Antibodies and compositions for use in detection or capture of polypeptide in sample, and methods for detecting or capturing polypeptide in sample

Abstract

To provide antibodies for use in detection or capture of polypeptides in samples, compositions and methods for detecting or capturing peptides in samples.SOLUTION: There is provided an isolated antibody that specifically binds to a modified IgG heavy chain constant region derived from any one of human IgG constant regions or a modified IgG heavy chain constant region derived from a chimeric constant region obtained from at least two selected human IgG constant regions, where the constant region of the human IgG is any of IgG4 constant regions having specific amino acid sequences, the modified IgG heavy chain constant region comprises a certain amino acid at a certain position, where the isolated antibody binds to a portion consisting of an amino acid sequence LHEALHAHYTRKE of the modified IgG heavy chain constant region, but substantially does not bind to one of the constant regions of human IgG and a chimeric constant region derived from at least two selected from the human IgG constant region.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antibodies and compositions for use in detecting or capturing polypeptides in a sample, 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 been widely used in many scientific fields (Non-Patent Document 1). Following the achievement of this technology, further efforts have been 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] Antibodies that specifically bind to modified Fc regions but not to wild-type Fc have been reported (Non-Patent Document 5; Patent Document 1). Antibodies against modified Fc regions have proven to be very useful for a variety of purposes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2017072210A1

Non-Patent Literature

[0006]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Summary of the Invention

[0007] The present inventors have provided several antibodies comprising modified IgG heavy chain constant regions derived from any one of the constant regions of naturally occurring human IgG or chimeric constant regions obtained from at least two selected from the constant regions of naturally occurring human IgG, including, for example, satralizumab, nemolizumab, emicizumab, SKY59 (crovalimab), AMY109, and GYM329. For example, one such modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). The present invention provides antibodies, compositions comprising the antibodies, and methods of using the antibodies that specifically bind to, detect, and / or capture a modified IgG heavy chain constant region or a polypeptide comprising an epitope thereof.

[0008] Specifically, the present invention relates to the following [1] to

[25] . [1] An isolated antibody that specifically binds to a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG, wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). [2] The antibody of [1], which does not substantially bind to any one of the constant regions of naturally occurring human IgG and a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG. [3] The antibody of [1] or [2], wherein the naturally occurring human IgG constant regions are an IgG1 constant region consisting of the amino acid sequence of SEQ ID NO: 106, an IgG2 constant region consisting of the amino acid sequence of SEQ ID NO: 107, an IgG3 constant region consisting of the amino acid sequence of SEQ ID NO: 108, and an IgG4 constant region consisting of the amino acid sequence of SEQ ID NO: 109. [4] The antibody of any one of [1] to [3], wherein the altered heavy chain constant region is derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and human IgG4. [5] Any one of the antibodies [1] to [4], wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). [6] Any one of the antibodies [1] to [5], wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system). [7] Any one of the antibodies [1] to [6], wherein the modified IgG heavy chain constant region includes Arg at position 235, and one or both of Arg at position 236 and Lys at position 239 (all positions according to the EU numbering system). [8] Any one of the antibodies of [1] to [7], which binds to a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO: 104) or RRGPS (SEQ ID NO: 117). [9] Any one of the antibodies [1] to [8], which contains any one of the following (a) to (f): (a) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93; (b) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 82, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 94; (c) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 61, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 73, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 85, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (d) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 98; (e) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 51, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 87, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99; and (f) A variable region comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 41, HVR-H2 having the amino acid sequence of SEQ ID NO: 53, HVR-H3 having the amino acid sequence of SEQ ID NO: 65, HVR-L1 having the amino acid sequence of SEQ ID NO: 77, HVR-L2 having the amino acid sequence of SEQ ID NO: 89, and HVR-L3 having the amino acid sequence of SEQ ID NO: 101.

[10] Any one of the antibodies [1] to [5], wherein the modified IgG heavy chain constant region comprises at least one selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[11] The antibody of any one of [1] to [5] and

[10] , wherein the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and optionally threonine at position 436 (all positions according to the EU numbering system).

[12] Any one of the antibodies of [1] to [5],

[10] , and

[11] , which binds to a portion consisting of the amino acid sequence LHEALHAHYTRKE (SEQ ID NO: 105) or LHEALHAHTTRKE (SEQ ID NO: 118) of the modified IgG heavy chain constant region.

[13] An antibody of any one of [1] to [5] and

[10] to

[12] , which comprises any one of the following (g) to (l): (g) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92; (h) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 47, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 95; (i) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 72, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 84, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96; (j) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 76, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 100; (k) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 42, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 78, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102; and (l) A variable region comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 43, HVR-H2 having the amino acid sequence of SEQ ID NO: 55, HVR-H3 having the amino acid sequence of SEQ ID NO: 67, HVR-L1 having the amino acid sequence of SEQ ID NO: 79, HVR-L2 having the amino acid sequence of SEQ ID NO: 91, and HVR-L3 having the amino acid sequence of SEQ ID NO: 103.

[14] An isolated antibody that binds to the same epitope as any one of the antibodies [1] to

[13] .

[15] An isolated antibody that specifically binds to a modified IgG heavy chain constant region, wherein the binding of the antibody to the modified IgG heavy chain constant region competes with that of any one of the antibodies [1] to

[14] , and the modified IgG heavy chain constant region is derived from any one of the constant regions of naturally occurring human IgG, or from a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG, and comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[16] A composition for use in detecting or capturing a polypeptide in a sample, comprising any one of the antibodies [1] to

[15] .

[17] The composition of

[16] , wherein the polypeptide comprises a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG, and the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[18] The composition of

[16] , wherein the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO: 104), an amino acid sequence consisting of RRGPS (SEQ ID NO: 117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO: 105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).

[19] The composition of

[18] , wherein the polypeptide comprises a modified IgG heavy chain constant region comprising any one of an amino acid sequence consisting of RRGPK (SEQ ID NO: 104), an amino acid sequence consisting of RRGPS (SEQ ID NO: 117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO: 105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).

[20] A method for detecting or capturing a polypeptide in a sample, comprising the step of contacting the sample with any one of the antibodies [1] to

[15] or any one of the compositions

[16] to

[19] .

[21] The method of

[20] , wherein the polypeptide comprises a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG, and the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[22] The method of

[20] , wherein the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO: 104), an amino acid sequence consisting of RRGPS (SEQ ID NO: 117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO: 105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).

[23] The method of

[22] , wherein the polypeptide comprises a modified IgG heavy chain constant region comprising any one of an amino acid sequence consisting of RRGPK (SEQ ID NO: 104), an amino acid sequence consisting of RRGPS (SEQ ID NO: 117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO: 105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).

[24] A method for measuring the concentration of a first antibody in a sample, wherein the first antibody is capable of binding to a first epitope of an antigen, the sample comprising the first antibody and the antigen, the method comprising: (A) contacting the sample with a plate or beads on which a second antibody is immobilized; (B) after (A), contacting a solution containing the antigen and not containing the first antibody and the second antibody with the plate or beads; and (C) After (B), the antigen captured on the plate or beads via the second antibody and the first antibody is detected by using a third antibody. Including, the first antibody comprises a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG; the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system); the second antibody is any one of the antibodies [1] to

[15] ; A method in which the third antibody is capable of binding to a second epitope of an antigen that is different from the first epitope and has an IgG heavy chain constant region that differs in amino acid sequence from those of the first antibody and the second antibody.

