variant is not fused to the antigen-binding domain fused to the first Fc region variant described above, or to any other antigen-binding domain capable of binding to the same antigen to which it binds, and / or not fused to the antigen-binding domain fused to the first Fc region variant described above, or to any other antigen-binding domain capable of binding to a different antigen (or epitope) to which it binds. In one embodiment, the first and second Fc region variants contained in the antigen-binding molecule of the present disclosure may include various amino acid mutations and modifications. In one embodiment, the first and second Fc region variants may be subjected to engineering techniques that control heavy-chain-heavy-chain association. In one embodiment, the antigen-binding domain is directly ligated to the N-terminus of the antibody CH2 domain in the first polypeptide chain. In another embodiment, the antigen-binding domain is ligated to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker region. In one embodiment, the first polypeptide chain and / or the second polypeptide chain contain only an Fc region, or contain only a linker region and an Fc region in that order from the N-terminal side. In a particular embodiment, the antigen-binding domain is ligated to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker region, and the second polypeptide chain contains only an Fc region.

[0104] In one aspect, the present disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region and a second Fc region, the first Fc region being fused to the first antigen-binding moiety, the second Fc region not being fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0105] In some embodiments of the above aspects, the antigen-binding portion (antigen-binding domain) is Fab, scFv, VHH, VL, VH, a single-domain antibody (sdAb), or a ligand. In one embodiment, the antigen-binding portion is Fab, scFv, or sdAb. In a particular preferred embodiment, the antigen-binding portion is Fab. In another particular preferred embodiment, the antigen-binding portion is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH. In yet another particular preferred embodiment, the antigen-binding portion is VHH.

[0106] In one embodiment, the antigen-binding molecule of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of the antigen of interest through two antigen-binding moieties contained within the antigen-binding molecule.

[0107] In one aspect, the present disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, provided that the second Fc region variant is not fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0108] In some embodiments of the aforementioned aspects, the antigen-binding portion is Fab, scFv, VHH, VL, VH, a single-domain antibody (sdAb), or a ligand. In one embodiment, the antigen-binding portion is Fab, scFv, or sdAb. In a particular embodiment, the antigen-binding portion is Fab. In another particular embodiment, the antigen-binding portion is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH. In yet another particular embodiment, the antigen-binding portion is VHH.

[0109] In one embodiment, the antigen-binding molecule of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of the antigen of interest through two antigen-binding moieties contained within the antigen-binding molecule.

[0110] In some embodiments, the disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, the second Fc region variant not being fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and further comprising a second antigen-binding moiety that specifically binds to an epitope on an antigen different from the epitope on the antigen bound by the first antigen-binding moiety.

[0111] In some embodiments, the antigen-binding molecule of the present invention is a bivalent antigen-binding molecule (one-armed bivalent antigen-binding molecule) comprising first and second antigen-binding moieties that specifically bind to the same epitope on the antigen.

[0112] In some embodiments, the Disclosure provides an antigen-binding molecule comprising: (i) a first antigen-binding moiety that specifically binds to an antigen; (ii) an Fc polypeptide; and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, the second Fc region variant not being fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and further comprising a second antigen-binding moiety that specifically binds to the same epitope on the antigen as the epitope on the antigen bound by the first antigen-binding moiety.

[0113] In one aspect, the present disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, provided that the second Fc region variant is not fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and which can accumulate in the kidneys compared to a parent antigen-binding molecule.

[0114] In one embodiment, the antigen-binding molecule of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of the antigen of interest through two antigen-binding moieties contained within the antigen-binding molecule.

[0115] In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety of the Disclosure comprises a Fab, scFv, VHH, VL, VH, or single-domain antibody or ligand. In more specific embodiments, the first antigen-binding moiety and the second antigen-binding moiety of the Disclosure comprises a VHH domain.

[0116] In a more preferred embodiment, the present disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region and a second Fc region, the first Fc region being fused to the first antigen-binding moiety, provided that the second Fc region is not fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0117] In some embodiments, the antigen-binding portion is Fab.

[0118] In some embodiments, the antigen-binding moiety is VHH.

[0119] In some embodiments, the antigen-binding portion is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH.

[0120] In one aspect, the present disclosure provides an antigen-binding molecule comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, provided that the second Fc region variant is not fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0121] In some embodiments, the antigen-binding portion is Fab.

[0122] In some embodiments, the antigen-binding moiety is VHH.

[0123] In some embodiments, the antigen-binding portion is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH.

[0124] In some embodiments, the one-armed antigen-binding molecules of the present disclosure include an Fc region variant comprising at least one amino acid modification to the parent Fc region. Examples of such amino acid modifications include, for example, the deletion of Gly at position 446 and Lys at position 447 of the EU numbering in the IgG-type Fc region. By deleting both of these amino acids, it is possible to reduce heterogeneity derived from the C-terminus of the Fc region.

[0125] According to any of the above embodiments, the components of the antigen-binding molecule (e.g., antigen-binding portion, Fc polypeptide, first Fc region (or "Fc domain"), second Fc region) may be fused directly or through various linkers, in particular peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n , or G4 (SG4) n A peptide linker is included, where n is generally a number from 1 to 10, typically from 2 to 4. For example, the first antigen-binding moiety may be fused to the N-terminus of the first Fc region variant, either directly or through a suitable linker. The second antigen-binding moiety may be fused to the N-terminus of the first antigen-binding moiety, either directly or through a suitable linker.

[0126] In a biparatope-like or bispecific antigen-binding molecule of the present disclosure having a first and a second antigen-binding moiety, the first and second antigen-binding moieties may be fused (directly or indirectly) to the same first Fc region variant to provide an antigen-binding molecule in a "one-armed" molecular format, or the first antigen-binding moiety may be fused to one of the first and second Fc region variants and the second antigen-binding moiety to the other to provide an antigen-binding molecule in a conventional "two-armed" molecular format. In any one embodiment of the one-armed or two-armed biparatope-like or bispecific antigen-binding molecule described above, comprising a first antigen-binding moiety and a second antigen-binding moiety, the second antigen-binding moiety can bind to a different epitope on the same antigen to which the first antigen-binding moiety binds or to a different antigen to which the first antigen-binding moiety binds.

[0127] In one aspect of polypeptide complexes, the disclosure provides a polypeptide complex comprising a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains comprises an Fc region, and neither or both of the first and second polypeptide chains contain an antibody hinge region capable of forming a disulfide bond, and a protein-binding molecule with a molecular weight of approximately 70 kDa or less (e.g., 50 kDa or less) is linked only to the first polypeptide chain, while the second polypeptide chain is not linked to such a protein-binding molecule. In one embodiment, the protein-binding molecule is an antigen-binding molecule, such as an antibody fragment. In a particular embodiment, the protein-binding molecule is Fab, scFv, or sdAb. In another embodiment, the protein-binding molecule is a ligand, such as a ligand that affects intracellular signaling in kidney tissue (e.g., a natural ligand). The ligand in the present invention is not particularly limited as long as it is a molecule that binds to a protein, but includes, for example, proteins, nucleic acids, lipids, carbohydrates, small molecule compounds, etc.

[0128] In embodiments of the polypeptide complex of the present disclosure, the antigen-binding molecule (also referred to as an antigen-binding moiety in relation to the polypeptide complex of the present disclosure) may be directly linked to the first polypeptide chain or indirectly linked via a linker region. In one embodiment, the antigen-binding molecule may be directly linked to the first polypeptide chain. In one embodiment, the antigen-binding molecule may be covalently linked to the first polypeptide chain using a peptide linker (peptide bond). In one embodiment, the antigen-binding molecule may be covalently linked to the first polypeptide chain using a GS linker. In one embodiment, the antigen-binding molecule may be covalently linked to the first polypeptide chain using a synthetic compound linker (e.g., a peptide bond). In one embodiment, the first polypeptide chain and the second polypeptide chain contained in the polypeptide complex of the present disclosure each contain a first Fc region variant and a second Fc region variant, and the polypeptide complex specifically binds to an antigen through an antigen-binding molecule (e.g., an antibody fragment, an antigen-binding site, an antigen-binding moiety, or any molecule having binding activity to the antigen) fused to the first Fc region variant. In certain examples of such embodiments, the second Fc region variant is not fused to the antigen-binding molecule fused to the first Fc region variant described above, nor to any other antigen-binding molecule capable of binding to the same antigen to which it binds, and / or not fused to any other antigen-binding molecule capable of binding to a different antigen (or epitope) than that to which the antigen-binding molecule fused to the first Fc region variant described above binds. In one embodiment, the antigen-binding molecule is directly ligated to the N-terminus of the antibody CH2 domain in the first polypeptide chain. In another embodiment, the antigen-binding molecule is ligated to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker region. In one embodiment, the first polypeptide chain and / or the second polypeptide chain contain only the Fc region, or contain only the linker region and the Fc region in that order from the N-terminal side.In a particular embodiment, the antigen-binding molecule is linked to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker region, and the second polypeptide chain contains only the Fc region.

