Antibodies, compositions, and methods for detecting or capturing polypeptides in a sample.

Abstract

The object of the present invention is to provide antibodies, compositions, and methods for detecting or capturing polypeptides in a sample for use in the detection or capture of polypeptides in a sample. [Solution] This disclosure provides an antibody that specifically binds to a constant region of an IgG heavy chain containing a specific sequence for use in detecting or capturing polypeptides in a sample, a composition thereof, and a method for detecting or capturing polypeptides in a sample.

Description

Technical Field

[0001] The present invention relates to antibodies, compositions for use in detecting or capturing polypeptides in a sample, and methods for detecting or capturing polypeptides in a sample.

Background Art

[0002] Hybridoma technology has enabled the production of monoclonal antibodies, and this monoclonal antibody technology has a wide range of applications in many scientific fields (Non-Patent Document 1). After the achievement of this technology, further efforts have been made in the fields of therapeutic antibodies and diagnostic antibodies. Thirty years have passed since the first approval of monoclonal antibody therapy in the United States (Non-Patent Document 2). More than 30 antibodies have been approved by the FDA, and a significant number of candidates are undergoing clinical and preclinical evaluations. To date, monoclonal antibodies continue to be standard therapeutic molecules and are used in various disease fields such as cancer, autoimmune diseases, respiratory diseases, infectious diseases, and neurological diseases (Non-Patent Document 3).

[0003] To increase the advantages of therapeutic antibodies, many different types of modified Fc modifications for improving functions have been identified, such as those for enhancing antibody-dependent cell-mediated cytotoxicity, those for enhancing complement-dependent cytotoxicity, those for extending the antibody half-life, those for modulating antigen clearance, and those for facilitating heavy-chain heterodimerization (Non-Patent Document 4).

[0004] In the process of producing therapeutic monoclonal antibodies, heterogeneity of antibody molecules can make it difficult to achieve consistent manufacturing and quality control (Non-Patent Literature 5). Heavy chain C-terminal heterogeneity in recombinant monoclonal antibodies, including wild-type heavy chains, C-terminal lysine-deficient heavy chains (also referred to as "ΔK"), and heavy chains ending in amidated proline (also referred to as "ΔGK amide"), is one such example that has been previously reported (Non-Patent Literature 6). Interestingly, this cleavage event is similar to the modification of endogenous antibody Fc. Generally, the C-terminal lysine of endogenous antibody heavy chains is cleaved in vivo by endogenous carboxypeptidases. Further modifications, such as C-terminal glycine cleavage and proline amidation, have also been reported. This is because peptidylglycine α-amidate monooxygenase (PAM) cleaves the C-terminal glycine and amidates the proline.

[0005] To overcome this C-terminal heterogeneity problem, scientists successfully identified the central residues and modified the antibody Fc by genetically deleting C-terminal lysine (K) (position 447 in the EU numbering system) and glycine (G) (position 446 in the EU numbering system) from the Fc region (also known as "ΔGK") (Patent Document 1).

[0006] Antibodies that specifically bind to modified Fc regions but not to wild-type Fc have been reported (Non-Patent Document 7; Patent Document 2). Antibodies against modified Fc regions have been proven to be very useful for various purposes. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2009041613A1 [Patent Document 2] WO2017072210A1 [Non-patent literature]

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0009] The inventors have provided several antibodies containing a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region, lacking both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retaining the amino acid at position 445 according to the EU numbering system. The set of modifications in such a modified IgG heavy chain constant region is referred herein to as "IgGΔGK," and antibodies containing a modified IgG heavy chain constant region with IgGΔGK include, for example, satralizumab, nemolizumab, emicizumab, SKY59, AMY109, and GYM329. The present invention provides antibodies, compositions containing the antibodies, and methods of using the antibodies for specifically binding to, detecting, and capturing such ΔGKs in a modified IgG heavy chain constant region.

