antigen-binding molecules

Abstract

One object of this disclosure is to provide an antigen-binding molecule. [Solution] An antigen-binding molecule comprising a first polypeptide and a second polypeptide is provided, wherein the first polypeptide comprises a heavy chain variable region (VH) and the CH4 domain (IgM-CH4) of a first human immunoglobulin M, and the second polypeptide comprises a light chain variable region (VL) and a second IgM-CH4, wherein the first IgM-CH4 and the second IgM-CH4 are mutually bound, and VH and VL form an antigen-binding site.

Description

[Technical Field]

[0001] This disclosure relates to antigen-binding molecules. [Background technology]

[0002] Bispecific antibodies, which can be used as diagnostic or therapeutic drugs, are attracting attention. While natural antibodies contain two identical antigen-binding sites and specifically recognize one epitope, bispecific antibodies contain two different antigen-binding sites and recognize two different epitopes on the same or different antigens. Since the antigen-binding site is formed by the heavy chain variable region (VH) and the light chain variable region (VL), bispecific antibodies require the correct combination of two different heavy chains and two different light chains.

[0003] When expressing two different heavy chains and two different light chains in a single cell to obtain a bispecific antibody, theoretically 16 combinations (10 types of molecules) are possible, but only one combination exhibits the desired bispecificity. Various techniques have been developed to obtain the desired bispecific antibody (Non-Patent Literature 1). For example, there are techniques to form hetero-Fc dimers such as knobs-into-holes (Genentech) and Duobody (Genmab). This prevents the dimerization of heavy chains containing the same heavy chain variable region and makes it possible to combine heavy chains containing different heavy chain variable regions. However, since four different light chain combinations can be generated for the desired heavy chain combination, the theoretical yield of the desired bispecific antibody remains at 25% (the light chain problem).

[0004] To address the light chain issue, modifying the binding site between the heavy and light chains has been explored. In natural antibodies, the heavy and light chains are bound via the CH1 domain of the heavy chain and the CL domain of the light chain. By replacing the CH1 domain of one heavy chain and the CL domain of the other light chain with other domains that can bind to each other, a bispecific antibody can be obtained in which the heavy and light chains are combined as intended. Specifically, it has been proposed to replace one of the CH1 / CL domains of the antibody with an IgG1 CH3 variant (Patent Document 1), an IgE / IgM CH2 variant (Patent Document 2), or a TCRα / β variant (Patent Document 3). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2016 / 087650 Brochure [Patent Document 2] International Publication No. 2017 / 106462 brochure [Patent Document 3] International Publication No. 2021 / 046072 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] Brinkmann U, Kontermann RE. The making of bispecific antibodies. MAbs. 2017 Feb / Mar;9(2):182-212 [Overview of the project] [Problems that the invention aims to solve]

[0007] The purpose of this disclosure is to provide a novel antigen-binding molecule that can solve the light chain problem. [Means for solving the problem]

[0008] The inventors conducted extensive research to solve the light chain problem and developed an antigen-binding molecule with a novel structure.

[0009] Accordingly, in some embodiments, the present disclosure relates to an antigen-binding molecule comprising a first polypeptide and a second polypeptide, The first polypeptide comprises a heavy chain variable region (VH) and the CH4 domain (IgM-CH4) of the first human immunoglobulin M. The second polypeptide comprises a light chain variable region (VL) and a second IgM-CH4. The first IgM-CH4 and the second IgM-CH4 are mutually bound. VH and VL form the antigen-binding site. Provides antigen-binding molecules. [Effects of the Invention]

[0010] This disclosure provides an antigen-binding molecule having a novel structure. This antigen-binding molecule is useful, for example, in the efficient production of bispecific molecules. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram of an antigen-binding fragment (Fab) containing IgM-CH4. [Figure 2] This paper compares the amino acid sequences of human IgM-CH4 and IgG1's Cκ or CH1, and shows the optimized amino acid sequence of IgM-CH4. [Figure 3] The structure of human IgM is shown. The upper left is a schematic diagram of the whole. The lower left shows the Fc pentamer. The upper right is a magnified view of two Fc molecules within the Fc pentamer. The lower right is a magnified view of the pentamer formation interface. The arrows indicate three amino acids present at the interaction interface between CH4 molecules that are different from human IgG1 CH3. [Figure 4]Size-exclusion chromatograms of crudely purified Trastuzumab Fab (Native) with CH1 and CL replaced by IgM-CH4 and the molecule with V547Y / E549Q mutation added thereto are shown. The solid line arrow indicates the peak corresponding to the monomeric Fab. The dashed line arrow indicates the peak shoulder of Native. [Figure 5] Size-exclusion chromatograms of crudely purified Trastuzumab Fab (Native) with CH1 and CL replaced by IgM-CH4 and the molecules with V547Y, V547Y / E549Q, or M489E / V547Y mutations added thereto are shown. The solid line arrow indicates the peak corresponding to the monomeric Fab. The dashed line arrow indicates the peak shoulder of Native. [Figure 6] Schematic diagrams of IgG-type bispecific antibodies (T4h-Ok and T4k-Oh) with CH1 and CL of one Fab replaced by IgM-CH4. [Figure 7] Chromatogram of size-exclusion chromatography performed immediately after separating T4h-Ok and T4k-Oh from cell culture supernatant by protein A. The solid line arrow indicates the fraction containing the monomer of the target bispecific antibody. [Figure 8] Chromatogram of cation-exchange chromatography performed after the separation shown in Fig. 7. The solid line arrow indicates the fraction containing the monomer of the target bispecific antibody. [Figure 9] Chromatogram of cation-exchange chromatography performed immediately after separating T4h-Ok and T4k-Oh from cell culture supernatant by protein A. The solid line arrow indicates the fraction containing the monomer of the target bispecific antibody. [Figure 10] Chromatogram of size-exclusion chromatography performed after the separation shown in Fig. 9. [Figure 11] Sensorgrams when Trastuzumab, T4h-Ok and T4k-Oh are immobilized on the sensor chip and recombinant HER2 protein is flowed in a dilution series of 0.3125 - 40 nM are shown. The gray dashed line indicates the actual data. The black thin line indicates the fitting curve. [Figure 12]The results of detecting each antibody by flow cytometry after conjugating OKT3, T4h-Ok, T4k-Oh, or trastuzumab to SK-OV-3 cells or Jurkat cells are shown. [Figure 13] The following shows the results of co-culturing Raji cells or Raji-HER2 cells with Jurkat-Lucia-NFAT cells in the presence of T4h-Ok or T4k-Oh, and measuring the activity of the NFAT pathway. [Modes for carrying out the invention]

[0012] Unless otherwise specified, terms used in this disclosure have the meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Some definitions of terms used in this disclosure are given below, but these definitions take precedence over general understandings in this disclosure.

[0013] In this disclosure, when a number is accompanied by the term "approximately," it is intended to include a range of ±10% of that value. For example, "approximately 20" includes "18 to 22." A range of numbers includes all numbers between the two endpoints and the numbers at both endpoints. The "approximately" in relation to a range applies to both endpoints of that range. Thus, for example, "approximately 20 to 30" includes "18 to 33."

[0014] Antibodies, also called immunoglobulins, are proteins that specifically bind to antigens. Human antibodies have five major isotypes with different functions: IgG, IgA, IgD, IgE, and IgM. IgG is further classified into four subtypes (IgG1, IgG2, IgG3, and IgG4). An antibody consists of two heavy chains and two light chains. The two heavy chains are linked by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. The heavy chains contain a variable heavy chain region (VH) and three constant regions (CH1, CH2, and CH3). The heavy chains of IgG, IgA, and IgD also contain a hinge region between CH1 and CH2. The light chains contain a variable light chain region (VL) and one constant region (CL). The VL and VH work together to specifically recognize the antigen. Heavy chains are linked by hinge regions (IgG, IgA, and IgD) or CH2 (IgM and IgE), while heavy and light chains are linked by CH1 and CL. The Fc region of the heavy chain, including CH2 and CH3, determines the biological function of the antibody. IgM contains an additional constant region (CH4) in its heavy chain, and can form a pentamer via CH4.