[0129] In one embodiment, the polypeptide complex of the present disclosure includes a ligand, the ligand may be directly linked to the first polypeptide chain or indirectly linked via a linker region. In another embodiment, the polypeptide complex of the present disclosure includes an antigen-binding molecule and a ligand, the ligand may be directly linked to the antigen-binding molecule or indirectly linked via a linker region, the ligand may be directly linked to the first polypeptide chain or indirectly linked via a linker region. In one embodiment, the ligand may be directly linked to the first polypeptide chain and / or the antigen-binding molecule. In one embodiment, the ligand may be covalently linked to the first polypeptide chain and / or the antigen-binding molecule using a peptide linker (peptide bond). In one embodiment, the ligand may be covalently linked to the first polypeptide chain and / or the antigen-binding molecule using a GS linker. In one embodiment, the ligand may be covalently linked to the first polypeptide chain and / or the antigen-binding molecule using a synthetic compound linker (e.g., a peptide bond). In one embodiment, the ligand linkage site in the polypeptide complex of the present disclosure may be any of the following locations, for example, the N-terminus of the antibody CH2 domain in the first polypeptide or the second polypeptide chain, the C-terminus of the antibody CH3 domain in the first polypeptide or the second polypeptide chain, the antigen-binding molecule, the N-terminus of the antigen-binding molecule, or the C-terminus of the antigen-binding molecule. Particularly preferred is the ligand linkage site to the C-terminus of the antibody CH3 domain in the first polypeptide or the second polypeptide chain.

[0130] In one aspect, the present disclosure provides a polypeptide complex comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region and a second Fc region, the first Fc region being fused to the first antigen-binding moiety, provided that the second Fc region is not fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0131] In some embodiments, the antigen-binding portion is Fab, scFv, VHH, VL, VH, a single-domain antibody (sdAb), or a ligand. In one embodiment, the antigen-binding portion is Fab, scFv, or sdAb. In a particular preferred embodiment, the antigen-binding portion is Fab. In another particular preferred embodiment, the antigen-binding portion is an antibody fragment in which one or both of the VL or VH chains in Fab are replaced with VHH. In yet another particular preferred embodiment, the antigen-binding portion is VHH.

[0132] In one embodiment, the polypeptide complex of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of a target antigen through two antigen-binding moieties contained in the antigen-binding molecule.

[0133] In one aspect, the present disclosure provides a polypeptide complex comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, provided that the second Fc region variant is not fused to the first antigen-binding moiety or to any other antigen-binding moiety that specifically binds to an antigen.

[0134] In one embodiment, the polypeptide complex of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of a target antigen through two antigen-binding moieties contained in the antigen-binding molecule.

[0135] In some embodiments, the disclosure provides a polypeptide complex comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, the second Fc region variant not being fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and further comprising a second antigen-binding moiety that specifically binds to an epitope on an antigen different from the epitope on the antigen bound by the first antigen-binding moiety.

[0136] In some embodiments, the polypeptide complex of the present invention is a bivalent antigen-binding molecule (one-armed bivalent antigen-binding molecule) comprising first and second antigen-binding moieties that specifically bind to the same epitope on an antigen.

[0137] In some embodiments, the disclosure provides a polypeptide complex comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, the second Fc region variant not being fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and further comprising a second antigen-binding moiety that specifically binds to the same epitope on the antigen as the epitope on the antigen bound by the first antigen-binding moiety.

[0138] In one aspect, the present disclosure provides a polypeptide complex comprising (i) a first antigen-binding moiety that specifically binds to an antigen, (ii) an Fc polypeptide, and (iii) a hinge region that does not form a disulfide bond (SS bond), wherein the Fc polypeptide comprises a first Fc region variant and a second Fc region variant, each comprising at least one amino acid modification to a parent Fc region, the first Fc region variant being fused to the first antigen-binding moiety, provided that the second Fc region variant is not fused to the first antigen-binding moiety or any other antigen-binding moiety that specifically binds to an antigen, and which can accumulate in the kidney compared to a parent polypeptide complex.

[0139] In one embodiment, the polypeptide complex of the present disclosure is a biparatope antigen-binding molecule (or a one-armed biparatope antigen-binding molecule), that is, it specifically binds to two different epitopes of the antigen of interest through two antigen-binding moieties contained in the antigen-binding molecule.

[0140] In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety of the Disclosure comprises Fab, scFv, VHH, VL, VH, or a single-domain antibody or ligand. In certain preferred embodiments, the first and second antigen-binding moieties of the Disclosure comprise Fab. In another particular preferred embodiment, the first and second antigen-binding moieties of the Disclosure comprise an antibody fragment in which either or both of the VL or VH chains in Fab are replaced with VHH. In yet another particular preferred embodiment, the first and second antigen-binding moieties of the Disclosure comprise VHH.

[0141] In some embodiments, the polypeptide complex of the present disclosure includes a modified Fc region comprising at least one amino acid modification relative to the parent Fc region. Examples of such amino acid modifications include, for example, the deletion of Gly at position 446 and Lys at position 447 of the EU numbering in the IgG-type Fc region. By deleting both of these amino acids, it is possible to reduce heterogeneity derived from the C-terminus of the Fc region.

[0142] According to any of the above embodiments, the components of the polypeptide complex (e.g., antigen-binding portion, Fc polypeptide, first Fc region (or "Fc domain"), second Fc region) may be fused directly or through various linkers, in particular peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n , or G4 (SG4) n A peptide linker is included, where n is generally a number from 1 to 10, typically from 2 to 4. For example, the first antigen-binding moiety may be fused to the N-terminus of the first Fc region variant, either directly or through a suitable linker. The second antigen-binding moiety may be fused to the N-terminus of the first antigen-binding moiety, either directly or through a suitable linker.

[0143] In a biparatope-like or bispecific polypeptide complex of the present disclosure having a first and a second antigen-binding moiety, the first and second antigen-binding moieties may be fused (directly or indirectly) to the same first Fc region variant to provide a polypeptide complex in a "one-armed" molecular format, or the first antigen-binding moiety may be fused to one of the first and second Fc region variants and the second antigen-binding moiety to the other to provide a polypeptide complex in a conventional "two-armed" molecular format. In any embodiment of the one-armed or two-armed biparatope-like or bispecific polypeptide complex described above, comprising a first antigen-binding moiety and a second antigen-binding moiety, the second antigen-binding moiety can bind to a different epitope on the same antigen to which the first antigen-binding moiety binds or to a different antigen to which the first antigen-binding moiety binds.

[0144] Linker Region In relation to the antigen-binding molecule or polypeptide complex of this disclosure, the “linker region” may be an antibody hinge region or a part thereof, or any other non-immunogenic linker, preferably a mobile linker, as long as the first polypeptide chain and the second polypeptide chain do not form a covalent bond (e.g., a disulfide bond) through the linker region. The linker used in the linker region of the antigen-binding molecule or polypeptide complex of this disclosure may be a peptide linker comprising one or more amino acids, typically about 2 to about 20 amino acid residues, which are described herein or known in the art. The length of the linker region included in the antigen-binding molecule or polypeptide complex of this disclosure is not particularly limited as long as the antigen-binding molecule or polypeptide complex of this disclosure is glomerular permeable, but may be, for example, about 5 to about 20 amino acid residues in length.