[0010] Specifically, the present invention relates to the following: [1] An antibody that specifically binds to a first modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, wherein the first modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remains in the first modified IgG heavy chain constant region. [2] The antibody described in [1] in which the amino acid at position 445 according to the EU numbering system in the first modified IgG heavy chain constant region is not amidated. [3] An antibody according to [2] that is substantially not bound to a second modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, wherein the second modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remains in the second modified IgG heavy chain constant region and is amidated. [4] An antibody described in any of [1] to [3] that does not substantially bind to (i) or (ii) below: (i) a third modified IgG heavy chain constant region or an unmodified human IgG heavy chain constant region derived from a human IgG heavy chain constant region, wherein the amino acids at positions 445, 446, and 447 according to the EU numbering system remain in the third modified IgG heavy chain constant region or the unmodified human IgG heavy chain constant region; and (ii) A fourth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, wherein the fourth modified IgG heavy chain constant region lacks lysine at position 447 according to the EU numbering system, and the amino acids at positions 445 and 446 according to the EU numbering system remain in the fourth modified IgG heavy chain constant region. [5] The antibody according to [3], wherein the binding activity of the antibody to a second modified IgG heavy chain constant region is less than the detection limit of the enzyme-linked immunoassay. [6] The antibody according to [4], wherein the binding activity of the antibody to at least one IgG heavy chain constant region selected from the group consisting of a third modified IgG heavy chain constant region, a fourth modified IgG heavy chain constant region, and an unmodified human IgG heavy chain constant region is less than the detection limit of the enzyme-linked immunoassay. [7] The antibody according to any one of [1] to [6], wherein the binding activity of the antibody to the first modified IgG heavy chain constant region is detectable by an enzyme-linked immunoassay. [8] (a) HVR-H1 containing the amino acid sequence of Sequence ID: 2; (b) HVR-H2 containing the amino acid sequence of Sequence ID: 3; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 containing the amino acid sequence of Sequence ID: 7; and (f) HVR-L3 containing the amino acid sequence of Sequence ID No. 8 An antibody according to any one of [1] to [7] that competes for binding to the first modified IgG heavy chain constant region with an antibody containing the above. [9] (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8 An antibody according to any one of [1] to [8], which binds to the same epitope as an antibody comprising

[10] A composition for use in detecting or capturing a polypeptide in a sample, comprising an antibody according to any one of [1] to [9], wherein the polypeptide comprises a fifth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, and the fifth modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remains in the fifth modified IgG heavy chain constant region.

[11] The composition according to

[10] , wherein the amino acid at position 445 according to the EU numbering system in the fifth modified IgG heavy chain constant region is not amidated.

[12] A method for detecting or capturing a polypeptide in a sample, comprising the step of contacting a sample with an antibody according to any one of [1] to [8], wherein the polypeptide comprises a sixth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, and the sixth modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remains in the sixth modified IgG heavy chain constant region.

[13] The method according to

[12] , wherein the amino acid at position 445 according to the EU numbering system in the sixth modified IgG heavy chain constant region is not amidated. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Figure 1] Shows the ELISA results of the primary screening. The identified single hit (positive) B cell clone was able to specifically bind to IgG1ΔGK and IgG4ΔGK, and did not bind to IgG1ΔK and IgG4ΔK. An anti-keyhole limpet hemocyanin (KLH) rabbit monoclonal antibody was used as an isotype control. [Figure 2] Shows the ELISA results of the secondary screening. The identified single hit (positive) B cell clone was able to specifically bind to IgG1ΔGK and IgG4ΔGK, and did not bind to IgG1ΔGK amide and IgG4ΔGK amide. An anti-KLH rabbit monoclonal antibody was used as an isotype control. [Figure 3] Shows the ELISA results of the purified monoclonal antibody. YG55 was able to specifically bind to IgG1ΔGK and IgG4ΔGK, and did not bind to IgG1ΔGK amide and IgG4ΔGK amide. An anti-KLH rabbit monoclonal antibody was used as an isotype control.