[0015] In this disclosure, “antigen-binding molecule” means a molecule capable of specifically binding to at least one antigen. The antigen-binding molecules of this disclosure comprise at least a first polypeptide and a second polypeptide. The first polypeptide comprises VH and the CH4 domain (IgM-CH4) of a first human immunoglobulin M, and the second polypeptide comprises VL and a second IgM-CH4.

[0016] The amino acid sequence of the human IgM heavy chain is registered, for example, as UniProt accession number P01871 (SEQ ID NO: 1), with positions 323 to 433 (SEQ ID NO: 2) corresponding to CH4. In this disclosure, IgM-CH4 includes the product of its naturally occurring allele, or a natural or artificial variant that can bind to another IgM-CH4 to form a dimer. Preferably, IgM-CH4 does not form any polymer other than a dimer. The first IgM-CH4 and the second IgM-CH4 may be the same, or they may be different as long as they can form a dimer.

[0017] In one embodiment, the first IgM-CH4 is VALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X1)QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR(X2)T(X3)RTVDKST(SEQ ID NO: 3) [(X1) is M or E, (X2) is V or Y, and (X3) is E or Q] The amino acid sequence consists of the amino acid sequence of SEQ ID NO: 3, or includes the amino acid sequence of SEQ ID NO: 3, or consists of a sequence in which 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, added, or inserted, preferably substituted, or consists of an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more identical to the amino acid sequence of SEQ ID NO: 3.

[0018] In one embodiment, the second IgM-CH4 is ALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X4)QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR(X5)T(X6)RTVDKST(SEQ ID NO: 4) [(X4) is M or E, (X5) is V or Y, and (X6) is E or Q] The amino acid sequence consists of the amino acid sequence of SEQ ID NO: 4, or includes the amino acid sequence of SEQ ID NO: 4, or consists of a sequence in which 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is deleted, substituted, added, or inserted, preferably substituted, or consists of an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more identical to the amino acid sequence of SEQ ID NO: 4.

[0019] The deletion, substitution, addition, or insertion of an amino acid may be at any position in the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the deletion or addition of an amino acid is at the C-terminus of the amino acid sequence of SEQ ID NO: 3 or 4. In one embodiment, the deletion, substitution, addition, or insertion of an amino acid is at a position other than X1-X6.

[0020] In this disclosure, amino acid substitutions may be conservative. A conservative substitution is the substitution of one amino acid with another amino acid having similar properties. Examples of conservative substitutions include substitutions within any of the following groups: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. Other examples of conservative substitutions include substitutions within the groups of nonpolar hydrophobic, polar neutral, basic, or acidic amino acids. Examples of nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Examples of polar neutral amino acids include glycine, serine, threonine, tyrosine, asparagine, and glutamine. Examples of basic amino acids include arginine, lysine, and histidine. Acidic amino acids include, for example, aspartic acid and glutamic acid. Alternatively, amino acid substitutions may be non-conservative.

[0021] In this disclosure, amino acid sequence identity is determined by comparing two sequences that are optimally aligned (maximum amino acid match) across the regions of the sequences to be compared. Various algorithms commonly available to those skilled in the art (e.g., BLAST algorithm, FASTA algorithm, etc.) can be used to obtain optimal alignment and sequence identity. Sequence identity can be determined using sequence analysis software such as BLAST or FASTA.

[0022] Preferably, the first and second IgM-CH4 do not form polymers other than dimers, such as pentamers. In natural IgM-CH4, the amino acid residue corresponding to (X1) in SEQ ID NO: 3 and (X4) in SEQ ID NO: 4 is M, the amino acid residue corresponding to (X2) in SEQ ID NO: 3 and (X5) in SEQ ID NO: 4 is V, and the amino acid residue corresponding to (X3) in SEQ ID NO: 3 and (X6) in SEQ ID NO: 4 is E. Although not limited by theory, these amino acid residues, particularly V corresponding to (X2) and (X5), are thought to be involved in pentamer formation, and the ability to form pentamers may be lost by substituting them with other amino acid residues, for example, the corresponding amino acid residue in CH3 of IgG.

[0023] In one embodiment, (X1) is M or E, (X2) is Y, and (X3) is E or Q (Sequence ID 5). In one embodiment, (X1) is M, (X2) is Y, and (X3) is Q (Sequence ID 6). In one embodiment, (X1) is M, (X2) is Y, and (X3) is E (Sequence ID 7). In one embodiment, (X1) is E, (X2) is Y, and (X3) is E (Sequence ID 8).

[0024] In one embodiment, (X4) is M or E, (X5) is Y, and (X6) is E or Q (Sequence ID 9). In one embodiment, (X4) is M, (X5) is Y, and (X6) is Q (Sequence ID 10). In one embodiment, (X4) is M, (X5) is Y, and (X6) is E (Sequence ID 11). In one embodiment, (X4) is E, (X5) is Y, and (X6) is E (Sequence ID 12).

[0025] The VH and VL of the antigen-binding molecules of this disclosure may be derived from any antibody. For example, the VH and VL may be derived from an existing antibody. Examples of existing antibodies include anti-Her2 antibodies (such as trastuzumab) and anti-CD3 antibodies (such as OKT3). The VH and VL may be from a human antibody, from an antibody of a non-human animal, or the complementarity-determining region (CDR) of the VH and VL of an antibody of a non-human animal may be transplanted to an appropriate position on the human VH and VL. Preferably, the VH and VL form an antigen-binding site that binds to the antigen with high affinity. For example, the antigen-binding site is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Binds to antigens with KD levels below M.

[0026] VH and the first IgM-CH4, and / or VL and the second IgM-CH4, may be directly bound or bound via a linker. The linker may be a peptide linker or a non-peptide linker. A peptide linker is one or more amino acid residues present between the domains of the antigen-binding molecule that can give the antigen-binding molecule a correctly folded structure. The length of the peptide linker is not limited and may consist of, for example, 1 to 30, 3 to 25, or 5 to 20 amino acids. In one embodiment, the peptide linker consists of glycine and / or serine.

[0027] For example, a linker containing the amino acid sequence shown in the table below can be used. [Table 1]

[0028] In one embodiment, the first polypeptide comprises trastuzumab-derived VH and consists of the amino acid sequence of any of SEQ ID NOs. 38-41, preferably any of SEQ ID NOs. 39-41, or includes the amino acid sequence of any of SEQ ID NOs. 38-41, preferably any of SEQ ID NOs. 39-41, or consists of an amino acid sequence having identity with any of SEQ ID NOs. 38-41, preferably any of SEQ ID NOs. 39-41, preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more.

[0029] In one embodiment, the second polypeptide comprises a trastuzumab-derived VL and consists of the amino acid sequence of any of SEQ ID NOs. 42-45, preferably any of SEQ ID NOs. 43-45, or contains the amino acid sequence of any of SEQ ID NOs. 42-45, preferably any of SEQ ID NOs. 43-45, or consists of an amino acid sequence having approximately 90% or more identity, particularly preferably approximately 95% or more, most preferably approximately 97%, approximately 98%, or approximately 99% or more identity with the amino acid sequence of any of SEQ ID NOs. These sequences may have a protease site and a purification tag, such as a TEV protease site and a His6-tag (ENLYFQGHHHHHH, SEQ ID NOs. 46), appended to the C-terminus.

[0030] The antigen-binding molecules of this disclosure may be in the form of an antibody or an antibody derivative. That is, the antigen-binding molecules of this disclosure may have a form in which at least one antigen-binding fragment (Fab) portion has CH1 and CL replaced with IgM-CH4. The Fab is a fragment with a molecular weight of approximately 50 kDa obtained by treating an immunoglobulin with the protease papain, and consists of a fragment containing the VH and CH1 domains of the heavy chain and the entire light chain linked via disulfide bonds. The antibody derivative may be any molecule that retains the antigen-binding properties of the antibody, and is not limited to such molecules. Numerous antibody derivatives containing the Fab moiety are known, including, for example, Fab, Fab', F(ab')2, Fab3, Fab-sc-Fv (bibody), Fab-sc-Fv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), TriFabs, Fab-scFv-Fc, and derivatives in the form of bispecific molecules, as described later. The antigen-binding molecule of this disclosure may also be in the form of a multispecific antibody derivative, for example, a bispecific antibody derivative.