[0145] In some embodiments, the first polypeptide chain includes a first linker region, and the second polypeptide chain includes a second linker region, and no disulfide bond is formed between the first and second linker regions. Here, "no disulfide bond" means that no disulfide bond is formed between the two hinge regions / between the two linker regions. In examples of such embodiments, a disulfide bond cannot be formed between the first and second linker regions. In certain embodiments, either or both of the first and second linker regions lack Cys residues, and therefore, no disulfide bond can be formed between them. In some embodiments, the amino acid sequences of the first and second linker regions are the same. In some other embodiments, the amino acid sequences of the first and second linker regions are different. In one embodiment, either or both of the first and second linker regions are variants of the hinge region or a part thereof of natural IgG. In one embodiment, either or both of the first and second linker regions are modified hinge regions (sometimes called modified antibody hinge regions) in which one or more Cys residues of the hinge region or a part of the natural IgG are replaced with amino acid residues other than Cys residues, as hinge regions that do not form disulfide bonds. In one embodiment, either or both of the first and second linker regions are modified hinge regions that are variants of any or a part of the hinge region of natural IgG1, natural IgG2, or natural IgG4, and have amino acid residues other than Cys residues at positions 226 (EU index) and 229 (EU index), and in the case of variants of the IgG2 hinge region, further have amino acid residues other than Cys residues at positions 219 and 220 (EU index).In one embodiment, either or both of the first and second linker regions are variants of any or a part of the hinge region of natural IgG1, natural IgG2, or natural IgG4, as a hinge region that does not form a disulfide bond, and are modified hinge regions having Ala or Ser at positions 226 (EU index) and 229 (EU index) (and, in the case of the variant of the IgG2 hinge region, further at positions 219 and 220 (EU index)). In one embodiment, the hinge region variant has amino acid residues at positions other than 226 (EU index) and 229 (EU index) (and, in the case of the variant of the IgG2 hinge region, further at positions 219 and 220 (EU index)) that differ from the natural IgG hinge region of the corresponding isotype, as a hinge region that does not form a disulfide bond.

[0146] In a particular embodiment, the amino acid sequences of either or both of the first and second linker regions are: (1) EPKSCDKTHTAPPAP (SEQ ID NO: 19); (2) EPKSCDKTHTSPPSP (SEQ ID NO: 20); (3) EPKSCDKTHTAPPSP (SEQ ID NO: 21); (4) EPKSCDKTHTSPPAP (SEQ ID NO: 22); (5) DKTHTAPPAP (SEQ ID NO: 24); (6) DKTHTSPPSP (SEQ ID NO: 25); (7) DKTHTAPPSP (SEQ ID NO: 26); (8) DKTHTSPPAP (SEQ ID NO: 27); (9) ERKCCVEAPPAP (SEQ ID NO: 29); (10) ERKCCVESPPSP (SEQ ID NO: 30); (11) ERKAAVEAPPAP (SEQ ID NO: 31); (12) ERKSSVEAPPAP (SEQ ID NO: 32); (13) It is one of the following: (14) ERKASVEAPPAP (SEQ ID NO: 33); (15) ERKSAVEAPPAP (SEQ ID NO: 34); (16) ERKAAVESPPSP (SEQ ID NO: 35); (17) ERKASVESPPSP (SEQ ID NO: 36); (18) ERKSAVESPPSP (SEQ ID NO: 38); (19) ESKYGPPAPPAP (SEQ ID NO: 40); (20) ESKYGPPAPSAP (SEQ ID NO: 41); (21) ESKYGPPSPSSP (SEQ ID NO: 42); (22) ESKYGPPSPPSP (SEQ ID NO: 43); (23) ESKYGPPAPPSP (SEQ ID NO: 45); (24) ESKYGPPSPPAP (SEQ ID NO: 46).

[0147] In one embodiment, the amino acid sequences of the first and second linker regions are not VPPPPP (Sequence ID: 49). In a particular embodiment, the amino acid sequence of one of the first and second linker regions is EPPPPP (Sequence ID: 50), and the sequence of the other is GPPPPP (Sequence ID: 51).

[0148] In some embodiments, either or both of the first and second linker regions are peptide linkers such as glycine polymer (G)n (where n is at least an integer of 1, e.g., 6 or 8), glycine-serine polymer (e.g., (GGGGS: SEQ ID NO: 53)n, where n is at least an integer of 1, e.g., 1 to 4), glycine-alanine polymer, alanine-serine polymer, etc. Examples of peptide linkers include: Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 53) (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 53))n (SEQ ID NO: 63) Gly Gly Gly Gly Ala (GGGGA, SEQ ID NO: 54) Gly Gly Gly Gly Glu (GGGGE, SEQ ID NO: 55) Gly Gly Gly Ser (GGGS, SEQ ID NO: 56) (Gly Gly Gly Ser (GGGS, SEQ ID NO: 56))n (SEQ ID NO: 66) Gly Gly Gly Ala (GGGA, SEQ ID NO: 57) Gly Gly Gly Glu (GGGE, SEQ ID NO: 58) Gln Gln Gln Gly (QQQG, SEQ ID NO: 59) Gln Gln Gln Gln Gly (QQQQG, SEQ ID NO: 60) Ser Ser Ser Gly (SSSG, SEQ ID NO: 61) Ser Ser Ser Ser Gly (SSSSG, SEQ ID NO: 62), (Glu Ala Ala Ala Lys)n ((EAAAK)n, SEQ ID NO: 107), Ala (Glu Ala Ala Ala Lys)n, Ala (A(EAAAK)nA, SEQ ID NO: 108) (where n is an integer greater than or equal to 1) may be included, but are not limited to these. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0149] In certain embodiments, either or both of the first and second linker regions are movable linkers (GS linkers) made of a glycine-serine polymer, for example, (G4S) n (SG4) n (G4S) n , or G4 (SG4) nIt is a peptide linker, where n is generally an integer between 1 and 10, typically between 2 and 4. Examples of GS linkers include: Ser Gly Ser (GS) Ser Gly (SG) Gly Gly Ser (GGS) 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, Sequence ID: 56) Gly Gly Ser Gly (GGSG, Sequence ID: 73) Gly Ser Gly Gly (GSGG, Sequence ID: 74) Ser Gly Gly Gly (SGGG, Sequence ID: 75) Gly Ser Ser Gly (GSSG, Sequence ID: 76) Gly Gly Gly Gly Ser (GGGGS, Sequence ID: 53) Gly Gly Gly Ser Gly (GGGSG, Sequence ID: 77) Gly Gly Ser Gly Gly (GGSGG, Sequence ID: 78) Gly Ser Gly Gly Gly (GSGGG, Sequence ID: 79) Gly Ser Gly Gly Ser (GSGGS, Sequence ID: 80) Ser Gly Gly Gly Gly (SGGGG, Sequence ID: 81) Gly Ser Ser Gly Gly (GSSGG, Sequence ID: 82) Gly Ser Gly Ser Gly (GSGSG, Sequence ID: 83) Ser Gly Gly Ser Gly (SGGSG, Sequence ID: 84) Gly Ser Ser Ser Gly (GSSSG, Sequence ID: 85) Gly Gly Gly Gly Gly Ser (GGGGGS, Sequence ID: 86) Ser Gly Gly Gly Gly Gly (SGGGGG, Sequence ID: 87) Gly Gly Gly Gly Gly Gly Ser (GGGGGGS, Sequence ID: 88) Ser Gly Gly Gly Gly Gly (SGGGGGG, Sequence ID: 89) (Gly Gly Gly Gly Ser (GGGGS, Sequence ID: 53))n (Sequence ID: 63) (Ser Gly Gly Gly Gly (SGGGG, Sequence ID: 81))n (Sequence ID: 98) (n is an integer greater than or equal to 1) may include, but is not limited to, these.However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0150] In some embodiments, either or both of the first and second linker regions are synthetic compound linkers (chemical crosslinking agents) commonly used for crosslinking peptides. Examples of synthetic compound linkers (chemical crosslinking agents) may include, but are not limited to, N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0151] In certain embodiments, only one of the first and second polypeptide chains includes a linker region linked to the N-terminus of the antibody CH2 domain. In some examples of such embodiments, the linker region is a native antibody hinge region or a variant thereof. In some examples of such embodiments, the amino acid sequences of the linker region are: 1. EPKSCDKTHTCPPCP (SEQ ID NO: 18); 2. EPKSCDKTHTAPPAP (SEQ ID NO: 19); 3. EPKSCDKTHTSPPSP (SEQ ID NO: 20); 4. EPKSCDKTHTAPPSP (SEQ ID NO: 21); 5. EPKSCDKTHTSPPAP (SEQ ID NO: 22); 6. DKTHTCPPCP (SEQ ID NO: 23); 7. DKTHTAPPAP (SEQ ID NO: 24); 8. DKTHTSPPSP (SEQ ID NO: 25); 9. DKTHTAPPSP (SEQ ID NO: 26); 10. DKTHTSPPAP (SEQ ID NO: 27); 11. ERKCCVECPPCP (SEQ ID NO: 28); 12. ERKCCVEAPPAP (SEQ ID NO: 29); 13. ERKCCVESPPSP (SEQ ID NO: 30); 14. ERKAAVEAPPAP (SEQ ID NO: 31); 15. ERKSSVEAPPAP (SEQ ID NO: 32); 16. ERKASVEAPPAP (SEQ ID NO: 33); 17. ERKSAVEAPPAP (SEQ ID NO: 34); 18. ERKAAVESPPSP (SEQ ID NO: 35); 19. ERKSSVESPPSP (SEQ ID NO: 36); 20. ERKASVESPPSP (SEQ ID NO: 37); 21. ERKSAVESPPSP (SEQ ID NO: 38); 22. ESKYGPPCPSCP (SEQ ID NO: 39); 23. ESKYGPPAPPAP (SEQ ID NO: 40); 24. ESKYGPPAPSAP (SEQ ID NO: 41); 25. ESKYGPPSPSSP (SEQ ID NO: 42); 26. ESKYGPPSPPSP (SEQ ID NO: 43); 27. ESKYGPPCPPCP (SEQ ID NO: 44); 28. ESKYGPPAPPSP (Sequence ID: 45); 29. ​​ESKYGPPSPPAP (Sequence ID: 46); 30. EPPPCP (Sequence ID: 47); 31.GPPPCP (SEQ ID NO: 48); 32. VPPPPP (SEQ ID NO: 49); 33. EPPPPP (SEQ ID NO: 50); 34. GPPPPP (SEQ ID NO: 51); 35. Glycine polymer; 36. Glycine-serine polymer; 37. Glycine-alanine polymer; 38. Alanine-serine polymer; 39. One of the following synthetic compound linkers.