Mode for Carrying Out the Invention

[0012] Description of Aspects I. Definition "Affinity" refers to the total strength of non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Unless otherwise indicated, the "binding affinity" or "binding activity" used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0013] The term "antibody that specifically binds to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region" refers to an antibody that can bind to a specific type of modified IgG heavy chain constant region with sufficient affinity to be useful as a detection agent, capture agent, or diagnostic agent targeting the modified IgG heavy chain constant region. In one embodiment, for an antibody that specifically binds to a first modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, the degree of binding of the antibody to other IgG heavy chain constant regions, such as the second, third, and fourth modified IgG heavy chain constant regions, and to the unmodified human IgG heavy chain constant region, is less than 10% of the binding to the first modified IgG heavy chain constant region, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, antibodies that bind to modified IgG heavy chain constant regions derived from human IgG heavy chain constant regions have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, an antibody that binds to a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region binds to the epitope of the modified IgG heavy chain constant region.

[0014] 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.

[0015] The "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.

[0016] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0017] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the 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.

[0018] 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).

[0019] The "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.

[0020] As used herein, the term “hypervariable region” or “HVR” refers to each region of the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used 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) A combination 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).

[0021] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced as necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0022] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0023] An antibody that "binds to the same epitope as the reference antibody" is defined as an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay. Conversely, the reference antibody blocks the binding of the aforementioned antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0024] II. Antibodies The antibody in this invention specifically binds to a first modified IgG heavy chain constant region derived from the human IgG heavy chain constant region. The first modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system. The amino acid at position 445 according to the EU numbering system remains in the first modified IgG heavy chain constant region.

[0025] In a further aspect of the present invention, the antibody that specifically binds to a first modified IgG heavy chain constant region derived from a human IgG heavy chain constant region is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1, IgG2, IgG3, and IgG4 antibody, or any other antibody class or isotype as defined herein.

[0026] A. First modified IgG heavy chain constant region In one embodiment, the first modified IgG heavy chain constant region may form a dimer, similar to the heavy chain constant region in naturally occurring IgG, or it may form a halfmer, similar to the heavy chain constant region in monomeric Fc, as reported by Ishino, T. et al., J. Biol. Chem. 288:16529-37 (2013).

[0027] In one embodiment, the first modified IgG heavy chain constant region may be derived from a portion of the human IgG heavy chain constant region. In a preferred embodiment, the first modified IgG heavy chain constant region includes at least a region corresponding to the CH3 region of the human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located within the range of the region corresponding to at least the CH3 region of the human IgG heavy chain. In a further preferred embodiment, the first modified IgG heavy chain constant region includes at least regions corresponding to the CH2 and CH3 regions of the human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located within the range of the region corresponding to at least the CH2 and CH3 regions of the human IgG heavy chain. In a further preferred embodiment, the first modified IgG heavy chain constant region includes at least a region corresponding to the Fc region of the human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located within the range of the region corresponding to at least the Fc region of the human IgG heavy chain.

[0028] In one embodiment, when the first modified IgG heavy chain constant region is a human modified IgG heavy chain, the human modified IgG heavy chain is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 heavy chains. In a preferred embodiment, the human IgG heavy chain is a human IgG1 or IgG4 heavy chain.

[0029] In a preferred embodiment, the amino acid at position 445 in the EU numbering system of the first modified IgG heavy chain constant region is not amidated.

[0030] A set of modifications in the constant region of a modified IgG heavy chain, such as that in the preferred first modified IgG heavy chain constant region, which retains a non-amidate amino acid at position 445 according to the EU numbering system, but lacks both glycine at position 446 according to the EU numbering system and lysine at position 447 according to the EU numbering system, is referred to herein as "IgGΔGK" or "ΔGK".

[0031] In a further preferred embodiment, the antibody substantially does not bind to the IgG heavy chain constant region containing a ΔGK amide, which is a second modified IgG heavy chain constant region derived from the human IgG heavy chain constant region. The second modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system. In the second modified IgG heavy chain constant region, the amino acid at position 445 according to the EU numbering system is present and amidated.