[25] A method for measuring the concentration of an antigen in a sample, wherein the sample comprises an antigen and a first antibody capable of binding to a first epitope of the antigen, the method comprising: (D) contacting the sample with a plate or beads on which a third antibody is immobilized; (E) after (D), contacting the plate or beads with a solution containing the first antibody and not containing the antigen and the third antibody; and (F) detecting the first antibody captured on the plate or beads via the third antibody and antigen by using a second antibody; Including, the first antibody comprises a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG; the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system); the second antibody is any one of the antibodies [1] to

[15] and has an IgG heavy chain constant region whose amino acid sequence differs from that of the first antibody and the third antibody; The method, wherein the third antibody is capable of binding to a second epitope of the antigen that is different from the first epitope. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 illustrates a sequence alignment of the five modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) described in Example 2. The germline sequences of human IgG heavy chain constant regions, i.e., IGHG1_01 (J00228) and IGHG4_01 (K01316), are also aligned for comparison. Dots indicate amino acids identical to SG115 at that position. [Figure 2-1]Figures 2-1 and 2-2 illustrate the binding of 12 anti-SG115 antibodies to five modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) in ELISA. As described in Example 2, SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127 showed selective binding to SG115v1, and SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171 showed selective binding to SG115v2. [Figure 2-2] Figure 2-2 is a continuation of Figure 2-1. [Figure 3] FIG. 3 illustrates the scheme of the Fc variant antibody detection assay. [Figure 4] FIG. 4 illustrates the scheme of the antigen detection assay. [Figure 5] FIG. 5 illustrates the scheme of the Simoa® assay. [Figure 6] Figure 6 shows sensorgrams of the dissociation of human C5 from anti-hC5 antibody captured by SKA0016 and SKA0117 at pH 7.4 and pH 6.0. Neither SKA0016 nor SKA0117 interfered with the pH-dependent interaction between anti-hC5 antibody and human C5. [Figure 7] 7 illustrates the sensorgram of the analysis of human Fc receptor binding with anti-hC5 antibody captured by SKA0016. SKA0016 did not block the binding between hFcRn and anti-hC5 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0010] 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.

[0011] The term "an isolated antibody that specifically binds to an engineered IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected of the constant regions of naturally occurring human IgG" refers to an antibody that can bind to a particular type of engineered IgG heavy chain constant region with sufficient affinity so as to be useful as a detection agent, capture agent, or diagnostic agent targeting the engineered IgG heavy chain constant region. In one embodiment, for an antibody that specifically binds to an engineered IgG heavy chain constant region, the extent of binding of the antibody to a non-engineered human IgG heavy chain constant region is less than about 10% of the binding of the engineered IgG heavy chain constant region, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to an engineered IgG heavy chain constant region has an 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 the modified IgG heavy chain constant region binds to an epitope within the modified IgG heavy chain constant region.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] As used herein, the term "constant region" refers to a region in an antibody that corresponds to any one of the IgG1 constant region consisting of the amino acid sequence of SEQ ID NO: 106, the IgG2 constant region consisting of the amino acid sequence of SEQ ID NO: 107, the IgG3 constant region consisting of the amino acid sequence of SEQ ID NO: 108, and the IgG4 constant region consisting of the amino acid sequence of SEQ ID NO: 109. The constant region consists of a CH1 region (positions 118 to 215 according to the EU numbering system), a hinge region (positions 216 to 230 according to the EU numbering system), a CH2 region (positions 231 to 340 according to the EU numbering system), and a CH3 region (positions 341 to 446 according to the EU numbering system).

[0016] 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.

[0017] 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).

[0018] "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.

[0019] 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).

[0020] "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.

[0021] 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.

[0022] 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.

[0023] II. Antibodies The antibody of the present invention is an isolated antibody that specifically binds to an altered IgG heavy chain constant region.

[0024] In one embodiment, the antibody does not substantially bind to the constant region of naturally occurring human IgG and a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG. In this embodiment, the binding activity of the antibody to the constant region of naturally occurring human IgG and a chimeric IgG consisting of at least two IgGs selected from naturally occurring human IgG is below the detection limit in an enzyme-linked immunoassay. Meanwhile, the binding activity of the antibody to the modified IgG heavy chain constant region is detectable in an enzyme-linked immunoassay.

[0025] In a further aspect of the invention, the antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as, for example, a complete IgG1, IgG2, IgG3, and IgG4 antibody, or any other antibody class or isotype defined herein.

[0026] A. Modified IgG heavy chain constant region In one embodiment, the modified IgG heavy chain constant region is derived from any one of the constant regions of naturally occurring human IgG, or from a chimeric constant region obtained from at least two selected from the constant regions of naturally occurring human IgG: the IgG1 constant region consisting of the amino acid sequence of SEQ ID NO: 106, the IgG2 constant region consisting of the amino acid sequence of SEQ ID NO: 107, the IgG3 constant region consisting of the amino acid sequence of SEQ ID NO: 108, and the IgG4 constant region consisting of the amino acid sequence of SEQ ID NO: 109.

[0027] In one embodiment, the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[0028] In a further embodiment, the modified IgG heavy chain constant region may be derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and human IgG4. In a preferred embodiment, the constant region is derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and human IgG4.

[0029] In a preferred embodiment, the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[0030] In one embodiment, the modified IgG heavy chain constant region may form a dimer like the heavy chain constant region of a naturally occurring IgG, or may form a halfmer like the heavy chain constant region of a monomeric Fc reported in Ishino, T. et al., J. Biol. Chem. 288:16529-37 (2013).

[0031] In one embodiment, when the modified IgG heavy chain constant region is present in a human modified IgG heavy chain, the human modified IgG heavy chain is selected from the group consisting of human modified heavy chains of IgG1, IgG2, IgG3, and IgG4, and chimeric IgG heavy chains thereof. In a preferred embodiment, the human IgG heavy chain is a human IgG1 heavy chain, a human IgG4 heavy chain, or a chimeric IgG heavy chain thereof.

[0032] B. Exemplary antibodies that specifically recognize modifications unique to the CH2 region of engineered IgG heavy chain constant regions Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system) are specifically present in the CH2 regions of SG115 and SG115v1 used in the Examples. Thus, the modified IgG heavy chain constant region of the exemplary antibody herein preferably comprises at least a region corresponding to the CH2 region of any one of the constant regions of naturally occurring human IgG, or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG.

[0033] In one embodiment, the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system). In this embodiment, the modified IgG heavy chain constant region comprises Arg at position 235, and one or both of Arg at position 236 and Lys at position 239 (all positions according to the EU numbering system).

[0034] In a preferred embodiment, the modified IgG heavy chain constant region comprises all three of these mutations, or an Arg at position 235 and an Arg at position 236 (both positions according to the EU numbering system). In that case, the antibody binds to a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO: 104) or RRGPS (SEQ ID NO: 117).

[0035] In one aspect, the invention provides an antibody that specifically binds to a modified IgG heavy chain constant region comprising at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system), a modified IgG heavy chain constant region comprising Arg at position 235, and one or both of Arg at position 236 and Lys at position 239 (all positions according to the EU numbering system), or a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO: 104) or RRGPS (SEQ ID NO: 117). or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0036] In another aspect, the present invention provides antibodies 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: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101. In a further embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53. In a further embodiment, the antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65.

