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

Abstract

To provide antibodies and compositions for use in detecting or capturing a polypeptide in a sample, and to provide methods for detecting or capturing a polypeptide in a sample.SOLUTION: The present disclosure provides an antibody that specifically binds to an IgG heavy chain constant region comprising a specific sequence for use in detecting or capturing a polypeptide in a sample; a composition thereof; and a method for detecting or capturing a polypeptide in a sample.SELECTED DRAWING: Figure 3

Description

[Technical Field]

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

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

[0003] To increase the benefits of therapeutic antibodies, many different types of altered Fc modifications have been identified to improve function, for example, to enhance antibody-dependent cell-mediated cytotoxicity, enhance complement-dependent cytotoxicity, extend antibody half-life, modulate antigen clearance, and facilitate heavy chain heterodimerization (Non-Patent Document 4).

[0004] During the production of therapeutic monoclonal antibodies, heterogeneity of antibody molecules can make consistent production and quality control difficult (Non-Patent Document 5). Heavy chain C-terminal heterogeneity for recombinant monoclonal antibodies, including wild-type heavy chains, C-terminal lysine-deleted heavy chains (also referred to as "ΔK"), and heavy chains ending in amidated proline (also referred to as "ΔGKamide"), is one such example that has been previously reported (Non-Patent Document 6). Interestingly, this cleavage event is similar to the modification of endogenous antibody Fc. In general, the C-terminal lysine of endogenous antibody heavy chains is cleaved in vivo by endogenous carboxypeptidases. Further modifications, C-terminal glycine cleavage and proline amidation, have also been reported. This is due to peptidylglycine α-amidating monooxygenase (PAM) cleaving the C-terminal glycine and amidating the proline.

[0005] To overcome this C-terminal heterogeneity problem, scientists successfully identified key residues and engineered antibody Fc by genetically deleting the 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 referred to 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 proven to be very useful for a variety of 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 present inventors have provided several antibodies comprising modified IgG heavy chain constant regions derived from human IgG heavy chain constant regions, lacking both glycine at position 446 according to the EU numbering system and lysine at position 447 according to the EU numbering system, while retaining the amino acid at position 445 according to the EU numbering system. Such a set of modifications in the modified IgG heavy chain constant region is referred to herein as "IgGΔGK." Antibodies comprising a modified IgG heavy chain constant region having an IgGΔGK include, for example, satralizumab, nemolizumab, emicizumab, SKY59, AMY109, and GYM329. The present invention provides antibodies that specifically bind to, detect, and capture such ΔGK in the modified IgG heavy chain constant region, compositions comprising the antibodies, and methods using the antibodies.

[0010] Specifically, the present invention relates to: [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 the glycine at position 446 according to the EU numbering system and the 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 according to [1], wherein 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] The antibody according to [2], which does not substantially bind 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 according to the EU numbering system 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] The antibody according to any one of [1] to [3], which does not substantially bind to the following (i) or (ii): (i) a third modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, or an unmodified 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 described in [3], wherein the binding activity of the antibody to the second modified IgG heavy chain constant region is below the detection limit of an enzyme-linked immunoassay. [6] The antibody described in [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 below the detection limit of an 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 enzyme-linked immunoassay. [8] (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 The antibody according to any one of [1] to [7], which competes for binding to the first modified IgG heavy chain constant region with an antibody comprising 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 The antibody according to any one of [1] to [8], which binds to the same epitope as an antibody comprising the compound.

[10] A composition for use in detecting or capturing a polypeptide in a sample, comprising the 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, the fifth modified IgG heavy chain constant region lacking both the glycine at position 446 according to the EU numbering system and the lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remaining in the fifth modified IgG heavy chain constant region.

[11] The composition described in

[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 a step of contacting the antibody according to any one of [1] to [8] with the sample, wherein the polypeptide comprises a sixth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, the sixth modified IgG heavy chain constant region lacking both the glycine at position 446 according to the EU numbering system and the lysine at position 447 according to the EU numbering system, and the amino acid at position 445 according to the EU numbering system remaining in the sixth modified IgG heavy chain constant region.