[0152] In some embodiments, each of the two monomeric Fc regions constituting the dimeric Fc region of the antigen-binding molecule or polypeptide complex of the present disclosure, namely the Fc region in the first polypeptide chain and the Fc region in the second polypeptide chain, has a different amino acid sequence.

[0153] In some embodiments, the antigen-binding molecule or polypeptide complex of the present disclosure is a heterodimer in which an antigen-binding domain or ligand is linked only to a first polypeptide chain containing one of the two monomeric Fc regions constituting the dimeric Fc region of the antigen-binding molecule or polypeptide complex, while the second polypeptide chain containing the other monomeric Fc region is not linked to either an antigen-binding domain or a ligand. Therefore, it is preferable that a heterodimer of the first polypeptide chain and the second polypeptide chain is efficiently formed, rather than a homodimer of the first polypeptide chain or a homodimer of the second polypeptide chain. In some embodiments, each of the two monomeric Fc regions constituting the dimeric Fc region of the antigen-binding molecule or polypeptide complex of the present disclosure, i.e., the Fc region in the first polypeptide chain and the Fc region in the second polypeptide chain, contains at least one amino acid modification that facilitates their association.

[0154] Techniques for facilitating the association between H chains having a desired combination, and between L chains and H chains, can be applied to the association between two monomeric Fc regions constituting a dimeric Fc region.

[0155] In a particular embodiment, techniques for suppressing undesirable H chain association by introducing electrostatic repulsion at the interface of the CH2 or CH3 region of an antibody H chain to efficiently produce a bispecific antibody that is a heterodimer molecule (see, for example, WO2006 / 106905) can be applied to the association of two monomeric Fc regions (with an antigen-binding domain linked to only one monomer) that constitute a dimeric Fc region in an antigen-binding molecule or polypeptide complex of the present disclosure. In one embodiment, one or more amino acid residues that form the interface between an Fc region in a first polypeptide chain and an Fc region in a second polypeptide chain are electrically repelling amino acid residues from each other.

[0156] In this disclosure, "interface" usually refers to the association surface during association (interaction), and the amino acid residues forming the interface usually refer to one or more amino acid residues contained in the polypeptide region that are subject to the association, more preferably amino acid residues that approach each other during the association and participate in the interaction. Specifically, such interactions include cases where the amino acid residues that approach each other during the association form hydrogen bonds, electrostatic interactions, salt bridges, etc.

[0157] In a technique for suppressing unintended H chain association by introducing electrostatic repulsion at the interface of the CH2 or CH3 region, examples of amino acid residues that come into contact at the interface of the other Fc region of the H chain include regions corresponding to the residues at EU numbering positions 356, 439, 357, 370, 399, and 409 in the CH3 region.

[0158] More specifically, an example of an antibody containing two types of H chain CH3 regions is one in which 1 to 3 pairs of amino acid residues selected from the following pairs of amino acid residues in the first H chain CH3 region have the same charge: (1) amino acid residues at EU numbering positions 356 and 439 in the H chain CH3 region, (2) amino acid residues at EU numbering positions 357 and 370 in the H chain CH3 region, and (3) amino acid residues at EU numbering positions 399 and 409 in the H chain CH3 region.

[0159] Furthermore, the antibody may be one in which a pair of amino acid residues in a second H chain CH3 region, distinct from the first H chain CH3 region, is selected from the pairs of amino acid residues (1) to (3), and 1 to 3 pairs of amino acid residues corresponding to the pairs of amino acid residues (1) to (3) having the same charge in the first H chain CH3 region have the opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

[0160] The amino acid residues shown in (1) to (3) above are close to each other when they associate. A person skilled in the art can find the positions corresponding to the amino acid residues in (1) to (3) above in a desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can modify the amino acid residues at these positions as appropriate.

[0161] In the above antibody, the "charged amino acid residue" is preferably selected from amino acid residues belonging to any one of the following groups: (a) glutamic acid (E) and aspartic acid (D), and (b) lysine (K), arginine (R), and histidine (H).

[0162] In the above antibodies, the phrase "having the same charge" means, for example, that any of the two or more amino acid residues are selected from amino acid residues belonging to either group (a) or (b) above. The phrase "having opposite charges" means, for example, that if at least one of the two or more amino acid residues is selected from amino acid residues belonging to either group (a) or (b) above, then the remaining amino acid residues are selected from amino acid residues belonging to the other group.

[0163] In the present invention, the amino acid residues to be modified are not limited to the amino acid residues in the antibody Fc region described above. Those skilled in the art can identify the amino acid residues forming the interface in the mutant polypeptide or heteropolymer using homology modeling with commercially available software, and then modify the amino acid residues at these positions to control their association.

[0164] Other known techniques can also be used for the association between two monomeric Fc regions constituting the dimeric Fc region of this disclosure. By substituting an amino acid side chain in one monomeric Fc region with a larger side chain (knob) and substituting an amino acid side chain in the other monomeric Fc region with a smaller side chain (hole), thereby enabling the placement of the knob within the hole, Fc region-containing polypeptides containing different amino acids can be efficiently associated with each other (WO1996 / 027011; Ridgway JB et al., Protein Engineering (1996) 9, 617-621; Merchant AM et al. Nature Biotechnology (1998) 16, 677-681; and US20130336973).

[0165] In addition, other known techniques can also be used for the association between the two monomeric Fc regions constituting the dimeric Fc region of this disclosure. By replacing a portion of the CH3 region of one monomeric Fc region with a corresponding IgA-derived sequence and introducing the corresponding IgA-derived sequence into the complementary portion of the CH3 region of the other monomeric Fc region, a strand-exchange engineered domain CH3 can be produced, which can efficiently induce the association of polypeptides having different sequences through complementary association of CH3 regions (Protein Engineering Design & Selection, 23; 195-202, 2010).

[0166] In addition, the formation of antigen-binding molecules or polypeptide complexes of this disclosure includes antibody production techniques using the association of CH1 and CL and VH and VL of antibodies, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb; 32(2):191-8; techniques for producing bispecific antibodies using a combination of separately prepared monoclonal antibodies, as described in WO2008 / 119353 and WO2011 / 131746 (Fab arm exchange); techniques for controlling the association between antibody heavy chain CH3s, as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two light chains and one heavy chain, as described in WO2012 / 023053; and Christoph et al. (Nature Biotechnology Vol. 31, p Techniques for producing bispecific antibodies using two bacterial cell lines that individually express one of the chains of an antibody, each containing one H chain and one L chain, as described in 753-758 (2013), may also be used.

[0167] Alternatively, even if the desired antigen-binding molecule or polypeptide complex cannot be efficiently formed, the antigen-binding molecule or polypeptide complex of this disclosure can be obtained by separating and purifying the desired antigen-binding molecule or polypeptide complex from the produced molecule. For example, in the antigen-binding molecule or polypeptide complex of this disclosure, the antigen-binding domain is linked to only one of the two monomeric Fc regions constituting the dimeric Fc region, so separation and purification can be performed by utilizing the difference in molecular weight between the two homomer forms and the desired heteromer molecule. Furthermore, as a method for purifying heteromer antigen-binding molecules, a method has been reported to date in which a heterodimer antibody containing a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A is purified using protein A (WO98050431 and WO95033844). Furthermore, by using H chains in which the amino acid residues at EU numbering positions 435 and 436, which are the IgG-protein A binding sites, are substituted with amino acid residues such as Tyr or His that result in different protein A affinity, or by using H chains with different protein A affinity, the interaction between each H chain and protein A can be altered, and then by using a protein A column, the heterodimeric antigen-binding molecule itself can be efficiently purified.