[0031] In one embodiment, the antigen-binding molecule of the present disclosure is a bispecific molecule further comprising a third polypeptide and a fourth polypeptide. The third polypeptide contains the CH1 domain (CH1) of the second VH and human immunoglobulin. The fourth polypeptide contains the second VL and the CL domain (CL) of human immunoglobulin. CH1 and CL are bonded to each other. The second VH and second VL form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site are linked via binding of the first polypeptide to the third polypeptide or the fourth polypeptide, or via binding of the second polypeptide to the third polypeptide or the fourth polypeptide.

[0032] CH1 may originate from any class of immunoglobulin, and may be derived from IgG (IgG1, IgG2, IgG3, or IgG4), IgM, IgD, IgA (IgA1, or IgA2), or IgE. The amino acid sequence of the human IgG1 heavy chain constant region is registered, for example, as UniProt accession number P01857 (sequence number 47), with positions 1 to 98 (sequence number 48) corresponding to CH1. The amino acid sequence of the human IgG2 heavy chain constant region is registered, for example, as UniProt accession number P01859 (sequence number 49), with positions 1 to 98 (sequence number 50) corresponding to CH1. The amino acid sequence of the human IgG3 heavy chain constant region is registered, for example, as UniProt accession number P01860 (sequence number 51), with positions 1 to 98 (sequence number 52) corresponding to CH1. The amino acid sequence of the human IgG4 heavy chain constant region is registered, for example, as UniProt accession number P01861 (sequence number 53), with positions 1 to 98 (sequence number 54) corresponding to CH1. The amino acid sequence of the human IgM heavy chain constant region is registered, for example, as UniProt accession number P01871 (sequence number 1), with positions 1 to 104 (sequence number 55) corresponding to CH1. The amino acid sequence of the human IgD heavy chain constant region is registered, for example, as UniProt accession number P01880 (sequence number 56), with positions 1 to 101 (sequence number 57) corresponding to CH1. The amino acid sequence of the human IgA1 heavy chain constant region is registered, for example, as UniProt accession number P01876 (sequence number 58), with positions 1 to 102 (sequence number 59) corresponding to CH1. The amino acid sequence of the constant region of the human IgA2 heavy chain is registered, for example, with UniProt accession number P01877 (sequence number 60), and positions 1 to 102 (sequence number 61) correspond to CH1. The amino acid sequence of the constant region of the human IgE heavy chain is registered, for example, with UniProt accession number P01854 (sequence number 62), and positions 1 to 103 (sequence number 63) correspond to CH1.In this disclosure, CH1 includes the products of these naturally occurring alleles, or natural or artificial variants that can bind to CL to form a dimer. In some embodiments, CH1 is derived from IgG, particularly IgG1.

[0033] In one embodiment, CH1 consists of the amino acid sequence of SEQ ID NOs. 48, 50, 52, 54, 55, 57, 59, 61 or 63, or contains the amino acid sequence of SEQ ID NOs. 48, 50, 52, 54, 55, 57, 59, 61 or 63, or has 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, and 1 amino acid in the amino acid sequence of SEQ ID NOs. 48, 50, 52, 54, 55, 57, 59, 61 or 63. The sequence consists of a deletion, substitution, addition, or insertion of 6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid, or an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more, identical to the amino acid sequence of SEQ ID NOs. The deletion, substitution, addition, or insertion of amino acids may be at any position in these amino acid sequences.

[0034] Immunoglobulin light chains consist of two types: κ and λ chains, and their constant regions (CLs) are called Cκ and Cλ, respectively. The amino acid sequence of human Cκ is registered, for example, as UniProt accession number P01834 (SEQ ID NO: 64). The amino acid sequence of human Cλ is registered, for example, as UniProt accession numbers P0CG04 (SEQ ID NO: 65), P0DOY2 (SEQ ID NO: 66), P0DOY3 (SEQ ID NO: 67), P0CF74 (SEQ ID NO: 68), or A0M8Q6 (SEQ ID NO: 69). In this disclosure, CLs include products of these naturally occurring alleles, or natural or artificial variants that can bind to CH1 to form dimers. In some embodiments, CL is Cκ.

[0035] In one embodiment, CL consists of any amino acid sequence of SEQ ID NOs. 64-69, or includes any amino acid sequence of SEQ ID NOs. 64-69, or consists of a sequence in which 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 amino acid is deleted, substituted, added, or inserted in any amino acid sequence of SEQ ID NOs. 64-69, or consists of an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more identical to any amino acid sequence of SEQ ID NOs. The deletion, substitution, addition, or insertion of amino acids may be at any position in these amino acid sequences.

[0036] The second antigen-binding site is different from the first antigen-binding site formed by the first VH contained in the first polypeptide and the first VL contained in the second polypeptide. The first and second antigen-binding sites may recognize different antigens, or they may recognize different epitopes on a single antigen. The second VH and second VL may be derived from any antibody and are defined in the same way as the first VH and first VL.

[0037] The second VH and CH1, and / or the second VL and CL, may be directly bonded or bonded via a linker. The linker is as described above for the first and second polypeptides.

[0038] The first polypeptide is bound to the third or fourth polypeptide, or the second polypeptide is bound to the third or fourth polypeptide, linking the first antigen-binding site to the second antigen-binding site and forming a bispecific molecule. The mode of binding is not limited as long as it does not hinder the binding of the first and second antigen-binding sites to their respective antigens. The binding may be direct or linker-mediated. In some embodiments, the linker is derived from the hinge region of the antibody. In some embodiments, the first polypeptide and the third polypeptide are bound together.

[0039] Typically, the bispecific molecules of this disclosure may be forms of bispecific molecules known in the art. An example of a bispecific molecule is disclosed, for example, in Brinkmann U, Kontermann RE. The making of bispecific antibody. MAbs. 2017 Feb / Mar;9(2):182-212 (Non-Patent Literature 1), which is part of this disclosure with due attribution.

[0040] The bispecific molecules of this disclosure include, for example, IgG, IgG(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), DuetMab, Duobody, LUZ-Y IgG, or FcFc * It may take the form of a bispecific molecule. In one embodiment, the bispecific molecule is a bispecific antibody. The bispecific antibody may be an antibody of any immunoglobulin class, for example, IgG, particularly IgG1. The bispecific molecule of this disclosure may have multispecificity by adding further antigen-binding sites. Such multispecific molecules may take the form of, for example, IgG(kih)-Fv, IgG(HA-TF-Fv, IgG(kih)-scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, DVI-Ig, IgG-HC-scFv, IgG-dAb, IgG-taFv, IgG-CrossFab, IgG-Fab, scFv-HC-IgG, tandem Fab-IgG, IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG, scFv4-IgG, F(ab')2-scFv2, DAF, DutaMab, or mAb2.

[0041] In one embodiment, the bispecific molecule is in the form of a bispecific antibody. That is, the first polypeptide further contains a first Fc domain (Fc) and forms a (VH)-(IgM-CH4)-(Fc) structure from the N-terminus, and the third polypeptide further contains a second Fc and forms a (VH)-(CH1)-(Fc) structure from the N-terminus, with the first Fc and the second Fc being mutually bound.

[0042] Fc may be derived from any class of immunoglobulin and may include a CH2 domain and a CH3 domain, and may also include a hinge region. The CH2 domain, CH3 domain, and / or hinge region may be replaced with the CH2 domain, CH3 domain, and / or hinge region of a different class of immunoglobulin.