[0037] In another aspect, the present invention provides antibodies comprising at least one, at least two, or all three VL HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101. In one embodiment, the antibody comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0038] In another aspect, the antibody of the present invention comprises at least one, at least two, or all three VHs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. and (II) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (iv) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0039] In another aspect, the present invention provides antibodies comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0040] 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: 9, 10, 13, 14, 15, or 17. 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 compared to the reference sequence, but an antibody described herein comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 9, 10, 13, 14, 15, or 17. In certain embodiments, the substitutions, insertions, or deletions are present in regions outside the HVRs (i.e., FRs). Optionally, the antibody comprises a VH sequence of SEQ ID NO: 9, 10, 13, 14, 15, or 17, including post-translational modifications of that sequence. In specific embodiments, the VH comprises one, two, or three HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. 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.

[0041] In another aspect, antibodies are 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: 21, 22, 25, 26, 27, or 29. 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 compared to the reference sequence, but an antibody comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 21, 22, 25, 26, 27, or 29. In certain embodiments, the substitutions, insertions, or deletions are present in regions outside the HVRs (i.e., FRs). Optionally, the antibody comprises a VL sequence of SEQ ID NO: 21, 22, 25, 26, 27, or 29, including post-translational modifications of that sequence. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (iv) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101. 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.

[0042] In another aspect, an antibody is provided that comprises a VH of any of the above-provided embodiments and a VL of any of the above-provided embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NOs: 9, 10, 13, 14, 15, or 17, and SEQ ID NOs: 21, 22, 25, 26, 27, or 29, respectively, and includes post-translational modifications of those 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.

[0043] In one aspect, an antibody is provided that competes for binding to a first modified IgG heavy chain constant region with an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0044] In one aspect, antibodies are provided that bind to the same epitope as an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101.

[0045] In specific embodiments, if an antibody specifically binds to a modified IgG heavy chain constant region comprising at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system), a modified IgG heavy chain constant region comprising Arg at position 235, and one or both of Arg at position 236 and Lys at position 239 (all positions according to the EU numbering system), or a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO: 104) or RRGPS (SEQ ID NO: 117), the antibody comprises any one of the following (a) to (f): (a) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 33, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 45, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 69, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 81, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 93; (b) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 34, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 46, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 70, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 82, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 94; (c) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 49, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 61, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 73, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 85, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (d) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 38, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 50, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 98; (e) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 39, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 51, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 75, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 87, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 99; and (f) A variable region comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 41, HVR-H2 having the amino acid sequence of SEQ ID NO: 53, HVR-H3 having the amino acid sequence of SEQ ID NO: 65, HVR-L1 having the amino acid sequence of SEQ ID NO: 77, HVR-L2 having the amino acid sequence of SEQ ID NO: 89, and HVR-L3 having the amino acid sequence of SEQ ID NO: 101.

[0046] C. Exemplary antibodies that specifically recognize unique modifications in the CH3 region of engineered IgG heavy chain constant regions Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system) are specifically present in the CH3 regions of SG115 and SG115v2 used in the Examples. Thus, the modified IgG heavy chain constant region of the exemplary antibody herein preferably comprises at least a region corresponding to the CH3 region of any one of the constant regions of naturally occurring human IgG, or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG.

[0047] In one embodiment, the modified IgG heavy chain constant region comprises at least one selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). In this embodiment, the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and optionally a threonine at position 436 (all positions according to the EU numbering system).

[0048] In a preferred embodiment, the modified IgG heavy chain constant region comprises all of these mutations, in which case the antibody binds to a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence LHEALHAHYTRKE (SEQ ID NO: 105) or LHEALHAHTTRKE (SEQ ID NO: 118).

[0049] In one aspect, the invention provides an antibody that specifically binds to a modified IgG heavy chain constant region comprising at least one selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system), a modified IgG heavy chain constant region comprising Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system), or a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence LHEALHAHYTRKE (SEQ ID NO: 105) or LHEALHAHTTRKE (SEQ ID NO: 118). Antibodies are provided that comprise at least one, two, three, four, five, or six HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0050] In one aspect, the present invention provides antibodies 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: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103. In a further embodiment, the antibody comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67, an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103, and an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55. In a further embodiment, the antibody comprises (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; and (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67.

[0051] In another aspect, the present invention provides antibodies comprising at least one, at least two, or all three VL HVR sequences selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103. In one embodiment, the antibody comprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0052] In another aspect, the antibody of the present invention comprises at least one, at least two, or all three VHs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67. and (II) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0053] In another aspect, the present invention provides antibodies comprising: (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; (iii) an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67; (iv) an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) an HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0054] 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: 8, 11, 12, 16, 18, or 19. 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 compared to the reference sequence, but an antibody described herein comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 8, 11, 12, 16, 18, or 19. In certain embodiments, the substitutions, insertions, or deletions are present in regions outside the HVRs (i.e., FRs). Optionally, the antibody comprises a VH sequence of SEQ ID NO: 8, 11, 12, 16, 18, or 19, including post-translational modifications of that sequence. In specific embodiments, the VH comprises one, two, or three HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67. 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.

[0055] In another aspect, antibodies are 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: 20, 23, 24, 28, 30, or 31. 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 compared to the reference sequence, but an antibody comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 20, 23, 24, 28, 30, or 31. In certain embodiments, the substitutions, insertions, or deletions are present in regions outside the HVRs (i.e., FRs). Optionally, the antibody comprises a VL sequence of SEQ ID NO: 20, 23, 24, 28, 30, or 31, including post-translational modifications of that sequence. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103. 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.

[0056] In another aspect, an antibody is provided that comprises a VH of any of the above-provided embodiments and a VL of any of the above-provided embodiments. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NOs: 8, 11, 12, 16, 18, or 19, and SEQ ID NOs: 20, 23, 24, 28, 30, or 31, respectively, and includes post-translational modifications of those 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.

[0057] In one aspect, an antibody is provided that competes for binding to a first modified IgG heavy chain constant region with an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0058] In one aspect, antibodies are provided that bind to the same epitope as an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103.

[0059] In specific embodiments, if an antibody specifically binds to a modified IgG heavy chain constant region comprising at least one selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system), a modified IgG heavy chain constant region comprising Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system), or a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence LHEALHAHYTRKE (SEQ ID NO: 105) or LHEALHAHTTRKE (SEQ ID NO: 118), the antibody comprises any one of the following (g) to (l): (g) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92; (h) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 47, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 95; (i) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 72, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 84, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96; (j) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 76, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 100; (k) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 42, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 78, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102; and (l) A variable region comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 43, HVR-H2 having the amino acid sequence of SEQ ID NO: 55, HVR-H3 having the amino acid sequence of SEQ ID NO: 67, HVR-L1 having the amino acid sequence of SEQ ID NO: 79, HVR-L2 having the amino acid sequence of SEQ ID NO: 91, and HVR-L3 having the amino acid sequence of SEQ ID NO: 103.

[0060] D. Other Aspects In one embodiment, the antibodies of the present invention include antibodies that bind to the same epitope as any one of the antibodies mentioned above in sections "A. Modified IgG heavy chain constant regions" to "C. Exemplary antibodies that specifically recognize modifications unique to the CH3 region of modified IgG heavy chain constant regions."