[0032] In all of these cases, the antibody can distinguish the modified IgG heavy chain constant region having a non-amidate amino acid at position 445 according to the EU numbering system from the modified IgG heavy chain constant region having a ΔGK amide.

[0033] In one embodiment, the binding activity of the antibody to the second modified IgG heavy chain constant region is below the detection limit in enzyme-linked immunoassays.

[0034] In another embodiment, the binding activity of the antibody to the first modified IgG heavy chain constant region can be detected by enzyme-linked immunoassay.

[0035] B. The constant region of the human IgG heavy chain where antibodies do not substantially bind. In one embodiment, the antibody substantially does not bind to (i) a third modified IgG heavy chain constant region having an "undeleted C-terminus" derived from the human IgG heavy chain constant region, or to an unmodified human IgG heavy chain constant region, or (ii) a fourth modified IgG heavy chain constant region derived from the human IgG heavy chain constant region containing "ΔK".

[0036] In (i), in the third modified IgG heavy chain constant region including the non-deleting C-terminus or the unmodified human IgG heavy chain constant region, the amino acids at positions 445, 446, and 447 according to the EU numbering system remain.

[0037] In (ii), the fourth modified IgG heavy chain constant region containing ΔK lacks lysine at position 447 according to the EU numbering system. In the fourth modified IgG heavy chain constant region in (ii), the amino acids at positions 445 and 446 according to the EU numbering system remain.

[0038] In all of these cases, the antibody can distinguish the first modified IgG heavy chain constant region from the modified IgG heavy chain constant region having a non-deleted C-terminus or ΔK, or from the unmodified human IgG heavy chain constant region.

[0039] In one embodiment, the binding activity of an antibody to at least one IgG heavy chain constant region selected from the group consisting of a third modified IgG heavy chain constant region, a fourth modified IgG heavy chain constant region, and an unmodified human IgG heavy chain constant region is below the detection limit in an enzyme-linked immunoassay.

[0040] C. YG55 Antibodies bind to their antigens through their variable region; therefore, the variable region is crucial for antibody binding specificity. Furthermore, it is generally known that the hypervariable region (HVR) is the most important region for antibody binding specificity.

[0041] The inventors obtained antibodies encompassing the aforementioned antibodies by screening, as described in the "Examples." One of these antibodies is named "YG55," but the scope of the present invention is not limited to this specific antibody, YG55. Based on the general knowledge described above, the inventors identified all HVRs of YG55. YG55 includes (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 3, (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4, (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6, (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 8.

[0042] In one aspect, the present invention provides an antibody comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0043] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4. In one aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4. In another aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8. In a further aspect, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3. In a further aspect, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4.

[0044] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0045] In another aspect, the antibody of the present invention comprises (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0046] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 8.

[0047] In another aspect, the antibodies described herein include a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence, but the antibodies described herein containing such sequence retain the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 1. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antibody contains the VH sequence in SEQ ID NO: 1, including post-translational modifications of said sequence. In certain embodiments, VH contains one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 3, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0048] In another aspect, an antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the antibody comprising such sequence retains the ability to bind to the first modified IgG heavy chain constant region. In a particular embodiment, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 5. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the antibody contains the VL sequence in SEQ ID NO: 5, including post-translational modifications of said sequence. In certain embodiments, the VL contains one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 8. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0049] In another aspect, an antibody is provided comprising VH in any of the above embodiments and VL in any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 1 and SEQ ID NO: 5, respectively, including those with post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modifications to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0050] In one situation, (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 containing the amino acid sequence of Sequence ID: 3; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 containing the amino acid sequence of Sequence ID: 7; and (f) HVR-L3 containing the amino acid sequence of Sequence ID No. 8 An antibody containing the first modified IgG heavy chain constant region is provided, which competes with the antibody for binding to the first modified IgG heavy chain constant region.