[0043] Fc may originate from IgG (IgG1, IgG2, IgG3, or IgG4), IgD, IgA, or IgE. In the amino acid sequence of the human IgG1 heavy chain of SEQ ID NO: 47, positions 99 to 330 (SEQ ID NO: 70) correspond to Fc. In the amino acid sequence of the human IgG2 heavy chain of SEQ ID NO: 49, positions 99 to 326 (SEQ ID NO: 71) correspond to Fc. In the amino acid sequence of the human IgG3 heavy chain of SEQ ID NO: 51, positions 102 to 377 (SEQ ID NO: 72) correspond to Fc. In the amino acid sequence of the human IgG4 heavy chain of SEQ ID NO: 53, positions 99 to 327 (SEQ ID NO: 73) correspond to Fc. In the amino acid sequence of the human IgD heavy chain of SEQ ID NO: 56, positions 102 to 430 (SEQ ID NO: 74) correspond to Fc. In the amino acid sequence of the human IgA1 heavy chain of SEQ ID NO: 58, positions 103 to 353 (SEQ ID NO: 75) correspond to Fc. In the amino acid sequence of the human IgA2 heavy chain of SEQ ID NO: 60, positions 103 to 340 (SEQ ID NO: 76) correspond to Fc. In the amino acid sequence of the human IgE heavy chain of SEQ ID NO: 62, positions 104 to 428 (SEQ ID NO: 77) correspond to Fc. In this disclosure, Fc includes the products of these naturally occurring alleles, or natural or artificial variants that can bind to another Fc to form a dimer. In some embodiments, Fc is derived from IgG, particularly IgG1. The first Fc and the second Fc may be the same, or they may be different, as long as they can bind to each other.

[0044] In one embodiment, Fc consists of any of the amino acid sequences of SEQ ID NOs: 70 to 77, includes any of the amino acid sequences of SEQ ID NOs: 70 to 77, consists of a sequence in which 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid is deleted, substituted, added or inserted in any of the amino acid sequences of SEQ ID NOs: 70 to 77, or consists of an amino acid sequence having an identity of preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98% or about 99% or more with any of the amino acid sequences of SEQ ID NOs: 70 to 77. The deletion, substitution, addition or insertion of an amino acid may be at any position of these amino acid sequences.

[0045] Fc may have a mutation that promotes heterodimerization of the heavy chains. Mutations that promote heterodimerization of the heavy chains are known in the art and are described, for example, in Brinkmann U, Kontermann RE. The making of bispecific antibodies. MAbs. 2017 Feb / Mar;9(2):182-212 (Non-Patent Document 1), which is incorporated herein by reference in its entirety. Examples of mutations that promote heterodimerization of the heavy chains include knob-into-hole (Y-T or CW-CSAV), HA-TF, ZW1 (VYAV-VLLW), CH3 charge pair (DD-KK), IgG1 hinge / CH3 charge pair (EEE-RRR), IgG2 hinge / CH3 charge pair (EEE-RRRR), EW-RVT, EW-RVT S-S , and include Biclonic, DuoBody (L-R), SEEDbody, BEAT, 7.8.60 (DMA-RRVV) and 20.8.34 (SYMV-GDQA).

[0046] In one embodiment, Fc has a knob-into-hole type mutation. Knob-into-hole technology is a technique that promotes heavy chain heterodimerization by replacing the amino acid side chain present in the CH3 of one heavy chain with a larger side chain (knob) and replacing the amino acid side chain present in the CH3 of the other heavy chain with a smaller side chain (hole), thereby positioning the knob within the hole (Ridgway JB, et al., 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 1996; 9:617-21, Atwell S, et al., Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol 1997; 270:26-35, and Merchant AM, et al., An efficient route to human bispecific IgG. Nat Biotechnol 1998; 16:677-681). For example, a knob-into-hole mutation could be a combination of a T366Y mutation (knob mutation) in one Fc and a Y407T mutation (hole mutation) in the other Fc, or a combination of a T366W mutation (knob mutation) in one Fc and T366S, L368A, and Y407V mutations (hole mutations) in the other Fc, or a combination of S354C and T366W mutations (knob mutations) in one Fc and Y349C, T366S, L368A, and Y407V mutations (hole mutations) in the other Fc. Here, the description of amino acid mutations is based on the amino acid position according to the EU index in the human Igγ1 constant region.

[0047] For example, the Fc of IgG1 with a knob-into-hole type knob mutation contains the amino acid sequence of SEQ ID NO: 78, and the Fc of IgG1 with a hole mutation contains the amino acid sequence of SEQ ID NO: 79. Knob:CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 78) Hall:CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 79)

[0048] In one embodiment, the Fc of the first polypeptide has a knob mutation, and the Fc of the third polypeptide has a hole mutation.

[0049] In the first polypeptide, IgM-CH4 and Fc may be directly bound or bound via a linker. In the third polypeptide, CH1 and Fc may be directly bound or bound via a linker. The linker is as described above for the first and second polypeptides.

[0050] In one embodiment, the first polypeptide comprises trastuzumab-derived VH and consists of the amino acid sequence of SEQ ID NO: 80, or comprises the amino acid sequence of SEQ ID NO: 80, or comprises an amino acid sequence having identity with the amino acid sequence of SEQ ID NO: 80, preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more. The third polypeptide comprises OKT3-derived VH and consists of the amino acid sequence of SEQ ID NO: 81, or comprises the amino acid sequence of SEQ ID NO: 81, or comprises an amino acid sequence having identity with the amino acid sequence of SEQ ID NO: 81, preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more.

[0051] In one embodiment, the first polypeptide comprises trastuzumab-derived VH and consists of the amino acid sequence of SEQ ID NO: 82, or comprises the amino acid sequence of SEQ ID NO: 82, or comprises an amino acid sequence having identity with the amino acid sequence of SEQ ID NO: 82, preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more. The third polypeptide comprises OKT3-derived VH and consists of the amino acid sequence of SEQ ID NO: 83, or comprises the amino acid sequence of SEQ ID NO: 83, or comprises an amino acid sequence having identity with the amino acid sequence of SEQ ID NO: 83, preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more.

[0052] In one embodiment, the second polypeptide comprises a trastuzumab-derived VL and consists of the amino acid sequence of SEQ ID NO: 43, or includes the amino acid sequence of SEQ ID NO: 43, or consists of an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more identical to the amino acid sequence of SEQ ID NO: 43.

[0053] In one embodiment, the fourth polypeptide comprises a VL derived from OKT3 and consists of the amino acid sequence of SEQ ID NO: 84, or includes the amino acid sequence of SEQ ID NO: 84, or consists of an amino acid sequence that is preferably about 90% or more, particularly preferably about 95% or more, most preferably about 97%, about 98%, or about 99% or more identical to the amino acid sequence of SEQ ID NO: 84.

[0054] In one embodiment, the present disclosure relates to a bispecific molecule comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, The first polypeptide comprises a first heavy chain variable region (VH) and a first human immunoglobulin M (IgM) CH4 domain (IgM-CH4), The second polypeptide comprises a first light chain variable region (VL) and a second IgM-CH4. The first IgM-CH4 and the second IgM-CH4 are mutually bound. The first VH and the first VL form the first antigen-binding site. The third polypeptide contains the CH1 domain (CH1) of the second VH and human immunoglobulin. The fourth polypeptide contains the second VL and the CL domain (CL) of human immunoglobulin. CH1 and CL are bonded to each other. The second VH and second VL form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site are linked via binding of the first polypeptide to the third polypeptide or the fourth polypeptide, or via binding of the second polypeptide to the third polypeptide or the fourth polypeptide. It provides a bispecific molecule.

[0055] The antigen-binding molecules or bispecific molecules of this disclosure may be modified. For example, the side chains of amino acid residues, the amino group of the N-terminal amino acid, or the carboxyl group of the C-terminal amino acid may be modified by esterification, alkylation, halogenation, phosphorylation, glycosylation, etc. The antigen-binding molecules or bispecific molecules of this disclosure may also include tags or labels. Examples of tags include His-tags, Myc-tags, FLAG-tags, GST-tags, MBP-tags, epitope tags, protein purification tags, etc. Examples of labels include radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzyme labels, and biotin. The antigen-binding molecules or bispecific molecules of this disclosure may be bound to physiologically active substances.