[13] The method described in

[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 explanation of the drawings]

[0011] [Figure 1] The ELISA results of the primary screening are shown. The identified single hit (positive) B cell clone was able to specifically bind to IgG1ΔGK and IgG4ΔGK, but not to IgG1ΔK or IgG4ΔK. Anti-keyhole limpet hemocyanin (KLH) rabbit monoclonal antibody was used as an isotype control. [Figure 2] The ELISA results of the secondary screening are shown. The identified single hit (positive) B cell clone was able to specifically bind to IgG1ΔGK and IgG4ΔGK, but not to IgG1ΔGKamide or IgG4ΔGKamide. An anti-KLH rabbit monoclonal antibody was used as an isotype control. [Figure 3] ELISA results for purified monoclonal antibodies are shown. YG55 specifically binds to IgG1ΔGK and IgG4ΔGK, but not to IgG1ΔGKamide or IgG4ΔGKamide. Anti-KLH rabbit monoclonal antibody was used as an isotype control. DETAILED DESCRIPTION OF THE INVENTION

[0012] Description of Aspects I. Definition "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" or "avidity," as used herein, refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative 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 particular type of modified IgG heavy chain constant region with sufficient affinity so as 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 that specifically binds to the first modified IgG heavy chain constant region to IgG heavy chain constant regions other than the first modified IgG heavy chain constant region, e.g., the second, third, and fourth modified IgG heavy chain constant regions, and the unmodified human IgG heavy chain constant region, is less than about 10% of the binding of the first modified IgG heavy chain constant region, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, antibodies that bind to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region have a binding affinity 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 (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, an antibody that binds to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region binds to an epitope of the modified IgG heavy chain constant region.

[0014] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so 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 major 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 the 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 population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0017] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from 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 is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0018] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. 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 specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0019] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically 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 an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (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) antigenic contacts 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), comprising 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" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0022] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against 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 matches by the sequence alignment program ALIGN-2 in its alignment of A and B, 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 will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0023] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen by 50% or more in a competition assay, or conversely, a reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0024] II. Antibodies The antibodies of the present invention specifically bind to a first modified IgG heavy chain constant region derived from a human IgG heavy chain constant region. The first modified IgG heavy chain constant region lacks both the glycine at position 446 (EU numbering system) and the lysine at position 447 (EU numbering system). The amino acid at position 445 (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 the 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 an 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, or IgG4 antibody, or any other antibody class or isotype 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 like the heavy chain constant region in a naturally occurring IgG, or may form a halfmer like the heavy chain constant region in a monomeric Fc reported in 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 a human IgG heavy chain constant region. In a preferred embodiment, the first modified IgG heavy chain constant region comprises at least a region corresponding to the CH3 region of a human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located at least within the region corresponding to the CH3 region of a human IgG heavy chain. In a further preferred embodiment, the first modified IgG heavy chain constant region comprises at least a region corresponding to the CH2 and CH3 regions of a human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located at least within the region corresponding to the CH2 and CH3 regions of a human IgG heavy chain. In a further preferred embodiment, the first modified IgG heavy chain constant region comprises at least a region corresponding to the Fc region of a human IgG heavy chain. In this embodiment, the epitope to which the antibody binds may be located at least within the region corresponding to the Fc region of a human IgG heavy chain.

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

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

[0030] The set of modifications in a modified IgG heavy chain constant region, i.e., one that retains a non-amidated amino acid at position 445 according to the EU numbering system, like those in the first preferred modified IgG heavy chain constant region, but lacks both a glycine at position 446 according to the EU numbering system and a 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 does not substantially bind to a second modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, the IgG heavy chain constant region comprising ΔGKamide. The second modified IgG heavy chain constant region lacks both the glycine at position 446 (EU numbering system) and the lysine at position 447 (EU numbering system). In the second modified IgG heavy chain constant region, the amino acid at position 445 (EU numbering system) remains and is amidated.

[0032] In all of them, the antibody can distinguish a modified IgG heavy chain constant region having a non-amidated amino acid at position 445 according to the EU numbering system in the human modified IgG heavy chain constant region from a modified IgG heavy chain constant region having ΔGKamide.

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

[0034] In another embodiment, the binding activity of the antibody to the first modified IgG heavy chain constant region is detectable in an enzyme-linked immunoassay.

[0035] B. Human IgG heavy chain constant region to which antibodies do not substantially bind In one embodiment, the antibody does not substantially bind to (i) a third modified IgG heavy chain constant region having a "non-deleted C-terminus" derived from the human IgG heavy chain constant region, or 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 and comprising a "ΔK".

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

[0037] The fourth modified IgG heavy chain constant region having ΔK in (ii) 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 them, the antibody can distinguish the first modified IgG heavy chain constant region from a modified IgG heavy chain constant region with a non-deleted C-terminus or ΔK, or from an unmodified human IgG heavy chain constant region.

[0039] In one embodiment, the binding activity of the antibody to at least one IgG heavy chain constant region selected from the group consisting of the third modified IgG heavy chain constant region, the 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 Since an antibody binds to its antigen via its variable region, the variable region is important for the binding specificity of the antibody. Furthermore, it is generally known that the hypervariable region (HVR) is the most important region for the binding specificity of an antibody.