[0061] In one embodiment, the antibodies of the present invention include antibodies that specifically bind to an engineered IgG heavy chain constant region, where the binding of the antibody to the engineered IgG heavy chain constant region competes with that of the antibodies mentioned above in sections "A. Engineered IgG Heavy Chain Constant Regions" through "C. Exemplary Antibodies that Specifically Recognize Unique Modifications in the CH3 Region of Engineered IgG Heavy Chain Constant Regions." In this embodiment, the engineered IgG heavy chain constant region is derived from any one of the constant regions of naturally occurring human IgG, or from a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG. The engineered IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). In that embodiment, the specific antibodies referred to herein are the same as those in the previous section.

[0062] E. Recombinant 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, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid 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 chain and / or heavy chain 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, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid 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 encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid 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 nucleic acid 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).

[0063] For recombinant production of the antibodies described herein, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid 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).

[0064] 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.

[0065] 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).

[0066] 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.

[0067] 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).

[0068] 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).

[0069] F. Assays 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.

[0070] G. 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.

[0071] In another aspect, a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG, comprising: Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Arg at position 440. A competition assay may be used to identify antibodies that compete with any one of the antibodies used in the Examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA0171) for binding to a modified IgG heavy chain constant region comprising at least one amino acid selected from the group consisting of Glu at positions according to the EU numbering system. In certain embodiments, such a competing antibody can bind to the same epitope (e.g., a linear or conformational epitope) as any one of the antibodies used in the Examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA0171). 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).

[0072] In an exemplary competitive assay, the immobilized modified IgG heavy chain constant region is incubated in a solution containing a labeled antibody that binds to the modified IgG heavy chain constant region and an unlabeled antibody to be tested for its ability to compete with the labeled antibody for binding to the immobilized modified IgG heavy chain constant region. The unlabeled antibody may be present in B cell or hybridoma supernatant. As a control, the immobilized modified IgG heavy chain constant region is incubated in a solution containing the labeled antibody but no unlabeled antibody. After incubation under conditions that allow binding of the labeled antibody to the immobilized modified IgG heavy chain constant region, excess unbound antibody is removed and the amount of label bound to the immobilized modified IgG heavy chain constant region is measured. A substantial decrease in the amount of label bound to the immobilized modified IgG heavy chain constant region in the test sample compared to the control sample indicates that the unlabeled antibody competes with the labeled antibody for binding to the immobilized 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).

[0073] 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, and comprises any one of the antibodies described in "II. Antibodies."

[0074] In another aspect, the composition of the present invention is a composition for use in the treatment or prevention of a disease. When the antibody is used for the treatment or prevention of any disease, the composition may be or comprise a cell expressing any one of the antibodies described in "II. Antibodies" or a fragment thereof that specifically binds to the modified IgG heavy chain constant region.

[0075] In a preferred embodiment, the polypeptide in the sample comprises a modified IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a modified IgG heavy chain constant region derived from at least two chimeric constant regions selected from the constant regions of naturally occurring human IgG. The modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system). Specific antibodies referred to herein are the same as those described in "II. Antibodies."

[0076] In another preferred embodiment, the polypeptide comprises any one of the amino acid sequences RRGPK (SEQ ID NO: 104), RRGPS (SEQ ID NO: 117), LHEALHAHYTRKE (SEQ ID NO: 105), and LHEALHAHTTRKE (SEQ ID NO: 118). As long as the polypeptide comprises one or more of these amino acid sequences, the polypeptide detected or captured by the composition is not particularly limited in terms of its structure. The polypeptide preferably comprises a modified IgG heavy chain constant region comprising one or more of these amino acid sequences.

[0077] In one embodiment, the polypeptide detected or captured by the composition can be an antibody, such as a human IgG1 molecule, a human IgG2 molecule, a human IgG3 molecule, or a human IgG4 molecule, an antibody fragment, a fusion protein, or any other type of polypeptide that comprises a modified IgG heavy chain constant region or an epitope thereof.

[0078] In cases where the polypeptide comprises an altered IgG heavy chain constant region, the altered IgG heavy chain constant region may comprise other amino acid substitutions or modifications, so long as it comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[0079] IV. Method In one aspect, the method of the present invention is a method for detecting or capturing a polypeptide in a sample, comprising contacting the sample with any one of the antibodies described in "II. Antibodies" or any one of the compositions described in "III. Compositions."

[0080] In a preferred embodiment, the polypeptide comprises a modified IgG heavy chain constant region. The modified IgG heavy chain constant region is derived from any one of the constant regions of naturally occurring human IgG, or from at least two chimeric constant regions selected from the constant regions of naturally occurring human IgG. The modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[0081] In another preferred embodiment, the polypeptide comprises any one of the amino acid sequence consisting of RRGPK (SEQ ID NO: 104), the amino acid sequence consisting of RRGPS (SEQ ID NO: 117), the amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO: 105), and the amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118). The polypeptide detected or captured by the method is not particularly limited in terms of its structure, as long as the polypeptide comprises one or more of these amino acid sequences. The polypeptide preferably comprises a modified IgG heavy chain constant region comprising one or more of these amino acid sequences.

[0082] In one embodiment, the polypeptide detected or captured by the method can be an antibody, such as a human IgG1 molecule, a human IgG2 molecule, a human IgG3 molecule, or a human IgG4 molecule, an antibody fragment, a fusion protein, or any other type of polypeptide comprising a modified IgG heavy chain constant region or an epitope thereof.

[0083] In cases where the polypeptide comprises an altered IgG heavy chain constant region, the altered IgG heavy chain constant region may comprise other amino acid substitutions or modifications, so long as it comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system).

[0084] A specific embodiment of the ELISA method is illustrated in FIG. 3 . The "rabbit anti-Fc mutant antibody" corresponds to one of the antibodies described in "II. Antibodies," and the "anti-hC5 antibody" in FIG. 3A and the "anti-IL-8 antibody" in FIG. 3B correspond to polypeptides containing modified IgG heavy chain constant regions. The rabbit anti-Fc mutant antibody immobilized on a plate captures the anti-hC5 antibody of FIG. 3A and the anti-IL-8 antibody of FIG. 3B in a sample. Next, in FIG. 3A, hC5 (human complement 5) as the antigen of the anti-hC5 antibody, "mouse anti-hC5" that binds to an epitope different from that bound by the anti-hC5 antibody, and anti-mouse POD are reacted in this order. In FIG. 3B, IL-8 as the antigen of the anti-IL-8 antibody, "mouse anti-IL-8" that binds to an epitope different from that bound by the anti-IL-8 antibody, and anti-mouse POD are reacted in this order. Finally, a POD substrate is added to the plate and the luminescence is measured. In this embodiment, when a certain amount of a polypeptide comprising a modified IgG heavy chain constant region is present in the sample, luminescence is detected by the luminometer.

[0085] In another embodiment, the antibodies described in "II. Antibodies" can be applied to detect antigens such as hC5 and IL-8, as shown in FIG. 4. In this embodiment, the "mouse anti-Fc mutant antibody" and the "rabbit anti-Fc mutant antibody" correspond to one of the antibodies described in "II. Antibodies," and the "anti-hC5 antibody" in FIG. 4A and the "anti-IL-8 antibody" in FIG. 4B correspond to polypeptides containing a modified IgG heavy chain constant region. In FIG. 4A, a rabbit anti-hC5 antibody immobilized on a plate captures hC5 in a sample. In FIG. 4B, a mouse anti-IL-8 antibody immobilized on a plate captures IL-8 in a sample. Then, in FIG. 4A, the anti-hC5 antibody, the mouse anti-Fc mutant antibody, and the anti-mouse POD are reacted in this order. In FIG. 4B, the anti-IL-8 antibody, the rabbit anti-Fc mutant antibody, and the anti-rabbit HRP are reacted in this order. Finally, a substrate for POD (peroxidase), such as HRP (horseradish peroxidase), is added to the plate, and the luminescence is measured. In this embodiment, when hC5 or IL-8 is present in a certain amount in the sample, luminescence is detected by the luminometer.