[0168] In one aspect of nucleic acid vectors, the Disclosure relates to isolated nucleic acids encoding antigen-binding molecules or polypeptide complexes of the Disclosure. In one aspect, the Disclosure relates to vectors supporting such nucleic acids.

[0169] The nucleic acids in this disclosure are typically loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (Stratagene) can be used as a cloning vector, and various commercially available vectors can also be used. When using a vector for the purpose of producing the polypeptides in this disclosure, expression vectors are particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in living organisms. For example, the pBEST vector (Promega) is used for in vitro expression, the pET vector (Invitrogen) is used for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is used for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472(1988)) is used for living organisms. Insertion of the DNA of this disclosure into a vector can be performed, for example, using the In-Fusion Advantage PCR Cloning Kit (Clontech).

[0170] The present disclosure further relates to host cells having the nucleic acids. The host cells are not particularly limited, and various host cells such as Escherichia coli and various animal cells may be used depending on the purpose. The host cells can be used, for example, as production systems for the production or expression of antibodies or polypeptides of the present disclosure. Production systems for polypeptide production include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

[0171] Examples of eukaryotic cells that can be used as host cells include animal cells, plant cells, and fungal cells. Examples of animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108: 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, C127, HEK293, Bowes melanoma cells, Vero, etc., amphibian cells such as African clawed frog oocytes (Valle et al., Nature (1981) 291: 338-340), and insect cells such as Drosophila S2, Sf9, Sf21, and Tn5. For the expression of the antibodies disclosed herein, CHO-DG44, CHO-DX11B, COS7 cells, and BHK cells are preferably used. In animal cells, CHO cells are used as an example when the goal is high-volume expression. Vector introduction into host cells can be performed using known methods such as the calcium phosphate method, the DEAE dextran method, the cationic ribosome DOTAP (Boehringer Mannheim), electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection, lipofectamine method (GIBCO-BRL), and microinjection. Furthermore, the HEK293 cell line can be used to perform gene transfer and polypeptide expression.

[0172] As for plant cells, for example, cells derived from Nicotiana tabacum are known as a protein production system, and the antibodies of this disclosure can be produced by callus culture of these cells. As for fungal cells, protein expression systems using yeast, such as cells of the genus Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces pombe), and filamentous fungi, such as cells of the genus Aspergillus (e.g., Aspergillus niger), are known and can be used as hosts for antibody production of this disclosure.

[0173] When using prokaryotic cells, there are production systems that utilize bacterial cells. In addition to the aforementioned Escherichia coli (E. coli), production systems using Streptococcus, Staphylococcus, Streptomyces, and Bacillus subtilis are known and can be used for antibody production as disclosed here.

[0174] In one aspect of a manufacturing method, the Disclosure provides a method for producing an antigen-binding molecule or polypeptide complex, comprising the step of culturing host cells of the Disclosure so as to produce an antigen-binding molecule or polypeptide complex. In one embodiment, the manufacturing method of the Disclosure further comprises the step of recovering the antigen-binding molecule or polypeptide complex from the host cells of the Disclosure. In one embodiment, some steps of the manufacturing method of the Disclosure can be carried out using known methods. In this Specification, “process” may be described as “step,” “stage,” etc.

[0175] To produce antigen-binding molecules or polypeptide complexes using the host cells of this disclosure, the host cells transformed with an expression vector containing a nucleic acid (polynucleotide) encoding the antigen-binding molecules or polypeptide complexes of this disclosure should be cultured and the nucleic acid expressed. Culturing can be carried out according to known methods. For example, when animal cells are used as the host, DMEM, MEM, RPMI1640, or IMDM can be used as the culture medium. In this case, serum supplementation such as FBS or fetal bovine serum (FCS) may be used in combination, or the cells may be cultured in serum-free culture. The pH during culture should be approximately 6 to 8 as an example. Culturing is usually carried out at approximately 30 to 40°C for approximately 15 to 200 hours, with the medium being changed, aerated, and stirred as needed.

[0176] On the other hand, examples of systems for producing polypeptides such as antigen-binding molecules or polypeptide complexes in vivo include production systems using animals and production systems using plants. A target nucleic acid (polynucleotide) is introduced into these animals or plants, causing them to produce polypeptides within their bodies, which are then recovered. In this disclosure, "host" encompasses these animals and plants.

[0177] To cause polypeptides expressed in host cells to be secreted into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretory signals can be incorporated into the target polypeptide. These signals may be endogenous or heterologous to the target polypeptide.

[0178] In the above-described manufacturing method, if the polypeptide, such as the antigen-binding molecule or polypeptide complex, is secreted into the culture medium, the culture medium is recovered. If the polypeptide, such as the antigen-binding molecule or polypeptide complex, is produced inside a cell, the cell is first lysed, and then the polypeptide is recovered.

[0179] When using animals, production systems can utilize mammals or insects. Mammals that can be used include goats, pigs, sheep, mice, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). Furthermore, when using mammals, transgenic animals can be employed.

[0180] For example, a nucleic acid (polynucleotide) encoding a polypeptide, such as an antigen-binding molecule or polypeptide complex, is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk, such as goat β-casein. Then, a nucleic acid fragment containing this fusion gene is injected into a goat embryo, and this embryo is transplanted into a female goat. The target antibody can be obtained from the milk produced by the transgenic goat born from the embryo-receiving goat or its offspring. To increase the amount of antibody-containing milk produced by the transgenic goat, hormones may be administered to the transgenic goat as appropriate (Ebert et al., Bio / Technology (1994) 12: 699-702).

[0181] Furthermore, silkworms can be used as insects to produce the antigen-binding molecules or polypeptide complexes of this disclosure. When using silkworms, the target antibody can be obtained from the body fluids of silkworms by infecting them with a baculovirus into which a polynucleotide encoding the target antibody has been inserted (Susumu et al., Nature (1985) 315: 592-4).

[0182] Furthermore, when using plants to produce polypeptides such as antigen-binding molecules or polypeptide complexes of this disclosure, tobacco can be used, for example. When using tobacco, the nucleic acid (polynucleotide) encoding the target polypeptide is inserted into a plant expression vector, such as pMON 530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This bacterium is then used to infect tobacco, such as Nicotiana tabacum, and the desired antibody can be obtained from the leaves of this tobacco (Ma et al., Eur. J. Immunol. (1994) 24: 131-8).

[0183] The polypeptides obtained in this way can be isolated from inside or outside the host cell (culture medium, milk, etc.) and purified as substantially pure and homogeneous antibodies. The isolation and purification of antibodies can be performed using any separation and purification method that is normally used for polypeptide purification, and are not limited in any way. For example, antibodies can be isolated and purified by appropriately selecting and combining methods such as ammonium sulfate or ethanol precipitation, acid extraction, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0184] Examples of chromatography include affinity chromatography, ion exchange chromatography such as anion exchange chromatography or cation exchange chromatography, phosphocellulose chromatography, hydrophobic (interaction) chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography, hydroxyl apatite chromatography, and lectin chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). These chromatography methods can be performed using liquid-phase chromatography, such as HPLC and FPLC. Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, columns using protein A include Hyper D, POROS, and Sepharose FF (manufactured by Pharmacia).

[0185] If necessary, the polypeptide can be modified or partially removed by treating it with an appropriate protein-modifying enzyme before or after purification. Examples of protein-modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, and glucosidase.

[0186] In some embodiments, the antigen-binding molecules or polypeptide complexes of the present disclosure pass through the renal glomeruli and are taken up by renal tissue. In one embodiment, the antigen-binding molecules or polypeptide complexes of the present disclosure accumulate in the kidney, for example, in the cells of the proximal tubules. Thus, in one embodiment, the antigen-binding molecules or polypeptide complexes of the present disclosure can be used to efficiently deliver molecules conjugated to them (particularly bioactive molecules) to the kidney.