[0041] As described in the "Examples" section, the present inventors have screened and obtained antibodies encompassed by the aforementioned antibodies. One of these antibodies is designated "YG55," but the scope of the present invention is not limited to this particular antibody, YG55. Based on the above general knowledge, the present inventors have identified all of the HVRs of YG55. YG55 includes (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.

[0042] In one aspect, the present invention provides antibodies 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 embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4. In another embodiment, 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 embodiment, 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 embodiment, 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 antibodies 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, an 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 comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibodies described herein comprising such sequences 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 regions outside the HVRs (i.e., within the FRs). Optionally, the antibody comprises the VH sequence in SEQ ID NO: 1, including post-translational modifications of that sequence. In certain embodiments, the VH comprises one, two, or three 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, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 4. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[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 to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but antibodies comprising such sequences retain the ability to bind to a 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: 5. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVR (i.e., within the FR). Optionally, the antibody comprises the VL sequence in SEQ ID NO: 5, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs 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. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

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

[0050] In one aspect, (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 and an antibody that competes for binding to the first modified IgG heavy chain constant region with an antibody comprising:

[0051] In one aspect, (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 is provided that binds to the same epitope as an antibody comprising:

[0052] D. Recombination methods and constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid(s) encoding an antibody described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid molecule encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid molecule encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid molecule encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, there is provided a method of making an antibody described herein, comprising culturing a host cell comprising a nucleic acid molecule encoding the antibody, as described above, under conditions suitable for expression of the antibody described herein, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0053] For recombinant production of the antibodies described herein, nucleic acid molecules encoding the antibodies (e.g., such as 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 may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[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, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0055] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0056] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing 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 transformation of Spodoptera frugiperda cells.

[0057] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0058] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., 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 characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0060] F. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, and the like.

[0061] In another aspect, a competition assay can 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 competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as that bound by YG55 used in the Examples. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0062] In an exemplary competitive 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 to be 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, the 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 binding of the first antibody to the first modified IgG heavy chain constant region, excess unbound antibody is removed and the amount of label bound to the immobilized IgG heavy chain constant region is measured. A substantial decrease in the amount of label bound to the immobilized IgG heavy chain constant region in the test sample compared to the control sample indicates that the second antibody competes 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 detecting or capturing a polypeptide in a sample. The composition includes an antibody described in "II. Antibodies." The polypeptide includes a fifth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region. The fifth modified IgG heavy chain constant region lacks both the glycine at position 446 (EU numbering system) and the lysine at position 447 (EU numbering system), while the amino acid at position 445 (EU numbering system) remains in the fifth modified IgG heavy chain constant region.

[0064] In a preferred embodiment, the amino acid at position 445 according to the EU numbering system in 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, so long as it includes the modification of lacking both the glycine at position 446 according to the EU numbering system and the lysine at position 447 according to the EU numbering system, while retaining the amino acid at position 445 according to the EU numbering system.

[0066] Furthermore, the structure of the polypeptide to be detected or captured using the composition of the present invention is not particularly limited, as long as the polypeptide contains the fifth modified IgG heavy chain constant region at its C-terminus. The polypeptide to be detected or captured using the composition of the present invention may be an antibody such as a human IgG1 molecule, IgG2 molecule, IgG3 molecule, or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide containing the fifth modified IgG heavy chain constant region.

[0067] IV. Method In one aspect, the method of the present invention is 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 includes a sixth modified IgG heavy chain constant region derived from a human IgG heavy chain constant region. The sixth modified IgG heavy chain constant region lacks both the glycine at position 446 (EU numbering system) and the lysine at position 447 (EU numbering system), while the amino acid at position 445 (EU numbering system) remains in the sixth modified IgG heavy chain constant region.

[0068] In a preferred embodiment, the amino acid at position 445 according to the EU numbering system in 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, so long as it includes the modification of lacking both the glycine at position 446 according to the EU numbering system and the lysine at position 447 according to the EU numbering system, while retaining the amino acid at position 445 according to the EU numbering system.

[0070] Furthermore, the polypeptide to be detected or captured by the method of the present invention is not particularly limited in terms of structure, as long as the polypeptide contains the sixth modified IgG heavy chain constant region at its C-terminus. The polypeptide to be detected or captured by the method of the present invention may be an antibody such as a human IgG1 molecule, IgG2 molecule, IgG3 molecule, or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide containing the sixth modified IgG heavy chain constant region. [Example]

[0071] Example 1 Preparation of ΔGK Fc The ΔGK Fc fragment derived from human IgG4 was expressed using the FreeStyle™ 293 Expression System (Invitrogen). The expressed Fc fragment was purified from the collected cell culture medium by affinity chromatography (MabSelect SuRe, GE). In the final step, the buffer was exchanged with D-PBS(-).