[0056] The antigen-binding molecules or bispecific molecules of this disclosure can be produced using methods well known in the art. For example, nucleic acids encoding the first and second polypeptides, or the first to fourth polypeptides, can be introduced into host cells by known methods such as the calcium phosphate method, the DEAE dextran method, electroporation, or lipofection. The nucleic acids encoding these polypeptides may be inserted into a vector suitable for expression in host cells, and the vector may be introduced into the host cells. The nucleic acids encoding each polypeptide may be inserted into the same vector or into separate vectors. Typically, the vector contains sequence elements that enable the expression of the inserted nucleic acids. Such vectors are well known in the art, and many are commercially available. Host cells into which nucleic acids or vectors have been introduced can be cultured under conditions that enable protein expression, and the antigen-binding molecules or bispecific molecules can be recovered from the cells or culture supernatant. The culture supernatant may be the culture supernatant of host cells containing all nucleic acids, the culture supernatant of multiple host cells containing one or more nucleic acids co-cultured, or the culture supernatant obtained by separately culturing multiple host cells containing one or more nucleic acids and mixing the resulting culture supernatants.

[0057] Eukaryotic cells such as animal cells, plant cells, and fungal cells can be used as host cells. Examples of animal cells include mammalian cells (e.g., CHO, COS, NIH3T3, Myeloma, BHK, HeLa, Vero, 293T, platE), amphibian cells (e.g., African clawed frog oocytes), or insect cells (e.g., Sf9, Sf21, Tn5). Examples of fungal cells include yeast (e.g., Saccharomyces genus, e.g., Saccharomyces cerevisiae) and filamentous fungi (e.g., Aspergillus genus, e.g., Aspergillus niger). Prokaryotic cells such as Escherichia coli (e.g., JM109, DH5α, HB101, etc.) and Bacillus subtilis can also be used as host cells.

[0058] The obtained antigen-binding molecules or bispecific molecules can be purified by appropriately combining methods known in this field, such as chromatography using a protein A column, ion exchange chromatography, size exclusion chromatography, hydrophobic chromatography, affinity chromatography, ammonium sulfate precipitation, gel filtration, etc.

[0059] Accordingly, in some embodiments, the Disclosure provides a nucleic acid encoding a first polypeptide of an antigen-binding molecule, a nucleic acid encoding a second polypeptide of an antigen-binding molecule, or a combination thereof. In some embodiments, the Disclosure provides a vector comprising such nucleic acid or combination. The vector may be a vector comprising a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, or a combination of a vector comprising a nucleic acid encoding a first polypeptide and a vector comprising a nucleic acid encoding a second polypeptide.

[0060] In some embodiments, the Disclosure provides cells comprising the nucleic acids or combinations of nucleic acids described above, or the vector described above. The cells may comprise a nucleic acid encoding a third polypeptide of the antigen-binding molecule, a nucleic acid encoding a fourth polypeptide of the antigen-binding molecule, or a combination thereof, or further comprise a vector comprising the nucleic acid encoding the third polypeptide, the nucleic acid encoding the fourth polypeptide, or a combination thereof. The cells may be maintained as a culture and stored at low temperatures, for example, by freezing. The cells alone may produce the antigen-binding molecules or bispecific molecules of the Disclosure and may be used, for example, as a master cell bank (MCB) or working cell bank (WCB) for pharmaceutical manufacturing. For example, cells expressing all of the first to fourth polypeptides of the Disclosure can produce the intended bispecific molecules without mismatching of each polypeptide.

[0061] The antigen-binding molecules or bispecific molecules, nucleic acids, vectors, or cells of this disclosure may be used as active ingredients in a pharmaceutical composition. Accordingly, in some embodiments, this disclosure provides a pharmaceutical composition comprising the above-mentioned antigen-binding molecules or bispecific molecules, nucleic acids, vectors, or cells. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient or carrier, and may also comprise a pharmaceutically acceptable formulation substance or other active ingredient. The pharmaceutical composition may be in any formulation known in the art.

[0062] In one embodiment, the present disclosure provides a method for producing the antigen-binding molecule of the present disclosure, comprising the following steps: (1) Introducing into a host cell (i) a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, (ii) a nucleic acid encoding a first polypeptide and a second polypeptide, (iii) a vector containing a nucleic acid encoding a first polypeptide and a vector containing a nucleic acid encoding a second polypeptide, or (iv) a vector containing a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide. (2) Culturing host cells under conditions suitable for the expression of the first polypeptide and the second polypeptide, (3) Recover antigen-binding molecules from host cells or culture supernatant. In one embodiment, a third polypeptide and a fourth polypeptide are further expressed in host cells to produce the bispecific molecules of this disclosure.

[0063] For example, the following embodiment is provided. [1] An antigen-binding molecule comprising a first polypeptide and a second polypeptide, The first polypeptide comprises a heavy chain variable region (VH) and the CH4 domain (IgM-CH4) of the first human immunoglobulin M. The second polypeptide comprises a light chain variable region (VL) and a second IgM-CH4. The first IgM-CH4 and the second IgM-CH4 are mutually bound. VH and VL form the antigen-binding site. Antigen-binding molecules. [2] The first IgM-CH4 is VALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X1)QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR(X2)T(X3)RTVDKST(Sequence ID 3) [(X1) is M or E, (X2) is V or Y, and (X3) is E or Q] It consists of the amino acid sequence of, or contains the amino acid sequence of SEQ ID NO: 3, or consists of a sequence in which 1 to 11 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 is ALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X4)QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR(X5)T(X6)RTVDKST(Sequence ID 4) [(X4) is M or E, (X5) is V or Y, and (X6) is E or Q] The amino acid sequence consists of, or contains the amino acid sequence of SEQ ID NO: 4, or consists of a sequence in which 1 to 11 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 4. The antigen-binding molecule described in item 1 above. [3] The first IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 3, or a sequence in which one to three amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3, and / or The antigen-binding molecule according to claim 2, wherein the second IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 4, or a sequence in which one to three amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 4. [4] The first IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 3, or a sequence in which one amino acid is deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3, and / or The antigen-binding molecule according to 2 or 3, wherein the second IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 4, or a sequence in which one amino acid is deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 4. [5] The first IgM-CH4 contains the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 contains the amino acid sequence of SEQ ID NO: 4. The antigen-binding molecule described in any of items 2 to 4 above.

[0064] [6] The first IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 4. The antigen-binding molecule described in any of items 2 to 5 above. [7] (X1) is M or E, (X2) is Y, (X3) is E or Q, and / or (X4) is M or E, (X5) is Y, and (X6) is E or Q. The antigen-binding molecule described in any of items 2 to 6 above. [8] (X1) is M, (X2) is Y, (X3) is Q, and / or (X4) is M, (X5) is Y, and (X6) is Q. The antigen-binding molecule described in any of items 2 to 7 above. [9] (X1) is M, (X2) is Y, (X3) is E, and / or (X4) is M, (X5) is Y, and (X6) is E. The antigen-binding molecule described in any of items 2 to 7 above.

[10] (X1) is E, (X2) is Y, (X3) is E, and / or (X4) is E, (X5) is Y, and (X6) is E. The antigen-binding molecule described in any of items 2 to 7 above.

[0065]

[11] An antigen-binding molecule according to any one of items 1 to 10 above, wherein VH and VL are derived from trastuzumab.

[12] The first polypeptide consists of an amino acid sequence that has approximately 90% or more identity with any of the amino acid sequences of SEQ ID NOs. 38-41, and / or The second polypeptide consists of an amino acid sequence that has approximately 90% or more identity with any of the amino acid sequences of sequence numbers 42-45. The antigen-binding molecule described in any of items 1 to 11 above.

[13] The first polypeptide contains any of the amino acid sequences of SEQ ID NOs. 38-41, and / or The second polypeptide contains one of the amino acid sequences of SEQ ID NOs. 42-45. The antigen-binding molecule described in any of items 1 to 12 above.

[14] The first polypeptide consists of any of the amino acid sequences of SEQ ID NOs. 38-41, and / or The second polypeptide consists of one of the amino acid sequences of SEQ ID NOs. 42-45. The antigen-binding molecule described in any of items 1 to 13 above.

[15] The first polypeptide contains any of the amino acid sequences of SEQ ID NOs. 39-41, and / or The second polypeptide contains one of the amino acid sequences of SEQ ID NOs. 43-45. The antigen-binding molecule described in any of items 1 to 13 above.