[0086] The above-described embodiments related to the ELISA method may be exchanged for the Simoa® assay. In one embodiment of the assay, the antibody described in "II. Antibodies" can be applied to detect an antigen such as IL-8, as shown in Figure 5. In this embodiment, the "rabbit anti-Fc mutant antibody" corresponds to one of the antibodies described in "II. Antibodies," and the "anti-IL-8 antibody" corresponds to a polypeptide containing a modified IgG heavy chain constant region. A mouse anti-IL-8 antibody immobilized on beads captures IL-8 in the sample. Then, the anti-IL-8 antibody, biotinylated anti-Fc mutant antibody, and streptavidin-β-galactosidase (SBG (Quanterix Corporation)) are reacted in this order. Finally, a substrate for β-galactosidase (RGB) is added to the reaction mixture, and the luminescence is measured. [Example]

[0087] Example 1 Preparation of antibodies containing constant regions with multiple mutations in the Fc region

[0088] Antibody expression and purification An antibody containing a constant region with multiple mutations in the Fc region was expressed using the FreeStyle293 expression system. The constant region used (SEQ ID NO: 1) is referred to as SG115 in WO2016098356A1. The harvested cell culture fluid (HCCF) was purified using rProtein A resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex200pg, GE). During SEC, the inventors exchanged the buffer to 20 mmol / L histidine, 150 mmol / L arginine-aspartic acid, pH 6.0. Finally, the antibody was concentrated to 143 mg / mL using ultrafiltration (UF).

[0089] Papain digestion For papain digestion, we used the Pierce Fab Preparation Kit (Pierce, Cat. No. 44985). The papain digestion procedure was as follows: - The antibody concentration was adjusted to 8.0 mg / mL with digestion buffer. - 0.5 mL of antibody solution was added to the spin column tube containing the equilibrated papain resin. A top cap and bottom plug were attached to the spin column. The digestion reaction solution was incubated at 37°C for 15 hours on a rotator. After incubation, the bottom cap was removed and the spin column was placed in a microcentrifuge tube. The column was centrifuged at 5000 x g for 1 minute. - The resin was washed with 0.5 mL of Dulbecco's PBS(-). The spin column was placed in a microcentrifuge tube. The column was centrifuged at 5000 x g for 1 minute. - The solutions from steps 4 and 5 were combined as the digested fraction. The total volume from one column was 1.0 mL.

[0090] Fc fragment purification The papain-digested sample was purified by rProtein A resin (MabSelect SuRe, GE) and size-exclusion chromatography (SEC, Superdex 200 pg, GE). During SEC, we removed intact IgG (undigested molecules) and exchanged the buffer with Dulbecco's PBS(-).

[0091] Example 2 Generation of antibodies that recognize mutations within SG115 Antibodies that recognize mutations within SG115, referred to as "anti-SG115 antibodies," were prepared, selected, and assayed as described below.

[0092] Ten-week-old New Zealand White rabbits were intradermally immunized with the Fc fragment of SG115 (50–100 micrograms / injection / rabbit). The administration was repeated five times over a two-month period, and blood was then collected from the immunized rabbits. Antigen-specific B cells were selected using a cell sorter, then seeded and cultured according to the method described in WO2016098356A1. After culture, the B cell culture supernatant was collected for further analysis, and the pellet was cryopreserved.

[0093] The ability to bind to SG115 was assessed by ELISA using B cell culture supernatant. To assess binding specificity, we evaluated binding to five modified IgG heavy chain constant regions: SG115 (SEQ ID NO: 1), SG115v1 (SEQ ID NO: 2), SG115v2 (SEQ ID NO: 3), G1m (SEQ ID NO: 4), and G4d (SEQ ID NO: 5). A sequence alignment of these five constant regions is shown in Figure 1.

[0094] A total of 10,560 B cell lines were screened for binding to the five modified IgG heavy chain constant regions. 186 lines that bound SG115 but not G1m or G4d, and also bound SG115v1 and / or SG115v2, were selected and designated SKA0001 to SKA0186. RNA from the selected lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, Cat. No. R1053). DNA encoding the antibody heavy chain variable region of the selected lines was amplified by reverse transcription PCR and recombined with DNA encoding the rbIgGv2 heavy chain constant region (SEQ ID NO: 6). DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR and recombined with DNA encoding the rbIgk light chain constant region (SEQ ID NO: 7). The antibodies were then purified using FreeStyle PCR. TM The antibodies were expressed in 293-F cells (Invitrogen) and purified from the culture supernatant. Further evaluation revealed that 12 clones were selected based on their binding ability and specificity in ELISA and the sequence diversity of the heavy chain CDR3. Of these clones, six (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127) showed selective binding to SG115v1 but not to SG115v2, while the other six (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171) showed selective binding to SG115v2 but not to SG115v1 (Figures 2-1 and 2-2). The VH and VL sequences of these 12 antibodies are listed in Table 1.

[0095] [Table 1]

[0096] Example 3 Detection of SG115 in samples using anti-SG115 antibodies The efficacy of the 12 monoclonal antibodies (hereafter referred to as "anti-Fc mutant antibodies" or "multiple anti-Fc mutant antibodies") was evaluated for the detection of antibodies containing Fc regions containing all or some of the Fc region mutations of SG115 (hereafter referred to as "Fc mutant antibodies" or "multiple Fc mutant antibodies") in biological samples. A specific anti-human C5 antibody containing SG115, hereafter referred to as "anti-hC5 antibody," was used as a model Fc mutant antibody in Examples 3 to 5.

[0097] Assay procedure Each well of a 96-well immunoplate was coated with rabbit anti-Fc mutant antibody and blocked with blocking buffer. Diluted serum samples were added to each well of the plate. Recombinant human C5 was added to each well of the plate. Mouse anti-hC5 antibody was added, followed by anti-mouse-POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate, and the OD was measured. The plate was washed between steps.

[0098] Reactivity testing for antibody selection Twelve of the anti-Fc mutant antibodies were tested. Anti-hC5 antibodies, including SG115, were diluted with pooled human serum and assayed using the 12 rabbit anti-Fc mutant antibody candidates. The signal-to-noise ratio was calculated. The measured ODs are shown in Table 2. Three candidates from each epitope type (six candidates in total) were selected for selectivity testing (Table 2). Specifically, SKA0009, SKA0052, and SKA0127 were selected as antibodies that specifically bind to SG115v1, and SKA0117, SKA0141, and SKA0171 were selected as antibodies that specifically bind to SG115v2.

[0099] [Table 2]

[0100] Selectivity tests for antibody selection Ten individual sera were measured with or without anti-hC5 antibody, as well as standard curve samples, using six candidate antibodies as capture reagents. Without anti-hC5 antibody, the measured concentrations of each individual sample were all below the lower limit of quantitation (BLQ) for any of the rabbit anti-Fc mutant antibodies. With anti-hC5 antibody, the relative error (RE) of the measured concentrations of each individual sample was within + / - 20% for any of the rabbit anti-Fc mutant antibodies (Table 3).