[0072] Example 2 Generation 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 (100-200 μg / injection / rabbit) derived from human IgG4 expressed in Example 1. After six repeated injections over a three-month period, blood and spleens were collected. For B cell selection, IgG4ΔGK antibody (an IgG4 antibody in which the IgG4 C-terminal GK has been genetically deleted) and wild-type IgG4 antibody were prepared. ΔGK-specific B cells were selected using a cell sorter and then seeded and cultured using the method described in WO2016098356A1. After culture, the B cell culture supernatant was collected for further analysis, and the corresponding B cell pellet was cryopreserved.

[0074] Specific binding to IgGΔGK was assessed by ELISA using B cell culture supernatants. In this primary screening, four types of antibodies were used as antigens to evaluate binding specificity to the ΔGK C-terminal sequence: an IgG1 antibody with a genetically deleted IgG1 C-terminal K (IgG1ΔK), an IgG1 antibody with a genetically deleted IgG1 C-terminal GK (IgG1ΔGK), an IgG4 antibody with a genetically deleted IgG4 C-terminal K (IgG4ΔK), and an IgG4 antibody with a genetically deleted 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 ΔGKamide Fc than to ΔK Fc. We also characterized the specific binding of ΔGK Fc and ΔGKamide Fc using the previously selected culture supernatants from positive B cell clones. IgG1ΔGKamide and IgG4ΔGKamide were prepared by PAM treatment of the previously described IgG1ΔK or IgG4ΔK 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 for the ΔGK C-terminal sequence: IgG1ΔGK, IgG1ΔGKamide, IgG4ΔGK, and IgG4ΔGKamide. Surprisingly, each B cell culture supernatant tested showed extremely high specificity for the ΔGK molecule (Figure 2).

[0076] Based on the results of these screenings, RNA of selected clones was extracted from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Catalog No. R1053). DNA encoding the antibody heavy chain variable region of the antibody produced by the selected clone was obtained and amplified by reverse transcription PCR, and then recombined with DNA encoding the rabbit IgG heavy chain constant region (SEQ ID NO: 9). DNA encoding the antibody light chain variable region was also obtained and amplified by reverse transcription PCR, and then recombined with DNA encoding the rabbit Igk light chain constant region (SEQ ID NO: 10). An anti-ΔGK antibody designated "YG55," having two heavy chains and two light chains, was produced from these recombinants. The sequences of the VH, VL, and HVR of the heavy and light chains are listed in Table 1. YG55 was expressed using the FreeStyle™ 293 Expression System and purified from the culture supernatant.

[0077] Example 3 Characterization of anti-ΔGK monoclonal antibody YG55 After gene cloning and antibody expression, the specificity of YG55 was evaluated by ELISA assay, as described above in the secondary screening. Antibody gene cloning was successful, resulting in YG55, which retained the same specificity as that exhibited 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. 1. A method for detecting or capturing a polypeptide in a sample, comprising: the polypeptide is a monoclonal antibody comprising a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, which lacks both glycine at position 446 according to the EU numbering system 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; The method comprises subjecting a sample to an antibody selected from the following (1) and (2): (1) (i) (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; Including, (ii) specifically binds to the modified IgG heavy chain constant region; antibody; (2) An antibody that competes with the antibody comprising (a) to (f) above for binding to the modified IgG heavy chain constant region, the antibody comprising a heavy chain variable domain sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable domain sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

5. The method comprises contacting in vitro with

2. The method of claim 1, wherein the modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, lacking both glycine at position 446 according to the EU numbering system 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 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. 3. The method of claim 2, wherein the antibody does not substantially bind to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, lacking both glycine at position 446 according to the EU numbering system 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. 4. The method of claim 1, wherein the antibody does not substantially bind to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region and retaining the amino acids at positions 445, 446, and 447 according to the EU numbering system or an unmodified human IgG heavy chain constant region, and does not substantially bind to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, 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.

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

6. 5. The method of 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 a 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 a human IgG heavy chain constant region that lacks lysine at position 447 according to the EU numbering system and retains the amino acids at positions 445 and 446 according to the EU numbering system; and an unmodified human IgG heavy chain constant region is below the detection limit of an enzyme-linked immunoassay.

7. 7. The method of claim 1, wherein the binding activity of the antibody to a modified IgG heavy chain constant region derived from a human IgG heavy chain constant region, which lacks both glycine at position 446 according to the EU numbering system 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 detectable in an enzyme-linked immunoassay.

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