[0051] In one situation, (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 2; (b) HVR-H2 containing the amino acid sequence of Sequence ID: 3; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 containing the amino acid sequence of Sequence ID: 7; and (f) HVR-L3 containing the amino acid sequence of Sequence ID No. 8 An antibody that binds to the same epitope as the antibody containing [the specified substance] is provided.

[0052] D. Recombination method and configuration For example, as described in U.S. Patent No. 4,816,567, antibodies can be produced using recombinant methods or configurations. In one embodiment, isolated nucleic acids (may be multiple) encoding the antibody described herein are provided. Such nucleic acids may encode an amino acid sequence containing the VL and / or VH of the antibody (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 acids are provided. In a further embodiment, host cells containing such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising nucleic acid molecules encoding an amino acid sequence containing the VL of the antibody and an amino acid sequence containing the VH of the antibody, or (2) a first vector comprising a nucleic acid molecule encoding an amino acid sequence containing the VL of the antibody and a second vector comprising a nucleic acid molecule encoding an amino acid sequence containing the VH of the antibody (e.g., transformed). In one embodiment, the host cell is 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 the antibody described herein is provided, comprising culturing host cells containing the nucleic acid molecule encoding the antibody as described above under conditions suitable for the expression of the antibody described herein, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0053] For the recombinant production of antibodies described herein, nucleic acid molecules encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid molecules 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 the antibody).

[0054] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, 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, for the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste into soluble fractions and further purified.

[0055] 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).

[0056] 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.

[0057] 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).

[0058] 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 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. 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).

[0059] E. Measurement method (assay) The antibodies provided herein may be identified, screened, or have their physical / chemical properties and / or biological activity elucidated by various assay methods known in the art.

[0060] F. Combined measurement methods and other measurement methods In one aspect, the antibody of the present invention is tested for its antigen-binding activity by known methods such as ELISA and Western blotting.

[0061] In another context, a competitive assay may be used to identify antibodies that compete with YG55 used in the "Examples" for binding to the first modified IgG heavy chain constant region. In certain embodiments, such competitive antibodies bind to the same epitopes (e.g., linear or conformational epitopes) as YG55 used in the "Examples." Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0062] In an exemplary competition assay, an immobilized first modified IgG heavy chain constant region is incubated in a solution containing a first labeled antibody that binds to the first modified IgG heavy chain constant region and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to the first modified IgG heavy chain constant region. The second antibody may be present in B cell or hybridoma supernatant. As a control, an immobilized first modified IgG heavy chain constant region is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to the first modified IgG heavy chain constant region, any excess unbound antibody is removed and the amount of label bound to the immobilized IgG heavy chain constant region is measured. If the amount of label bound to the immobilized IgG heavy chain constant region is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the first modified IgG heavy chain constant region. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0063] III. Composition In one aspect, the composition of the present invention is a composition for use in the detection or capture of polypeptides in a sample. The composition comprises the antibody described in "II. Antibodies". The polypeptide comprises a fifth modified IgG heavy chain constant region derived from the human IgG heavy chain constant region. The fifth modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, while the amino acid at position 445 according to the EU numbering system remains in the fifth modified IgG heavy chain constant region.

[0064] In a preferred embodiment, the amino acid at position 445 in the EU numbering system of the fifth modified IgG heavy chain constant region is not amidated.

[0065] The fifth modified IgG heavy chain constant region may include other amino acid substitutions or modifications, insofar as it includes a modification that lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retains the amino acid at position 445 according to the EU numbering system.

[0066] Furthermore, the polypeptides detected or captured using the compositions of the present invention are not particularly limited in terms of structure, as long as the polypeptides contain a fifth modified IgG heavy chain constant region at their C-terminus. The polypeptides detected or captured using the compositions of the present invention may be antibodies such as human IgG1, IgG2, IgG3, or IgG4 molecules, antibody fragments, fusion proteins, or other forms of polypeptides containing a fifth modified IgG heavy chain constant region.