[0101] [Table 3] BLQ: below the lower limit of quantification

[0102] Selected antibodies According to the results from the reactivity test and the selectivity test, SKA0141, which had the highest signal-to-noise ratio, was selected.

[0103] Example 4 Evaluation of a method for measuring anti-hC5 antibodies in human serum (Fc mutant antibody detection assay) Assay procedure A 96-well immunoplate was coated with rabbit anti-Fc mutant antibody (SKA0141) and blocked with blocking buffer. Diluted serum samples containing anti-hC5 antibody were added to each well of the plate. Recombinant human C5 was added to each well of the plate. Mouse anti-hC5 monoclonal antibody was added, followed by anti-mouse-POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate, and the OD was measured. The plate was washed between steps.

[0104] Method evaluation Reproducibility was tested. The intra-batch accuracy (RE) and precision (CV) were -16.3% to -5.1% and 1.6% to 4.4%, respectively (Table 4). The inter-batch accuracy (RE) and precision (CV) were -10.1% to -4.0% and 2.7% to 6.9%, respectively (Table 5).

[0105] [Table 4]

[0106] [Table 5]

[0107] Selectivity was tested. Without the addition of anti-hC5 antibody, the measured concentrations of individual samples were all BLQ. With the addition of anti-hC5 antibody, the RE of the measured concentrations of individual samples was -15.2% to 2.2% (Table 6).

[0108] [Table 6]

[0109] Dilution linearity was tested: 1 mg of anti-hC5 antibody per ml could be measured at a dilution factor of 50,000, and no prozone effect was observed (Table 7).

[0110] [Table 7] ALQ: Quantitative upper limit exceeded

[0111] Interference from C5 was tested and no interference from C5 was observed (Table 8).

[0112] [Table 8]

[0113] A method for measuring Fc mutant antibodies in human serum using anti-Fc mutant antibodies was established. The assay scheme is illustrated in Figure 3.

[0114] Example 5 The present inventors also attempted to establish an assay for detecting antigens recognized by Fc mutant antibodies in biological samples, and in this evaluation, anti-hC5 antibody was used as a model for Fc mutant antibodies.

[0115] Evaluation of a method (antigen detection assay) for measuring C5 in human serum. Assay procedure A 96-well immunoplate was coated with rabbit anti-hC5 monoclonal antibody and blocked with blocking buffer. Diluted serum samples were added to each well of the plate. The anti-hC5 antibody was added to each well of the plate. A mouse anti-Fc mutant antibody obtained by substituting the Fc region of SKA0141 with a mouse Fc region was added to each well of the plate, followed by the addition of anti-mouse POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate, and the OD was measured. The plate was washed between steps.

[0116] Method evaluation Reproducibility was tested. The intra-batch accuracy (RE) and precision (CV) were -8.2% to 4.2% and 2.3% to 6.2%, respectively (Table 9). The inter-batch accuracy (RE) and precision (CV) were -6.8% to 1.3% and 3.5% to 6.2%, respectively (Table 10).

[0117] [Table 9]

[0118] [Table 10]

[0119] Parallelism was tested. Individual sera from 10 individuals were serially diluted from 325 to 2600 times and assayed. Measured concentrations were recovered at every dilution factor (Table 11).

[0120] [Table 11]

[0121] Dilution linearity was tested: 1130 micrograms of C5 per milliliter could be measured at a dilution factor of 26,000, and no prozone effect was observed (Table 12).

[0122] [Table 12] ALQ: Quantitative upper limit exceeded

[0123] Interference from anti-hC5 antibodies was tested and no interference from anti-hC5 antibodies was observed (Table 13).

[0124] [Table 13]

[0125] A method for measuring antigens in human serum using anti-Fc mutant antibodies was established. The assay scheme is illustrated in Figure 4.

[0126] Example 6 Evaluation of a method for measuring anti-IL-8 antibodies in human plasma (Fc mutant antibody detection assay) Assay procedure Hereinafter, a specific anti-human IL-8 antibody containing a modified IgG heavy chain constant region containing some of the mutations in the Fc region of SG115 (sequence number: 110), referred to as the "anti-IL-8 antibody," was used as a model for Fc mutant antibodies in Examples 6 to 8.

[0127] A 96-well immunoplate was coated with one of the rabbit anti-Fc mutant antibodies (SKA0117) and blocked with blocking buffer. Diluted plasma samples were added to each well of the plate. Recombinant human IL-8 (SEQ ID NO: 111) was added to each well of the plate. Mouse anti-IL-8 monoclonal antibody (heavy chain variable region, SEQ ID NO: 112; light chain variable region, SEQ ID NO: 113; heavy chain constant region, SEQ ID NO: 114; light chain constant region, SEQ ID NO: 115) was added, followed by anti-mouse POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate, and the OD was measured. The plate was washed between steps.

[0128] Method evaluation Reproducibility was tested by measuring known concentrations of anti-IL-8 antibody (50.0 ng / mL (REP-LL), 100 ng / mL (REP-L), 400 ng / mL (REP-M), 2400 ng / mL (REP-H), and 3200 ng / mL (REP-UL)). The within-batch accuracy (RE) and precision (CV) were -14.2% to -9.7% and 4.9% to 7.3%, respectively (Table 14).

[0129] [Table 14]

[0130] The batch-to-batch accuracy (RE) and precision (CV) were −10.3% to −6.6% and 7.0% to 10.1%, respectively (Table 15).

[0131] [Table 15]

[0132] Selectivity was tested. When no anti-IL-8 antibody was added (SEL-O or SEL-EM-O), the measured concentrations of each sample were all BLQ. When anti-IL-8 antibody was added (50.0 ng / mL (SEL-LL or SEL-EM-LL)), the RE of each measured concentration of each sample was -23.2% to -4.3% (Table 16).

[0133] [Table 16]

[0134] Dilution linearity was tested: 1.6 milligrams of anti-IL-8 antibody per milliliter could be measured at a 10,000-fold dilution, and no prozone effect was observed (Table 17).

[0135] [Table 17]

[0136] Interference from IL-8 was tested. At an anti-IL-8 antibody concentration of 2400 ng / mL, up to 50.0 ng / mL of IL-8 did not interfere with the assay, and at an anti-IL-8 antibody concentration of 50.0 ng / mL, up to 1.00 ng / mL of IL-8 did not interfere with the assay (Table 18).

[0137] [Table 18]

[0138] The effectiveness of the method for measuring anti-IL-8 antibodies in plasma was confirmed.

[0139] Example 7 Evaluation of a method for measuring IL-8 in human plasma using ELISA (antigen detection assay) Assay procedure A 96-well streptavidin immunoplate was blocked with blocking buffer and then coated with biotinylated mouse anti-IL-8 monoclonal antibody. The anti-IL-8 antibody was added to the diluted plasma sample (reaction solution) in a 96-well polypropylene plate. After incubation, the reaction solution was transferred to each well of the streptavidin plate. One of the rabbit anti-Fc mutant antibodies (SKA0001) was added, followed by anti-rabbit-HRP (Southern Biotechnology Associates Inc.). Finally, POD substrate was added to each well of the plate, and the OD was measured. The streptavidin plate was washed between steps.

[0140] Method evaluation Reproducibility was tested, and the within-batch trueness (RE) and precision (CV) were -6.4% to -1.9% and 1.5% to 2.9%, respectively (Table 19).