[0187] In one aspect, the Disclosure provides an antigen-binding molecule or polypeptide complex of the Disclosure for use as a reagent for renal delivery. In one aspect, the Disclosure provides an antigen-binding molecule or polypeptide complex of the Disclosure for delivery to and / or accumulation in the kidney. In one aspect, the Disclosure provides an antigen-binding molecule or polypeptide complex of the Disclosure for delivery to and / or accumulation in the kidney. In one aspect, the Disclosure provides a composition or pharmaceutical composition for delivery to and / or accumulation of a physiologically active molecule in the kidney, comprising an antigen-binding molecule or polypeptide complex of the Disclosure. In one aspect, the Disclosure provides the use of an antigen-binding molecule or polypeptide complex of the Disclosure in the manufacture of a pharmaceutical composition for renal delivery. In one aspect, the Disclosure provides the use of an antigen-binding molecule or polypeptide complex of the Disclosure in the manufacture of a composition or pharmaceutical composition for delivery to and / or accumulation of a physiologically active molecule in the kidney. In one aspect, the Disclosure provides a method for delivery to and / or accumulation of an antigen-binding molecule or polypeptide complex in the kidney. In one aspect, the Disclosure provides a method for delivering and / or accumulating a bioactive molecule in the kidney, comprising the step of administering an antigen-binding molecule or polypeptide complex of the Disclosure conjugated thereto. In certain embodiments of these aspects, the antigen-binding molecule or polypeptide complex of the Disclosure, or a bioactive molecule conjugated thereto, is delivered to and / or accumulated in proximal tubular cells, podocytes, distal tubular cells, or collecting duct cells. In certain embodiments, the bioactive molecule is a cytotoxic substance, nucleic acid, peptide (including cyclic peptides), or small molecule compound.

[0188] In one aspect of renal imaging, the Disclosure provides an antigen-binding molecule or polypeptide complex of the Disclosure for use in renal imaging. In another aspect, the Disclosure provides the use of an antigen-binding molecule or polypeptide complex of the Disclosure in the manufacture of reagents for renal imaging. In another aspect, the Disclosure provides a renal imaging method comprising the step of administering an antigen-binding molecule or polypeptide complex of the Disclosure. In certain embodiments of these aspects, the antigen-binding molecule or polypeptide complex used is labeled (e.g., fluorescently labeled or radioactively labeled).

[0189] In one therapeutic aspect, the Disclosure provides the antigen-binding molecule or polypeptide complex of the Disclosure for use as a pharmaceutical product. In another aspect, the Disclosure provides the use of the antigen-binding molecule or polypeptide complex of the Disclosure in the manufacture of a pharmaceutical product.

[0190] In one aspect, the Disclosure provides an antigen-binding molecule or polypeptide complex of the Disclosure for use in the treatment of kidney disease. In another aspect, the Disclosure provides the use of an antigen-binding molecule or polypeptide complex of the Disclosure in the manufacture of a pharmaceutical product for the treatment of kidney disease. In another aspect, the Disclosure provides a method for treating kidney disease, comprising the step of administering an antigen-binding molecule or polypeptide complex of the Disclosure.

[0191] In one aspect, the Disclosure provides a pharmaceutical formulation comprising an antigen-binding molecule or polypeptide complex of the Disclosure and a pharmaceutically acceptable carrier. In one aspect, the Disclosure provides a pharmaceutical composition comprising an antigen-binding molecule or polypeptide complex of the Disclosure and a pharmaceutically acceptable carrier. In one aspect, the Disclosure provides a pharmaceutical composition for the treatment of kidney disease comprising an antigen-binding molecule or polypeptide complex of the Disclosure and a pharmaceutically acceptable carrier. In one aspect, the Disclosure provides a composition for kidney imaging comprising a fluorescently labeled or radiolabeled antigen-binding molecule or polypeptide complex of the Disclosure. In one aspect, the Disclosure provides a pharmaceutical composition for kidney delivery comprising an antigen-binding molecule or polypeptide complex of the Disclosure and a pharmaceutically acceptable carrier.

[0192] In one aspect of the kit, the Disclosure provides a kit comprising an antigen-binding molecule or polypeptide complex of the Disclosure and a pharmaceutically acceptable carrier. In one embodiment, the Kit of the Disclosure is a kit for the treatment of kidney disease. In one embodiment, the Kit of the Disclosure is a kit for kidney delivery. In one embodiment, the Kit of the Disclosure is a kit for kidney imaging. In one embodiment, the antigen-binding molecule or polypeptide complex of the Disclosure may be fluorescently labeled or radioactively labeled. In one embodiment, the Kit of the Disclosure may include instructions for carrying out the method of the Disclosure.

[0193] All references cited herein are incorporated herein by reference.

[0194] The following are examples of the antigen-binding molecule, composition, and method of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0195] [Example 1] Evaluation of antibodies with different presence or absence of inter-heavy chain disulfide bonds and number of antigen-binding domains Example 1-1. Production of antibodies with different presence or absence of inter-heavy chain disulfide bonds and number of antigen-binding domains Antibodies were produced with different presence or absence of inter-heavy chain disulfide bonds and number of antigen-binding domains of the anti-KLH IgG1 antibody Ab_1 (heavy chain variable region: IC17HdK (SEQ ID NO: 1), heavy chain constant region: G1T7 (SEQ ID NO: 2), light chain variable region: IC17L (SEQ ID NO: 3), light chain constant region: k0 (SEQ ID NO: 4)). To reduce the number of antigen-binding domains in the antibody, a knobs-into-holes (KiH) mutation was introduced into the CH3 region of Ab_1, and Ab_1_KiH (heavy chain constant region 1: G1T7k (SEQ ID NO: 5), heavy chain constant region 2: G1T7h (SEQ ID NO: 6)) which forms a heterodimer was produced. Furthermore, by deleting the CH1 region of heavy chain constant region 2 of Ab_1_KiH, Ab_2 (heavy chain constant region 1: G1T7k (SEQ ID NO: 5), heavy chain constant region 2: G1T7h.dCH1 (SEQ ID NO: 7)) with one antigen-binding domain was created. To eliminate the formation of inter-heavy chain disulfide bonds in the antibodies, modifications were introduced in which Cys in the hinge region of Ab_1 and Ab_2 was replaced with Ala, or the hinge region was changed from IgG1 type to IgA2 type, or the hinge region was deleted, resulting in the heavy chain constant region variants shown in Table 1. Expression vectors for the antibodies shown in Table 2 were prepared by methods known to those skilled in the art, and the antibodies shown in Table 2 were expressed and purified using the HEK293 cell line by methods known to those skilled in the art. The molecular forms of the antibodies shown in Table 2 are shown in Figure 1, and their hinge region amino acid sequences are shown in Table 3.

[0196]

[0197]

[0198]

[0199] Example 1-2. Labeling of antibodies with different presence or absence of inter-heavy chain disulfide bonds and number of antigen-binding domains. To evaluate the in vivo dynamics of the antibodies prepared in Example 1-1, the lysine residues of the antibodies were labeled with the fluorescent dye VivoTag 680XL (Perkin. Elmer). The antibodies were mixed with VivoTag 680XL in a pH 8.3 sodium bicarbonate solution and incubated for 2 hours at room temperature under light shielding. To remove unreacted VivoTag 680XL, the labeled antibodies were purified using a 40K Zeba Spin Desalting Column (Thermo Fisher Scientific). Labeling efficiency and antibody concentration were calculated by measuring the absorbance at 280 nm and 668 nm using NanoDrop One (Thermo Fisher Scientific).

[0200] Examples 1-3. Evaluation of kidney accumulation of antibodies with different heavy chain disulfide bond presences and antigen-binding domain numbers. Fluorescently labeled antibodies prepared in Examples 1-2 were administered to C57BL / 6J mice (male) via tail vein at a dose of 5 mg / kg. Jugular vein blood was collected at time points from 5 minutes to 2 days after administration. The obtained blood was centrifuged (12,000 rpm, 4°C, 5 minutes) to recover the plasma. Two days after administration, PBS was perfused into the heart by inserting a catheter under isoflurane anesthesia. After systemic blood drainage, the kidneys were removed from the mice.

[0201] Plasma antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Anti-human IgG heavy and light chain antibody (Bethyl Laboratories) was added to the plate as the capture antibody. After adding phosphate-buffered saline with Tween20 (Sigma-Aldrich) containing 1% bovine serum albumin (Sigma-Aldrich) as a blocking solution, diluted plasma samples were added. Subsequently, biotin-labeled anti-ΔGK antibody (WO2019 / 112027) was added as the detection antibody, and Streptavidin-labeled polyHRP80 (Stereospecific Detection Technologies) was added as the secondary detection antibody. Finally, tetramethylbenzidine (Surmodics) was added as the substrate, and the absorbance at 650 nm was measured using a CLARIOstar spectrophotometer (BMG Labotech). The plasma antibody concentration profile is shown in Figure 2. All antibodies showed similar PK profiles.