[0067] IV. Method In one aspect, the present invention provides a method for detecting or capturing a polypeptide in a sample. The method includes contacting the sample with an antibody described in "II. Antibodies". The polypeptide comprises a sixth modified IgG heavy chain constant region derived from the human IgG heavy chain constant region. The sixth modified IgG heavy chain constant region lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, while the amino acid at position 445 according to the EU numbering system remains in the sixth modified IgG heavy chain constant region.

[0068] In a preferred embodiment, the amino acid at position 445 in the EU numbering system of the sixth modified IgG heavy chain constant region is not amidated.

[0069] The sixth modified IgG heavy chain constant region may include other amino acid substitutions or modifications, insofar as it includes a modification that lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retains the amino acid at position 445 according to the EU numbering system.

[0070] Furthermore, the polypeptides detected or captured by the methods of the present invention are not particularly limited in terms of structure, as long as the polypeptide contains a sixth modified IgG heavy chain constant region at its C-terminus. The polypeptides detected or captured by the methods of the present invention may be antibodies such as human IgG1, IgG2, IgG3, or IgG4 molecules, antibody fragments, fusion proteins, or other forms of polypeptides containing a sixth modified IgG heavy chain constant region. [Examples]

[0071] Example 1 Preparation of ΔGK Fc Human IgG4-derived ΔGK Fc fragments were expressed using the FreeStyle® 293 expression system (Invitrogen). The expressed Fc fragments were purified from the collected cell culture medium by affinity chromatography (MabSelect SuRe, GE). In the final step, the buffer was replaced with D-PBS(-).

[0072] Example 2 Production of anti-ΔGK antibodies Anti-ΔGK antibodies were prepared, selected, and assayed as described below.

[0073] NZW rabbits were intradermally immunized with the ΔGK Fc fragment derived from human IgG4 expressed in Example 1 (100-200 μg / dose / rabbit). After six doses over a 3-month period, blood and spleen samples were collected. For B cell selection, IgG4ΔGK antibody (IgG4 antibody in which the IgG4 C-terminal GK is genetically deleted) and wild-type IgG4 antibody were prepared. ΔGK-specific B cells were sorted using a cell sorter, then seeded and cultured according to the method described in WO2016098356A1. After culturing, the B cell culture supernatant was collected for further analysis, and the corresponding B cell pellets were cryopreserved.

[0074] Specific binding to IgGΔGK was evaluated using ELISA with B cell culture supernatant. In this primary screening, four types of antibodies were used as antigens to evaluate binding specificity to the ΔGK C-terminal sequence: IgG1 antibody with genetic deletion of IgG1 C-terminal K (IgG1ΔK), IgG1 antibody with genetic deletion of IgG1 C-terminal GK (IgG1ΔGK), IgG4 antibody with genetic deletion of IgG4 C-terminal K (IgG4ΔK), and IgG4 antibody with genetic deletion of IgG4 C-terminal GK (IgG4ΔGK). The results showed that only one culture supernatant sample from a single B cell clone showed specific binding to both IgG1ΔGK and IgG4ΔGK (Figure 1).

[0075] ΔGK Fc is structurally more similar to ΔGK amide Fc than to ΔK Fc. The inventors also characterized specific binding to ΔGK Fc and ΔGK amide Fc using the aforementioned selected culture supernatants from positive B cell clones. IgG1ΔGK amide and IgG4ΔGK amide were prepared by PAM treatment of IgG1ΔK or IgG4ΔK as described above and purified by conventional methods. In this secondary screening, four types of antibodies were used as antigens in an ELISA assay to evaluate binding specificity to the ΔGK C-terminal sequence: IgG1ΔGK, IgG1ΔGK amide, IgG4ΔGK, and IgG4ΔGK amide. Surprisingly, the single B cell culture supernatant tested showed extremely high specificity to the ΔGK molecule (Figure 2).