[0066]

[16] The first polypeptide consists of any of the amino acid sequences of SEQ ID NOs. 39-41, and / or The second polypeptide consists of one of the amino acid sequences of SEQ ID NOs. 43-45. The antigen-binding molecule described in any of items 1 to 15 above.

[17] Furthermore, the antigen-binding molecule according to any one of 1 to 16 above is a bispecific molecule containing a third polypeptide and a fourth polypeptide, The third polypeptide contains the CH1 domain (CH1) of the second VH and human immunoglobulin. The fourth polypeptide contains the second VL and the CL domain (CL) of human immunoglobulin. CH1 and CL are bonded to each other. The second VH and second VL form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site are linked via binding of the first polypeptide to the third polypeptide or the fourth polypeptide, or via binding of the second polypeptide to the third polypeptide or the fourth polypeptide. Antigen-binding molecules.

[18] The antigen-binding molecule described in 17 above, wherein CH1 is the CH1 of IgG.

[19] The antigen-binding molecule according to 17 or 18, wherein CH1 is CH1 of IgG1.

[20] An antigen-binding molecule according to any one of items 17 to 19 above, wherein CL is Cκ.

[0067] [twenty one] An antigen-binding molecule according to any one of 17 to 20, wherein a first antigen-binding site and a second antigen-binding site are linked via the binding of a first polypeptide and a third polypeptide. [twenty two] The first polypeptide further contains a first Fc domain (Fc), and from the N-terminus, it forms the structure (VH)-(IgM-CH4)-(Fc). The third polypeptide further contains a second Fc, forming a (VH)-(CH1)-(Fc) structure from the N-terminus. The first Fc and the second Fc are bonded to each other. The antigen-binding molecule described in any of items 17 to 21 above. [twenty three] The antigen-binding molecule according to 22, wherein the first Fc and / or the second Fc is the Fc of IgG. [twenty four] The antigen-binding molecule according to 22 or 23, wherein the first Fc and / or the second Fc is the Fc of IgG1. [twenty five] An antigen-binding molecule according to any one of 22 to 24, wherein the first Fc and / or the second Fc have mutations that promote heterodimerization.

[0068]

[26] The antigen-binding molecule according to 25, wherein the mutation that promotes heterodimerization is a knob-into-hole type mutation.

[27] (i) The first polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 80, and the third polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 81, or (ii) The first polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 82, and the third polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 83. The antigen-binding molecule described in any of items 17 to 26 above.

[28] (i) The first polypeptide contains the amino acid sequence of SEQ ID NO: 80, and the third polypeptide contains the amino acid sequence of SEQ ID NO: 81, or (ii) The first polypeptide contains the amino acid sequence of SEQ ID NO: 82, and the third polypeptide contains the amino acid sequence of SEQ ID NO: 83. The antigen-binding molecule described in any of items 17 to 27 above.

[29] (i) The first polypeptide consists of the amino acid sequence of SEQ ID NO: 80, and the third polypeptide consists of the amino acid sequence of SEQ ID NO: 81, or (ii) The first polypeptide consists of the amino acid sequence of SEQ ID NO: 82, and the third polypeptide consists of the amino acid sequence of SEQ ID NO: 83. The antigen-binding molecule described in any of items 17 to 28 above.

[30] An antigen-binding molecule according to any one of 17 to 29, wherein the second polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 43, and / or the fourth polypeptide consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO: 84.

[0069]

[31] An antigen-binding molecule according to any one of claims 17 to 30, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO: 43, and / or the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 84.

[32] An antigen-binding molecule according to any one of 17 to 31, wherein the second polypeptide consists of the amino acid sequence of SEQ ID NO: 43, and / or the fourth polypeptide consists of the amino acid sequence of SEQ ID NO: 84.

[33] An antigen-binding molecule according to any one of items 1 to 32, in the form of an antibody or an antibody derivative.

[34] The antigen-binding molecule described in 33 above, which is in the form of Fab, Fab', F(ab')2, Fab3, Fab-sc-Fv (bibody), Fab-sc-Fv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), TriFabs, or Fab-scFv-Fc.

[35] An antigen-binding molecule according to any one of items 1 to 33 above, which is a bispecific molecule.

[36] IgG, IgG(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), DuetMab, Duobody, LUZ-Y IgG or FcFc * The antigen-binding molecule described in 35 above, which is in the form of the antigen-binding molecule described in 35 above.

[37] An antigen-binding molecule according to any one of items 1 to 33 above, which is a form of a multispecific molecule.

[38] The antigen-binding molecule described in 37 above, which is in the form of IgG(kih)-Fv, IgG(HA-TF-Fv, IgG(kih)-scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, DVI-Ig, IgG-HC-scFv, IgG-dAb, IgG-taFv, IgG-CrossFab, IgG-Fab, scFv-HC-IgG, tandemFab-IgG, IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG, scFv4-IgG, F(ab')2-scFv2, DAF, DutaMab, or mAb2.

[39] A nucleic acid encoding a first polypeptide as described in any of items 1 to 38 above, a nucleic acid encoding a second polypeptide as described in any of items 1 to 36 above, or a combination thereof.

[40] A vector comprising the nucleic acid or combination described in item 39 above.

[0070]

[41] A cell containing the nucleic acid or combination described in 39 above, or the vector described in 40 above.

[42] A nucleic acid encoding a third polypeptide as described in any of items 17 to 38 above, a nucleic acid encoding a fourth polypeptide as described in any of items 17 to 38 above, or a combination thereof, A vector comprising a nucleic acid encoding a third polypeptide as described in any of items 17 to 38 above, a nucleic acid encoding a fourth polypeptide as described in any of items 17 to 38 above, or a combination thereof. The cells described in 41, further comprising:

[43] A pharmaceutical composition comprising an antigen-binding molecule according to any one of items 1 to 38, a nucleic acid or combination according to item 39, a vector according to item 40, or a cell according to item 41 or 42.

[44] A pharmaceutical composition comprising an antigen-binding molecule as described in any of items 1 to 38 above.

[0071] All references cited herein are included as part of this specification upon proper attribution. The above description is non-limiting, and the present invention is defined in the appended claims and can be modified in various ways without departing from its technical spirit. The present invention will be described in more detail below by examples, but the present invention is not limited to these examples. [Examples]

[0072] [1] Design of Fab containing IgM-CH4 and acquisition of expression vector A molecule (Fab-IgM) was designed in which the CH1 and Cκ of the antigen-binding fragment (Fab) derived from IgG1 were replaced with the CH4 of IgM (Figure 1). The sequences of the junctions between the variable regions (VH and VL) of the heavy and light chains and the constant region (CH1 and Cκ) were compared with the IgM-CH4 sequence. Referring to the findings in Schaefer et al, Proc. Natl. Acad. Sci. 108, 11187-11192 (2011), and referencing IMGT Repertoire (https: / / www.imgt.org / ), a junctioning method that does not distort the β-sheet connections of each domain was investigated. As shown in Figure 2, the junction sequences of Fab-IgM were optimized separately for the light and heavy chains so that the number of amino acids between strand A, which forms the β-barrel structure of the constant region, and the variable region matched that of the IgG1-derived Fab.

[0073] Specifically, the CH1 of Fab derived from IgG1 was replaced with the IgM-CH4 subregion shown in Sequence ID No. 85, and the Cκ was replaced with the IgM-CH4 subregion shown in Sequence ID No. 86. (Sequence ID 85) VALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKST (Sequence No. 86) ALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKST

[0074] Using the variable region of trastuzumab as a model, we designed the heavy chain shown in SEQ ID NO: 38 and the light chain shown in SEQ ID NO: 87. A TEV protease site and a His6-tag (underlined) were introduced at the C-terminus of the light chain for purification. (Sequence No. 38) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTL VTVSSVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKST (Sequence ID 87) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR ALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKST ENLYFQGHHHHHH The polynucleotides encoding the amino acid sequences of SEQ ID NOs. 38 and 87 were introduced into pcDNA3.4, which had previously contained a secretion signal sequence.