[0202] The excised kidneys were mixed with PBS containing NP-40 alternative detergent (Millipore) and Complete Mini Protease Inhibitor Cocktail (Roche), and 5 mm stainless steel beads (Qiagen). The kidneys were then homogenized using Tissue Lyser II (Qiagen). The kidney homogenates were rotated at 4°C for 60 minutes, and the homogenate supernatant was collected by centrifugation (15000 rpm, 4°C, 20 min). The fluorescence intensity of the collected kidney homogenate samples was measured using a CLARIOstar spectrophotometer, and the antibody concentrations in the kidneys were calculated. The calculated kidney concentrations of each antibody are shown in Figure 3. Since the VivoTag concentration in urine does not vary much depending on the molecular weight and molecular shape of the antibody, it is thought that antibodies that have permeated the glomeruli are reabsorbed in the renal tubules. Therefore, in evaluating the renal transfer capacity of antibodies, the results of the amount of antibody accumulated in the kidney are important. As shown in Figure 2, each antibody showed equivalent blood concentrations. On the other hand, as shown in Figure 3, Ab_6, Ab_7, and Ab_8 showed higher renal concentrations than the other antibodies. These results suggest that antibodies possessing only one antigen-binding domain and lacking inter-heavy-chain disulfide bonds in the hinge region, as shown in Figure 4, can efficiently permeate the glomeruli due to their high mobility between Fc cells and narrow shape, thereby achieving high renal concentrations.

[0203] [Example 2] Evaluation of antibodies with one antigen-binding domain (one-armed antibody) that differ in the presence or absence of inter-heavy-chain disulfide bonds, hinge region sequence, and glycosylation sequence. Example 2-1. Production of antibodies with one antigen-binding domain (one-armed antibody) that differ in the presence or absence of inter-heavy-chain disulfide bonds, hinge region sequence, and glycosylation sequence. Antibodies were produced that differ in the presence or absence of inter-heavy-chain disulfide bonds, hinge region sequence, and glycosylation sequence of the anti-KLH IgG1 antibody Ab_9 (heavy chain variable region 1: IC17HdK (SEQ ID NO: 1), heavy chain constant region 1: G1T7k (SEQ ID NO: 5), heavy chain constant region 2: G1T7h.dCH1 (SEQ ID NO: 7), light chain variable region: IC17L (SEQ ID NO: 3), light chain constant region: k0 (SEQ ID NO: 4)), which has only one antigen-binding domain, in terms of the presence or absence of inter-heavy-chain disulfide bonds, hinge region sequence, and glycosylation sequence. To change the sequence of the antibody hinge region from IgG1 type, modified versions were created in which the hinge region of Ab_9 was changed to IgG2 type (heavy chain constant region 1: G1T7k.G2hinge (SEQ ID NO: 109), heavy chain constant region 2: G1T7h.dCH1.G2hinge (SEQ ID NO: 110)), or modified versions in which the hinge region of Ab_9 was changed to IgG4 type (heavy chain constant region 1: G1T7k.G4hinge (SEQ ID NO: 111), heavy chain constant region 2: G1T7h.dCH1.G4hinge (SEQ ID NO: 112)). To eliminate the glycosylation sequence present in the native antibody, modified versions were created in which Asn was replaced with Ala at position 297 (EU index) of Ab_9 (heavy chain constant region 1: G1T7k.N297A (SEQ ID NO: 113), heavy chain constant region 2: G1T7h.dCH1.N297A (SEQ ID NO: 114)). To eliminate the formation of interheavy chain disulfide bonds in the antibody, modified versions of Ab_9, modified versions of the hinge region of Ab_9, and modified versions of the glycosylation sequence of Ab_9 were created in which Cys in the hinge region was replaced with Ala, resulting in the heavy chain constant region modifications shown in Table 4. The antibody expression vectors shown in Table 5 were prepared by methods known to those skilled in the art, and the antibodies shown in Table 5 were expressed and purified using the HEK293 cell line by methods known to those skilled in the art. The molecular forms of the antibodies shown in Table 5 are shown in Figure 5, and their hinge region amino acid sequences are shown in Table 6.

[0204]

[0205]

[0206]

[0207] Example 2-2. Labeling of one-armed antibodies with a single antigen-binding domain, differing in the presence or absence of inter-heavy chain disulfide bonds, hinge region sequence, and glycosylation sequence. To evaluate the in vivo dynamics of the antibodies produced in Example 2-1, the lysine residues of the antibodies were labeled with the fluorescent dye VivoTag 680XL (Perkin. Elmer). The antibodies were mixed with VivoTag 680XL in a pH 8.3 sodium bicarbonate solution and incubated for 2 hours at room temperature under light shielding. To remove unreacted VivoTag 680XL, the labeled antibodies were purified using a 40K Zeba Spin Desalting Column (Thermo Fisher Scientific). Labeling efficiency and antibody concentration were calculated by measuring the absorbance at 280 nm and 668 nm using NanoDrop One (Thermo Fisher Scientific).

[0208] Example 2-3. Evaluation of the accumulation of antibodies with one antigen-binding domain (one-armed antibodies) differing in the presence or absence of inter-heavy-chain disulfide bonds, hinge region sequence, and glycosylation sequence in the kidneys. Fluorescently labeled antibodies prepared in Example 2-2 were administered to C57BL / 6J mice (male) at a dose of 5 mg / kg via the tail vein, and jugular vein blood was collected at time from 5 minutes to 2 days later. The obtained blood was centrifuged (12,000 rpm, 4°C, 5 minutes) and plasma was recovered. Two days after administration, PBS was perfused into the heart by inserting a catheter under anesthesia. After systemic blood drainage, the kidneys were removed from the mice.

[0209] Plasma antibody concentrations were measured by electrochemiluminescence ligand binding assay. Anti-human IgG heavy and light chain antibody (Sigma-Aldrich) was added to a plate (Meso Scale Discovery) as the capture antibody. After adding phosphate-buffered saline with Tween20 (Sigma-Aldrich) containing 1% bovine serum albumin (Sigma-Aldrich) as a blocking solution, diluted plasma samples were added. Subsequently, biotin-labeled anti-human IgG (SouthernBiotech) was added as the detection antibody, and SULFO-TAG-labeled Streptavidin (Meso Scale Discovery) was added as a secondary detection reagent. Finally, after adding Read buffer (Meso Scale Discovery), the electrochemiluminescence signal was measured using MESO SECTOR S 600 (Meso Scale Discovery). The plasma antibody concentration profile is shown in Figure 6. All antibodies showed similar PK profiles. The excised kidneys were placed in 3 mL freeze-grinding tubes, and Cell extraction buffer (Thermo Fisher Scientific), Complete Mini Protease Inhibitor Cocktail (Roche), and a metal cone were added. The kidneys were then homogenized using a multi-bead shocker (Yasui Kikai). The kidney homogenates were rotated at 4°C for 60 minutes, and the homogenate supernatant was collected by centrifugation (14000 rpm, 4°C, 15 min). The fluorescence intensity of the collected kidney homogenate samples was measured using a CLARIOstar spectrophotometer, and the kidney antibody concentrations were calculated. The calculated kidney concentrations of each antibody are shown in Figure 7.

[0210] As shown in Figure 6, each antibody showed similar blood concentrations. On the other hand, as shown in Figure 7, Ab_12 and Ab_14 showed higher renal concentrations than Ab_11 and Ab_13, respectively. These results suggest that even antibodies with IgG2 or IgG4 hinges, if they have only one antigen-binding domain and lack inter-heavy-chain disulfide bonds in the hinge region, can achieve high renal concentrations. Also, as shown in Figure 7, Ab_16 showed a higher renal concentration than Ab_15. These results suggest that even antibodies without glycosylated sequences, if they have only one antigen-binding domain and lack inter-heavy-chain disulfide bonds in the hinge region, can achieve high renal concentrations.

[0211] Despite containing a dimeric Fc region, the antigen-binding molecule of this disclosure can efficiently permeate the glomerulus and achieve high concentrations in the kidney. Having these characteristics, the antigen-binding molecule of this disclosure can, for example, cause the accumulation of a cytotoxic substance conjugated to it in the kidney, and can be used in the treatment of kidney diseases such as renal tumors.