[0141] [Table 19]

[0142] The batch-to-batch accuracy (RE) and precision (CV) were −8.0% to 2.1% and 3.0% to 5.2%, respectively (Table 20).

[0143] [Table 20]

[0144] Dilution linearity was tested: 1 microgram of IL-8 per milliliter (spiked) could be measured at a dilution factor of 20,000, and no prozone effect was observed (Table 21).

[0145] [Table 21]

[0146] Interference from anti-IL-8 antibodies was tested and no interference from anti-IL-8 antibodies (100 μg / mL in plasma) was observed (Table 22).

[0147] [Table 22]

[0148] The method for measuring IL-8 in human plasma using ELISA was validated.

[0149] Example 8 Evaluation of a method for measuring IL-8 in human plasma using Simoa® (Simoa® assay) Assay procedure The assay was performed automatically using the Simoa® system (Quanterix Corporation). The diluted sample, anti-IL-8 antibody, and mouse anti-IL-8 monoclonal antibody coated on beads were mixed. The beads were loaded into the microwells of the array disk. A biotinylated anti-Fc mutant antibody (SKA0028) was added to the disk, followed by streptavidin-β-galactosidase SBG (Quanterix Corporation). Finally, RGB, a substrate for β-galactosidase, was added, and the fluorescence intensity was measured.

[0150] Method evaluation Reproducibility was tested, and within-batch precision (CV) ranged from 1.3% to 14.3% (Table 23).

[0151] [Table 23]

[0152] The batch-to-batch precision (CV) ranged from 8.6% to 24.7%, respectively (Table 24).

[0153] [Table 24-1]

[0154] [Table 24-2]

[0155] Parallelism was tested. Three individual plasma samples were serially diluted 20- to 40-fold and assayed. The assay concentrations were recovered at every dilution factor (Table 25).

[0156] [Table 25]

[0157] Dilution linearity was tested: 3.48 micrograms of IL-8 per milliliter could be measured at a dilution factor of 50,000, and no prozone effect was observed (Table 26).

[0158] [Table 26]

[0159] Interference from anti-IL-8 antibodies was tested and no interference from anti-IL-8 antibodies (100 μg / mL in plasma) was observed (Table 27).

[0160] [Table 27]

[0161] The method for measuring IL-8 in human plasma using the Simoa® assay was validated.

[0162] Example 9 Evaluation of the affinity of anti-Fc mutant antibodies that selectively bind to SG115v1 for anti-hC5 antibodies K values ​​at pH 7.4 of anti-Fc mutant antibodies (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127) that selectively bind to SG115v1 versus anti-hC5 antibody DValues ​​were determined at 25°C using a Biacore T200 machine (GE Healthcare).

[0163] Mouse anti-rabbit IgG (Fc) antibody (Abbexa) (hereafter referred to as anti-rabbit IgG) was immobilized on flow cells (FC) 1 and 2 of a CM5 sensor chip using an amine coupling kit (GE Healthcare). For immobilization of anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After immobilization, the running buffer was exchanged for phosphate pH 7.4 buffer (50 mM phosphate buffer, pH 7.4, containing 150 mM NaCl and 0.05% w / v P-20). Each anti-Fc mutant antibody was captured by anti-rabbit IgG on FC2 of the sensor chip. The amount of captured anti-Fc mutant antibody was adjusted to obtain 100 resonance units (RU). Anti-hC5 antibody was injected at 10 μL / min at 0 nM, 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM. After each cycle, the sensor surface was regenerated with 10 mM glycine HCl, pH 2.0, injected at a flow rate of 30 μL / min. K was measured using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). D The association rate (ka), dissociation rate (kd), and dissociation constant (K D ) are shown in Table 28.

[0164] [Table 28] * : Due to the slow dissociation rate, the data may be unreliable.

[0165] Example 10 Evaluation of the affinity of anti-Fc mutant antibodies that selectively bind to SG115v1 for anti-IL-8 antibodies To confirm the binding ability of the anti-Fc mutant antibodies that selectively bind to SG115v1, the K DValues ​​were determined using a Biacore T200 machine (GE Healthcare) at 25° C. The sequence of the Fc region of the anti-IL-8 antibody has high similarity to the sequence of the anti-hC5 antibody.

[0166] Anti-rabbit IgG (Abbexa) was immobilized on FC1 and FC2 of a CM5 sensor chip using an amine coupling kit (GE Healthcare). For anti-rabbit IgG immobilization, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After immobilization, the running buffer was exchanged for phosphate pH 7.4 buffer (50 mM phosphate buffer, pH 7.4, containing 150 mM NaCl and 0.05% w / v P-20). Each anti-Fc mutant antibody was captured on FC2 of the sensor chip using anti-rabbit IgG. The amount of captured anti-Fc mutant antibody was adjusted to obtain 100 resonance units (RU). Anti-IL-8 antibody was injected at 0, 100, 400, and 800 nM concentrations at 10 μL / min. After each cycle, the sensor surface was regenerated with 10 mM glycine-HCl, pH 2.0, injected at a flow rate of 30 μL / min. Using Biacore T200 Evaluation software, version 2.0 (GE Healthcare), D got the value.

[0167] ka, kd, and K D are listed in Table 29. The amino acid at position 239 according to the EU numbering system of the anti-hC5 antibody was mutated from Ser to Lys, while the amino acid at the corresponding position of the anti-IL-8 antibody was not mutated. Under these circumstances, the anti-Fc mutant antibody was able to bind to the anti-IL-8 antibody. This means that the two mutations in SG115v1 shared by both the anti-hC5 antibody and the anti-IL-8 antibody, i.e., L235R and G236R (both at positions according to the EU numbering system), are essential for the selective binding of the anti-Fc mutant antibody to SG115v1.

[0168] [Table 29] * : Due to the slow dissociation rate, the data may be unreliable.

[0169] Example 11 Evaluation of the affinity of anti-Fc mutant antibodies that selectively bind to SG115v2 against anti-DENV E protein antibodies K values ​​at pH 7.4 of anti-Fc mutant antibodies (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171) that selectively bind to SG115v2 against an anti-DENV E protein antibody containing a modified IgG heavy chain constant region (SEQ ID NO: 116) as an Fc mutant antibody. D The values ​​were determined at 25°C using a Biacore T200 machine (GE Healthcare). Anti-rabbit IgG was immobilized on FCs 3 and 4 of a CM5 sensor chip using an amine coupling kit (GE Healthcare). For immobilization of anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After immobilization, the running buffer was exchanged for phosphate pH 7.4 buffer. Each antibody was captured on FC4 of the sensor chip using anti-rabbit IgG. The amount of anti-Fc mutant antibody captured was adjusted to obtain 100 resonance units (RU). Anti-DENV E protein antibodies were injected at 0, 12.5, 50, and 400 nM at 10 μL / min. The sensor surface was regenerated at each cycle with 10 mM glycine HCl, pH 2.0, injected at a flow rate of 30 μL / min. Using Biacore T200 Evaluation software, version 2.0 (GE Healthcare), D The values ​​obtained were ka, kd, and K D is shown in Table 30.

[0170] [Table 30] * : Due to the slow dissociation rate, the data may be unreliable.