[0075] [2] Mutation introduction to CH4 The IgM-CH4 domain forms homo-associations at two different interfaces. The Fc of IgM containing CH3 and CH4 is a dimer, which then forms a pentamer to create the structure of decameral IgM (Figure 3). To prevent the polymerization of Fab-IgM, we attempted to eliminate the pentamerization ability of the IgM-CH4 domain. Using the structure of IgM obtained by cryo-electron microscopy particle analysis (PDB ID: 8BPE), we analyzed the interface for pentamerization using the PISA server (https: / / www.ebi.ac.uk / pdbe / pisa / ). We found that the amino acids M489, G492, N545, V547, T548, and E549 contribute to pentamerization. When we compared the sequences with those of the human IgG1 CH3 domain, which has high homology to the IgM-CH4 domain and almost no ability to polymerize on its own, we found that G492 and N545 were conserved in both. Therefore, mutations M489E, V547Y, and E549Q, in which IgM-CH4 M489, V547, and E549 are substituted with the corresponding amino acids of human IgG1 CH3, were selected as candidates for causing loss of pentamer formation ability.

[0076] These mutations were introduced into the expression vector obtained in [1] using the KOD Plus Mutagenesis Kit (Toyobo). Alternatively, synthetic DNA containing these mutations was cloned into the same vector. The sequences of the mutant IgM-CH4 used are shown in the table below. The introduced mutations are indicated in underline and bold. [Table 2]

[0077] [3] Obtaining trastuzumab Fab protein containing IgM-CH4 or its variants 25 μg each of the heavy chain and light chain encoding plasmids obtained in [1] and [2] above were transfected using the Expi293F transfection kit (Thermo Fisher Scientific) in 1.5 × 10⁶ units. 8Cells were transfected with Expi293F (Thermo Fisher Scientific). These were cultured for 7 days at 37°C under 8% CO2 conditions with shaking. The supernatant was passed through a 0.2 μm filter, and the Fab protein in the supernatant was adsorbed onto 1 mL of cOmplete His-Tag Purification Resin (Roche Diagnostics). After washing with a buffer containing 20 mM or 50 mM imidazole, the Fab protein was eluted with a buffer containing 200 mM or 500 mM imidazole, dialyzed overnight with a buffer of 20 mM Tris-HCl, 500 mM NaCl, pH 7.5, and size exclusion chromatography was performed using the same buffer with a HiLoad Superdex200 26 / 600 pg (Cytiva) (Figure 4).

[0078] When using IgM-CH4 without mutations (SEQ ID NOs: 85 and 86), Fab-like molecules were obtained, but the peak corresponding to the Fab-like molecule monomer was not separated from the peak considered to be the dimer, resulting in a complex elution profile (Figure 4, dashed line). When using IgM-CH4 with the two mutations V547Y / E549Q (SEQ ID NOs: 6 and 10), the peak corresponding to the Fab-like molecule monomer was obtained with relatively good separation (Figure 4, solid line).

[0079] As an alternative protocol, 1.25 μg of plasmid is divided into 7.5 × 10⁻⁶ units. 6 Cells were transfected with Expi293F, cultured, and the supernatant was filtered. The supernatant was then adsorbed onto 1 mL of HisTrap FF (Cytiva), and the Fab protein was eluted using a 50 mM to 500 mM imidazole gradient. The solution was dialyzed, concentrated to 500 μL using Vivaspin Turbo 4 MWCO 10 kDa (Sartorius), and then size exclusion chromatography was performed using Superdex200 Increase 10 / 300 GL (Cytiva) (Figure 5).

[0080] In the latter protocol, although aggregates were formed during the enrichment process, similar results were obtained. In addition to V547Y / E549Q, when IgM-CH4 with the mutations V547Y (sequence numbers 7 and 11) and M489E / V547Y (sequence numbers 8 and 12) was used, separated peaks corresponding to Fab-like molecular monomers were obtained (Figure 5).

[0081] [4] Design and acquisition of IgG-type asymmetric bispecific antibodies in which one CH1 / Cκ pair of IgG is replaced with IgM-CH4. Using the Fab-like molecules described above, we obtained the IgG-type asymmetric bispecific antibodies shown in Figure 6. One variable region was derived from trastuzumab, which recognizes HER2, and the other variable region was derived from OKT3, which recognizes CD3. The trastuzumab-derived Fab was modified to incorporate IgM-CH4 with the V547Y / E549Q mutation obtained in [3]. For heterodimerization of Fc, we used the knob-into-hole mutant reported in Merchant et al, Nat. Biotechnol., 16, 677-681 (1998). We designed bispecific antibodies in which the heavy chain forming the trastuzumab-derived variable region is a hole mutant (Figure 6 left, T4h-Ok) and a knob mutant (Figure 6 right, T4k-Oh).

[0082] The amino acid sequence of T4h-Ok is shown in the table below. [Table 3]

[0083] The amino acid sequence of T4k-Oh is shown in the table below. [Table 4]

[0084] The polynucleotides encoding the above amino acid sequences were each introduced into pcDNA3.4, which had previously contained a secretion signal sequence. 6.25 μg of each of the four plasmids was transfected using the Expi293F transfection kit (Thermo Fisher Scientific) at a rate of 7.5 × 10⁶ 7 Cells were transfected with Expi293F (Thermo Fisher Scientific). These were cultured for 7 days at 37°C under 8% CO2 conditions with shaking. The supernatant was passed through a 0.2 μm filter, and the antibody in the supernatant was adsorbed onto 1 mL of rProtein A Sepharose Fast Flow (Cytiva) equilibrated with phosphate-buffered saline (PBS). After washing with 10 mL of PBS, the antibody was eluted with 3 mL of 100 mM sodium citrate (pH 3.0).

[0085] First purification method: The eluted fraction from the Protein A purification was mixed with 0.5 mL of 2 M Tris-HCl, pH 8.0, and dialyzed overnight with PBS. Subsequently, size exclusion chromatography was performed using a HiLoad Superdex200 16 / 600 pg (Cytiva) (Figure 7). The fraction containing the monomer of the target bispecific antibody was further dialyzed overnight with 20 mM MES-NaOH, pH 5.4, and cation exchange chromatography was performed with A: 20 mM MES-NaOH, pH 6.0 and B: 20 ​​mM MES-NaOH, 1 M NaCl, pH 5.4. The antibody was adsorbed onto a Mono S 5 / 50 column (Cytiva) and eluted by a 0→50% B gradient (Figure 8). All peaks of the target product were above 50%, suggesting the presence of the target molecule at a high proportion. The target peaks were dialyzed overnight with PBS. Transient expression on a 30 mL scale yielded T4h-Ok at 10 mg / L and T4k-Oh at 15 mg / L.

[0086] Second purification method: Since scaling up the first purification method was difficult, another purification method was also considered. The eluted fraction from the Protein A purification was mixed with 0.5 mL of 2 M Tris-HCl, pH 8.0, dialyzed overnight with 20 mM MES-NaOH, pH 5.4, and the resulting insoluble components were removed by centrifugation. The supernatant was passed through a 0.2 μm filter, and cation exchange chromatography was performed using A: 20 mM MES-NaOH, pH 6.0 and B: 20 ​​mM MES-NaOH, 1 M NaCl, pH 5.4. The antibody was adsorbed onto a Mono S 5 / 50 column (Cytiva) and eluted using a 20 → 30% B gradient (Figure 9). All target peaks were above 50%, suggesting the presence of the target molecule at a high proportion. The target peaks were dialyzed overnight with PBS. Size exclusion chromatography was performed using HiLoad Superdex200 16 / 600 pg (Cytiva) to obtain T4h-Ok and T4k-Oh (Figure 10).