Claims

1. An antigen-binding molecule comprising a dimeric Fc region and an antigen-binding domain, wherein either or both of the first polypeptide chain comprising one monomer of the Fc region and the second polypeptide chain comprising the other monomer of the Fc region do not contain an antibody hinge region capable of forming a disulfide bond, and the antigen-binding domain is linked only to the first polypeptide chain, and not to the second polypeptide chain.

2. The antigen-binding molecule according to claim 1, wherein there is only one antigen-binding domain linked to the first polypeptide chain.

3. The antigen-binding molecule according to claim 1 or 2, wherein the second polypeptide chain comprises only an Fc region, or comprises only a linker region and an Fc region in that order from the N-terminus.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the first polypeptide chain comprises a first linker region, the second polypeptide chain comprises a second linker region, and in the antigen-binding molecule, a disulfide bond cannot be formed between the first linker region and the second linker region.

5. The antigen-binding molecule according to claim 4, wherein the C-terminus of the first linker region is linked to the N-terminus of an Fc region contained in the first polypeptide chain.

6. The antigen-binding molecule according to claim 4 or 5, wherein the C-terminus of the second linker region is linked to the N-terminus of an Fc region contained in the second polypeptide chain.

7. The antigen-binding molecule according to any one of claims 4 to 6, wherein the amino acid sequence of the first linker region and the amino acid sequence of the second linker region are the same.

8. The antigen-binding molecule according to any one of claims 4 to 7, wherein the first linker region and the second linker region are variants of the hinge region of natural IgG.

9. The antigen-binding molecule according to any one of claims 4 to 8, wherein the first linker region and the second linker region are modified hinge regions in which one or more Cys amino acids in the hinge region of natural IgG are replaced with amino acids other than Cys.

10. The antigen-binding molecule according to any one of claims 4 to 7, wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having an amino acid other than Cys at positions 226 (EU index) and 229 (EU index).

11. The antigen-binding molecule according to any one of claims 4 to 7, wherein the first linker region and the second linker region are variants of the hinge region of natural IgG1, the hinge region of natural IgG2, or the hinge region of natural IgG4, and are modified hinge regions having Ala or Ser at positions 226 (EU index) and 229 (EU index).

12. The antigen-binding molecule according to claim 10 or 11, wherein the variant of the hinge region further has different amino acids from the native IgG hinge region of the corresponding isotype at positions other than 226 (EU index) and 229 (EU index).

13. The antigen-binding molecule according to claims 10 to 12, wherein the variant of the hinge region of IgG2 is a modified hinge region having an amino acid other than Cys at positions 219 and 220 (EU index).

14. The sequence of the first linker region and the second linker region is as follows: (1) EPKSCDKTHTAPPAP (Sequence ID: 19); (2) EPKSCDKTHTSPPSP (Sequence ID: 20); (3) EPKSCDKTHTAPPSP (Sequence ID: 21); (4) EPKSCDKTHTSPPAP (Sequence ID: 22); (5) DKTHTAPPAP (Sequence ID: 24); (6) DKTHTSPPSP (Sequence ID: 25); (7) DKTHTAPPSP (Sequence ID: 26); (8) DKTHTSPPAP (Sequence ID: 27); (9) ERKCCVEAPPAP (Sequence ID: 29); (10) ERKCCVESPPSP (Sequence ID: 30); (11) ERKAAVEAPPAP (Sequence ID: 31); (12) ERKSSVEAPPAP (Sequence ID: 32); (13) An antigen-binding molecule according to any one of claims 4 to 8, which is any of the following: (14) ERKASVEAPPAP (SEQ ID NO: 33); (15) ERKSAVEAPPAP (SEQ ID NO: 34); (16) ERKSSVESPPSP (SEQ ID NO: 36); (17) ERKASVESPPSP (SEQ ID NO: 37); (18) ERKSAVESPPSP (SEQ ID NO: 38); (19) ESKYGPPAPPAP (SEQ ID NO: 40); (20) ESKYGPPAPSAP (SEQ ID NO: 41); (21) ESKYGPPSPSSP (SEQ ID NO: 42); (22) ESKYGPPSPPSP (SEQ ID NO: 43); (23) ESKYGPPAPPSP (SEQ ID NO: 45); (24) ESKYGPPSPPAP (SEQ ID NO: 46).

15. The antigen-binding molecule according to any one of claims 4 to 7, wherein the sequence of the first linker region and the second linker region is not VPPPPP (Sequence ID: 49).

16. The antigen-binding molecule according to claim 4, wherein the sequence of one of the first linker region and the second linker region is EPPPPP (SEQ ID NO: 50) and the sequence of the other is GPPPPP (SEQ ID NO: 51).

17. The antigen-binding molecule according to any one of claims 4 to 7, wherein the arrangement of the first linker region and the second linker region is: (1) a glycine polymer; (2) a glycine-serine polymer; (3) a glycine-alanine polymer; (4) an alanine-serine polymer; or (5) a synthetic compound linker.

18. The antigen-binding molecule according to any one of claims 1 to 3, wherein only one of the first polypeptide chain and the second polypeptide chain includes a linker region linked to the N-terminus of the antibody CH2 domain.

19. The antigen-binding molecule according to claim 18, wherein the linker region is a natural antibody hinge region or a variant thereof.

20. The sequence of the linker region is as follows: (1) EPKSCDKTHTCPPCP (Sequence ID: 18); (2) EPKSCDKTHTAPPAP (Sequence ID: 19); (3) EPKSCDKTHTSPPSP (Sequence ID: 20); (4) EPKSCDKTHTAPPSP (Sequence ID: 21); (5) EPKSCDKTHTSPPAP (Sequence ID: 22); (6) DKTHTCPPCP (Sequence ID: 23); (7) DKTHTAPPAP (Sequence ID: 24); (8) DKTHTSPPSP (Sequence ID: 25); (9) DKTHTAPPSP (Sequence ID: 26); (10) DKTHTSPPAP (Sequence ID: 27); (11) ERKCCVECPPCP (Sequence ID: 28); (12) ERKCCVEAPPAP (Sequence ID: 29); (13) ERKCCVESPPSP (Sequence ID: 30); (14) ERKAAVEAPPAP (SEQ ID NO: 31); (15) ERKSSVEAPPAP (SEQ ID NO: 32); (16) ERKASVEAPPAP (SEQ ID NO: 33); (17) ERKSAVEAPPAP (SEQ ID NO: 34); (18) ERKAAVESPPSP (SEQ ID NO: 35); (19) ERKSSVESPPSP (SEQ ID NO: 36); (20) ERKASVESPPSP (SEQ ID NO: 37); (21) ERKSAVESPPSP (SEQ ID NO: 38); (22) ESKYGPPCPSCP (SEQ ID NO: 39); (23) ESKYGPPAPPAP (SEQ ID NO: 40); (24) ESKYGPPAPSAP (SEQ ID NO: 41); (25) ESKYGPPSPSSP (SEQ ID NO: 42); (26) ESKYGPPSPPSP (SEQ ID NO: 43); (27) ESKYGPPCPPCP (SEQ ID NO: 44); (28) ESKYGPPAPPSP (SEQ ID NO: 45); (29) ESKYGPPSPPAP (SEQ ID NO: 46); (30) EPPPCP (SEQ ID NO: 47); (31) GPPPCP (SEQ ID NO: 48); (32) VPPPPP (SEQ ID NO: 49); (33) EPPPPP (SEQ ID NO: 50); (34) GPPPPP (SEQ ID NO: 51); (35) Glycine polymer; (36) Glycine-serine polymer; (37) Glycine-alanine polymer;(38) an alanine-serine polymer; (39) a synthetic compound linker; the antigen-binding molecule according to claim 18.

21. The antigen-binding domain is linked to the N-terminus of the antibody CH2 domain in the first polypeptide chain without a linker, and the second polypeptide chain comprises only the Fc region, according to claim 1 or 2.

22. The antigen-binding molecule according to any one of claims 1 to 21, wherein the molecular weight of the antigen-binding domain is 50 kDa or less.

23. The antigen-binding molecule according to any one of claims 1 to 22, wherein the antigen-binding domain is Fab, scFv, or sdAb.

24. The antigen-binding molecule according to any one of claims 1 to 23, wherein the Fc region contained in the first polypeptide chain and the Fc region contained in the second polypeptide chain have different sequences.

25. The antigen-binding molecule according to any one of claims 1 to 24, wherein the Fc region is the Fc region of natural IgG or a variant thereof.

26. The antigen-binding molecule according to any one of claims 1 to 24, wherein the Fc region is the Fc region of natural IgG or a variant thereof, and does not have a glycosylation sequence.

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