[0171] Example 12 Evaluation of the affinity of anti-hC5 antibodies to human C5 using SKA0016 and SKA0117 as capture molecules K of anti-hC5 antibody against human C5 at pH 7.4 D The values ​​were determined at 37°C using a Biacore T200 machine (GE Healthcare). SKA0016 was immobilized on FC1 and 2 of a CM5 sensor chip, and SKA0117 was immobilized on FC3 and 4 using an amine coupling kit (GE Healthcare). For immobilization of SKA0016 and SKA0117, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After immobilization, the running buffer was exchanged for phosphate pH 7.4 buffer. Anti-hC5 antibody was captured by SKA0016 and SKA0117 on FC2 and FC4 of the sensor chip. The amount of anti-hC5 antibody captured was adjusted to 35 resonance units (RU). Human C5 was injected at 10 μL / min at 0 nM, 2 nM, 4 nM, 8 nM, 16 nM, and 32 nM. In each cycle, the sensor surface was regenerated with 100 mM glycine HCl, pH 2.0, followed by 25 mM NaOH, both injected at a flow rate of 30 μL / min. The K was measured using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). D The values ​​obtained were ka, kd, and K D are listed in Table 31.

[0172] [Table 31]

[0173] Example 13 Qualitative analysis of pH-dependent interactions of anti-hC5 antibodies with human C5 using SKA0016 and SKA0117 as immobilized molecules The pH-dependent interaction between anti-hC5 antibody and human C5 at pH 7.4 and pH 6.0 was evaluated at 37°C using a Biacore T200 machine (GE Healthcare). Anti-hC5 antibody was captured on FC2 and FC4 of the CM5 chip prepared in Example 12. The amount of captured anti-hC5 antibody was adjusted to obtain 35 resonance units (RU). To confirm the association between anti-hC5 antibody and human C5 at pH 7.4, 32 nM human C5 in phosphate pH 7.4 buffer was injected into all FCs. The dissociation phase was then monitored in phosphate pH 7.4 buffer or phosphate pH 6.0 buffer (50 mM phosphate buffer containing 150 mM NaCl and 0.05 w / v% P-20, pH 6.0) as the running buffer. After monitoring the dissociation phase, the sensor chip was regenerated by injecting 100 mM Gly-HCl, pH 2.0, followed by 25 mM NaOH, both injected at a flow rate of 30 μL / min. The pH-dependent interaction of the anti-hC5 antibody with human C5 was analyzed by comparing the dissociation phases of the sensorgrams at pH 7.4 and pH 6.0 using Biacore T200 Evaluation software, version 2.0. Each sensorgram was normalized by subtracting FC1 from the sensorgram at FC2 and FC3 from the sensorgram at FC4, and adjusting the human C5 binding response 5 seconds before the completion of the human C5 injection to a value of "100."

[0174] Regardless of the capture molecule, dissociation of human C5 from the anti-hC5 antibody was more rapid at pH 6.0 than at pH 7.4 (Figure 6; anti-hC5 antibody was immobilized with (a) SKA0016 and (b) SKA0117). Therefore, both SKA0016 and SKA0117 are considered effective for monitoring the pH-dependent interaction between anti-hC5 antibody and human C5.

[0175] Example 14 Evaluation of binding between human Fc receptor (hFcRn) and anti-hC5 antibody captured by SKA0016 The ability of human FcRn to bind to anti-hC5 antibody captured by SKA0016 at pH 6.0 was evaluated using a Biacore T200 machine (GE Healthcare). Anti-hC5 antibody was captured on FC2 by SKA0016 immobilized on a CM5 chip using the same procedure as in Example 9. Phosphate pH 6.0 buffer was used as the running buffer. The amount of anti-hC5 antibody captured was adjusted to 400 resonance units (RU). hFcRn was injected at 0 nM, 26.3 nM, 52.5 nM, 105 nM, 210 nM, and 420 nM at 10 μL / min using a single-cycle kinetic method. The sensor surface was regenerated with 100 mM glycine HCl, pH 2.0, followed by 25 mM NaOH, both injected at a flow rate of 30 μL / min. The increase in binding response of hFcRn was confirmed using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0176] Figure 7 shows the sensorgrams of FC1 (dashed line) and FC2 (solid line). The FC2 sensorgram (solid line) reveals a concentration-dependent increase in the binding response of hFcRn. SKA0016 did not inhibit the binding between hFcRn and anti-hC5 antibody. However, because the sensorgram of FC1 also showed an increased binding response (dashed line), it appears that hFcRn binds to the Fc region of SKA0016. This undesired binding of human FcRn to the capture molecule can be eliminated by introducing amino acid substitutions into SKA0016 that abolish the binding to human FcRn.

Claims

1. An isolated antibody that specifically binds to an altered IgG heavy chain constant region derived from any one of the constant regions of naturally occurring human IgG or a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG, the naturally occurring human IgG constant region is any one of an IgG1 constant region consisting of the amino acid sequence of SEQ ID NO: 106, an IgG2 constant region consisting of the amino acid sequence of SEQ ID NO: 107, an IgG3 constant region consisting of the amino acid sequence of SEQ ID NO: 108, and an IgG4 constant region consisting of the amino acid sequence of SEQ ID NO: 109; the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system); binds to a portion of the modified IgG heavy chain constant region consisting of the amino acid sequence LHEALHAHYTRKE (SEQ ID NO: 105), An isolated antibody that does not substantially bind to any one of the constant regions of the naturally occurring human IgG and a chimeric constant region obtained from at least two selected from the constant regions of the naturally occurring human IgG.

2. 2. The antibody of claim 1, wherein the modified IgG heavy chain constant region is derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and human IgG4.

3. 3. The antibody of claim 1 or 2, wherein the modified IgG heavy chain constant region further comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions according to the EU numbering system).

4. 4. The antibody of any one of claims 1 to 3, wherein the modified IgG heavy chain constant region further comprises Arg at position 235, and one or both of Arg at position 236 and Lys at position 239 (all positions according to the EU numbering system).

5. The antibody of any one of claims 1 to 4, comprising any one of the following (a) to (f): (a) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 44, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 68, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 80, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 92; (b) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 47, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 71, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 83, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 95; (c) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 48, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 72, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 84, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 96; (d) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 52, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 64, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 76, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 100; (e) a variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 42, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 54, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 66, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 78, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 90, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 102; and (f) A variable region comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 43, HVR-H2 having the amino acid sequence of SEQ ID NO: 55, HVR-H3 having the amino acid sequence of SEQ ID NO: 67, HVR-L1 having the amino acid sequence of SEQ ID NO: 79, HVR-L2 having the amino acid sequence of SEQ ID NO: 91, and HVR-L3 having the amino acid sequence of SEQ ID NO:

103.

6. An isolated antibody that specifically binds to an engineered IgG heavy chain constant region, wherein the binding of the antibody to the engineered IgG heavy chain constant region competes with the antibody of any one of claims 1 to 5, and the engineered IgG heavy chain constant region is derived from any one of the constant regions of naturally occurring human IgG or from a chimeric constant region obtained from at least two selected constant regions of naturally occurring human IgG; the naturally occurring human IgG constant region is any one of an IgG1 constant region consisting of the amino acid sequence of SEQ ID NO: 106, an IgG2 constant region consisting of the amino acid sequence of SEQ ID NO: 107, an IgG3 constant region consisting of the amino acid sequence of SEQ ID NO: 108, and an IgG4 constant region consisting of the amino acid sequence of SEQ ID NO: 109; the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions according to the EU numbering system); An isolated antibody that does not substantially bind to any one of the constant regions of the naturally occurring human IgG and a chimeric constant region obtained from at least two selected from the constant regions of the naturally occurring human IgG.

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