[0087] [5] Evaluation of binding ability The obtained bispecific antibody contains IgM-CH4 instead of CH1 / Cκ of trastuzumab, so the binding affinity of Fab to HER2 may differ from that of conventional trastuzumab. Therefore, the affinity of conventional trastuzumab and the bispecific antibody obtained by the second purification method to recombinant HER2 protein was compared using Biacore T200 (Cytiva). Using HBS-EP+ as the running buffer, approximately 9000 RU of anti-human IgG was immobilized on a Series S Sensor Chip CM5 (Cytiva) using the Human Antibody Capture Kit, type 2 (Cytiva). Each antibody was prepared to 5 μg / mL and captured approximately 2500 RU of each antibody on the chip by flowing at 10 μL / min for 120 seconds. Recombinant HER2 protein was bound in a dilution series of 0.3125–40 nM under conditions of contact 180 seconds, dissociation 420 seconds, and 30 μL / min. The resulting sensorgrams were fitted using a 1:1 binding ratio (Figure 11). The parameters shown in the table below were calculated. No significant changes in affinity were observed when using IgM-CH4. [Table 5]

[0088] Furthermore, flow cytometry confirmed that the bispecific antibody obtained by the first purification method could bind to both antigens. SK-OV-3 cells were used as HER2-expressing cells, and Jurkat cells were used as CD3-expressing cells. These cells were subcultured in RPMI-1640 medium containing 10% FBS and 1% PS. SK-OV-3 cells were isolated by 0.25% trypsin / 1 mM EDTA. The two cell types were suspended in FACS buffer (0.1% sodium azide, 5% FBS, PBS solution) and 1 × 10⁶ wells were placed in a 96-well V-bottom plate. 5 Cells / well (SK-OV-3) or 3 × 10 5 The solution was dispensed to a cell / well ratio (Jurkat). The supernatant was removed by centrifugation at 400×g for 5 minutes. OKT3 (a chimeric antibody with a human constant region), T4h-Ok, T4k-Oh, or trastuzumab dissolved at 15 μg / mL in FACS buffer was added, and the mixture was left on ice for 30 minutes. After washing twice with FACS buffer, R-PE modified Goat Anti-Human IgG (Jackson ImmunoResearch) was added at a 1 / 400 dilution, and the mixture was left on ice for 30 minutes. After washing twice with FACS buffer, R-PE fluorescence was quantified by flow cytometry (Figure 12). The bispecific antibody bound to both types of cells, meaning that a bispecific antibody with the intended heavy and light chain combination was obtained, and it was shown to bind to both HER2 and CD3.

[0089] [6] Evaluation of bispecific bonds The ability of the bispecific antibody obtained by the first purification method to simultaneously bind to two antigens was evaluated using reporter cells. 3.6 × 10 6 50 μL of Raji cells or Raji-HER2 cells (InvivoGen, forced expression strain) at a cell / mL ratio, T4h-Ok or T4k-Oh prepared to 1, 10, or 100 nM in PBS, or PBS (10 μL), and 4.5 × 106 Jurkat-Lucia-NFAT cells (40 μL) were mixed at a cell / mL ratio and cultured at 37°C under 5% CO2 conditions for 21 hours. 40 μL of the culture was taken, centrifuged at 400 × g for 5 minutes, and 50 μL of QUANTI-Luc solution (InvivoGen) was added to 20 μL of the supernatant, and luminescence was scanned (Figure 13). The NFAT pathway in Jurkat-Lucia-NFAT cells showed little activation even at the highest concentration of the bispecific antibody in co-culture with Raji cells, but was strongly activated in co-culture with Raji-HER2 cells. This result indicates that the NFAT pathway was activated by CD3 co-stimulation in a HER2 expression-specific and antibody concentration-dependent manner, suggesting that a bispecific antibody with the intended heavy and light chain combination was obtained, and that it simultaneously bound to HER2 and CD3. [Industrial applicability]

[0090] This disclosure provides a novel antigen-binding molecule that can be used in the pharmaceutical field.

Claims

1. An antigen-binding molecule comprising a first polypeptide and a second polypeptide, The first polypeptide comprises a heavy chain variable region (VH) and the CH4 domain (IgM-CH4) of the first human immunoglobulin M. The second polypeptide comprises a light chain variable region (VL) and a second IgM-CH4, The first IgM-CH4 and the second IgM-CH4 are bonded to each other. VH and VL form the antigen-binding site. Antigen-binding molecules.

2. The first IgM-CH4 is VALHRPDVYLLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X １ ) QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR(X ２ )T(X ３ )RTVDKST (Sequence No. 3) [(X １ ) is M or E, and (X ２ ) is V or Y, and (X ３ ) is E or Q] It consists of the amino acid sequence of, or contains the amino acid sequence of SEQ ID NO: 3, or consists of a sequence in which 1 to 11 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 is ALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQW(X ４ )QRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHALPNR(X ５ )T(X ６ )RTVDKST (SEQ ID NO: 4) [(X ４ ) is M or E, and (X ５ ) is V or Y, and (X ６ ) is E or Q] It consists of the amino acid sequence of, or contains the amino acid sequence of SEQ ID NO: 4, or consists of a sequence in which 1 to 11 amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO:

4. The antigen-binding molecule according to claim 1.

3. The first IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 3, or a sequence in which one to three amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 4, or a sequence in which one to three amino acids are deleted, substituted, added, or inserted in the amino acid sequence of SEQ ID NO:

4. The antigen-binding molecule according to claim 2.

4. The first IgM-CH4 consists of the amino acid sequence of SEQ ID NO: 3, and / or The second IgM-CH4 consists of the amino acid sequence of SEQ ID NO:

4. The antigen-binding molecule according to claim 2.

5. (X １ ) is M or E, and (X ２ ) is Y, and (X ３ ) is E or Q, and / or (X ４ ) is M or E, and (X ５ ) is Y, and (X ６ ) is E or Q, The antigen-binding molecule according to claim 4.

6. (X １ ) is M, and (X ２ ) is Y, and (X ３ ) is Q and / or, (X ４ ) is M, and (X ５ ) is Y, and (X ６ ) is Q, The antigen-binding molecule according to claim 4.

7. (X １ ) is M, and (X ２ ) is Y, and (X ３ ) is E and / or, (X ４ ) is M, and (X ５ ) is Y, and (X ６ ) is E, The antigen-binding molecule according to claim 4.

8. (X １ ) is E, and (X ２ ) is Y, and (X ３ ) is E and / or, (X ４ ) is E, and (X ５ ) is Y, and (X ６ ) is E, The antigen-binding molecule according to claim 4.

9. Furthermore, the antigen-binding molecule according to any one of claims 1 to 8 is a bispecific molecule comprising a third polypeptide and a fourth polypeptide, The third polypeptide contains the CH1 domain (CH1) of the second VH and human immunoglobulin. The fourth polypeptide contains the second VL and the CL domain (CL) of human immunoglobulin. CH1 and CL are bonded to each other. The second VH and second VL form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site are linked via the binding of the first polypeptide to the third polypeptide or the fourth polypeptide, or via the binding of the second polypeptide to the third polypeptide or the fourth polypeptide. Antigen-binding molecules.

10. The antigen-binding molecule according to claim 9, wherein CH1 is the CH1 of IgG.

11. The antigen-binding molecule according to claim 9, wherein CL is Cκ.

12. The antigen-binding molecule according to claim 9, wherein the first antigen-binding site and the second antigen-binding site are linked via the binding of the first polypeptide and the third polypeptide.

13. The first polypeptide further contains a first Fc domain (Fc), and from the N-terminus, it forms a structure of (VH)-(IgM-CH4)-(Fc). The third polypeptide further contains a second Fc, forming a (VH)-(CH1)-(Fc) structure from the N-terminus. The first Fc and the second Fc are bonded to each other. The antigen-binding molecule according to claim 9.

14. The antigen-binding molecule according to claim 13, wherein the first Fc and / or the second Fc have mutations that promote heterodimerization.

15. A nucleic acid encoding the first polypeptide according to claim 1, a nucleic acid encoding the second polypeptide according to claim 1, or a combination thereof.

16. A vector comprising the nucleic acid or combination described in claim 15.

17. A cell comprising the nucleic acid or combination described in claim 15, or the vector described in claim 16.

18. A nucleic acid encoding the third polypeptide according to claim 9, a nucleic acid encoding the fourth polypeptide according to claim 9, or a combination thereof, A vector comprising a nucleic acid encoding the third polypeptide according to claim 9, a nucleic acid encoding the fourth polypeptide according to claim 9, or a combination thereof. The cell according to claim 15, further comprising:

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