[0076] Based on these screening results, RNA from selected clones was extracted from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, catalog number R1053). Reverse transcription PCR was used to obtain and amplify the DNA encoding the antibody heavy chain variable region of the antibody produced by the selected clones, and then recombinate it with DNA encoding the rabbit IgG heavy chain constant region (SEQ ID NO: 9). Reverse transcription PCR was also used to obtain and amplify the DNA encoding the antibody light chain variable region, and then recombinate it with DNA encoding the rabbit Igk light chain constant region (SEQ ID NO: 10). An anti-ΔGK antibody named "YG55," having two heavy chains and two light chains, was constructed from these recombinants. The VH, VL, and HVR sequences of the heavy and light chains are shown in Table 1. YG55 was expressed using the FreeStyle® 293 expression system and purified from the culture supernatant.

[0077] Example 3 Characterization of the anti-ΔGK monoclonal antibody YG55 Following gene cloning and antibody expression, the specificity of YG55 was evaluated by ELISA assay, as described above in the secondary screening section. Antibody gene cloning was successful, yielding YG55 with the same specificity as that demonstrated by the hit (positive) B cell clone (Figure 3). This highly specific binding was also confirmed by surface plasmon resonance assay. The specific binding motif and its epitope were identified by crystal structure analysis.

[0078] [Table 1]

Claims

1. A method for detecting or capturing polypeptides in a sample, The polypeptide is a monoclonal antibody containing a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region, lacking both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retaining the amino acid at position 445 according to the EU numbering system. The method involves applying an antibody selected from (1) and (2) below to the sample: (1) (i) (a) HVR-H1 containing the amino acid sequence of Sequence ID No. 2; (b) HVR-H2 containing the amino acid sequence of Sequence ID: 3; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 4; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 6; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 7; and (f) Sequence ID: HVR-L3 containing the amino acid sequence of 8, Includes, (ii) Specifically binds to the constant region of the modified IgG heavy chain, antibody; (2) An antibody that competes for binding between the antibody comprising (a) to (f) above and the modified IgG heavy chain constant region, comprising a heavy chain variable domain sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and a light chain variable domain sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5, A method including the step of bringing into contact with in vitro.

2. The method according to claim 1, wherein the modified IgG heavy chain constant region, derived from the human IgG heavy chain constant region, lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retains the amino acid at position 445 according to the EU numbering system, is a modified IgG heavy chain constant region in which the amino acid at position 445 according to the EU numbering system is not amidated.

3. The method according to claim 2, wherein the antibody is derived from the human IgG heavy chain constant region and substantially does not bind to a modified IgG heavy chain constant region that lacks both glycine at position 446 and lysine at position 447 according to the EU numbering system, and in which the amino acid at position 445 according to the EU numbering system is amidated.

4. The method according to any one of claims 1 to 3, wherein the antibody is derived from the human IgG heavy chain constant region and does not substantially bind to a modified IgG heavy chain constant region or an unmodified human IgG heavy chain constant region that has amino acids at positions 445, 446, and 447 according to the EU numbering system, and does not substantially bind to a modified IgG heavy chain constant region that is derived from the human IgG heavy chain constant region, lacks lysine at position 447 according to the EU numbering system, and has amino acids at positions 445 and 446 according to the EU numbering system.

5. The method according to claim 3, wherein the binding activity of the antibody to a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region, lacking both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retaining and amidating the amino acid at position 445 according to the EU numbering system, is below the detection limit of an enzyme-linked immunoassay.

6. The method according to claim 4, wherein the binding activity of the antibody to at least one IgG heavy chain constant region selected from the group consisting of a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region and retaining the amino acids at positions 445, 446, and 447 according to the EU numbering system, a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region and lacking the lysine at position 447 according to the EU numbering system and retaining the amino acids at positions 445 and 446 according to the EU numbering system, and an unmodified human IgG heavy chain constant region is less than the detection limit of the enzyme-linked immunoassay.

7. The method according to any one of claims 1 to 6, wherein the binding activity of the antibody to a modified IgG heavy chain constant region derived from the human IgG heavy chain constant region, lacking both glycine at position 446 and lysine at position 447 according to the EU numbering system, and retaining the amino acid at position 445 according to the EU numbering system, is detectable by an enzyme-linked immunoassay.

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