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

Abstract

This disclosure provides antibodies, compositions, and methods for detecting or capturing peptides in a sample.

Description

Technical Field

[0001] This invention relates to antibodies and compositions for detecting or capturing peptides in samples, and methods for detecting or capturing peptides in samples. Background Technology

[0002] Hybridoma technology enabled the production of monoclonal antibodies, a technology widely used in many scientific fields (NPL1). Following this technological achievement, further efforts were made in the fields of therapeutic and diagnostic antibodies. Thirty years have passed since the first monoclonal antibody therapy was approved in the United States (NPL2). More than 30 antibodies have been approved by the FDA, and a large number of candidate antibodies are undergoing clinical and preclinical evaluation. To date, monoclonal antibodies remain standard therapeutic molecules and are used in a variety of disease areas, such as cancer, autoimmune diseases, respiratory diseases, infectious diseases, and neurological diseases (NPL3).

[0003] To enhance the value of therapeutic antibodies, many different types of engineered Fc modifications have been identified to improve function, such as those for antibody-dependent cell-mediated cytotoxicity enhancement, complement-dependent cytotoxicity enhancement, antibody half-life prolongation, antigen clearance regulation, and promotion of heavy chain heterodimerization (NPL4).

[0004] Antibodies that specifically bind to engineered Fc regions but not to wild-type Fc have been reported (NPL5, PTL1). Antibodies targeting engineered Fc regions have proven to be very useful for a variety of purposes.

[0005] Reference List

[0006] Patent documents

[0007] [PTL1]WO2017072210A1

[0008] Non-patent literature

[0009] [NPL1]Kohler,G.et al.,Nature 256:495-497(1975)

[0010] [NPL2]Reichert,JMet al.,Curr.Pharm.Biotechnol.9:423-430(2008)

[0011] [NPL3]Lagasse HAD et al.F1000Research 2017,6(F1000 Faculty Rev):113

[0012] [NPL4]Mimoto et al., Curr.Pharm.Biotechnol.17:1298-1314(2016)

[0013] [NPL5]Yu et al.,Antimicrob Agents Chemother.61(2016) Summary of the Invention

[0014] We provide antibodies comprising modified IgG heavy chain constant regions derived from any one constant region of naturally occurring human IgG or from a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG. Antibodies include, for example, satralizumab, nemolizumab, emicizumab, SKY59 (crovallimab), AMY109, and GYM329. For example, the modified IgG heavy chain constant region in one of them 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 are numbered according to the EU numbering system). The present invention provides antibodies that specifically bind to, detect, and / or capture polypeptides or epitopes therein comprising modified IgG heavy chain constant regions, compositions comprising the antibodies, and methods of using the antibodies.

[0015] Specifically, the present invention relates to the following [1] to

[25] .

[0016] [1] An isolated antibody that specifically binds to a modified IgG heavy chain constant region, the modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG, or a chimeric constant region derived from at least two constant regions selected from 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 are numbered according to the EU numbering system).

[0017] [2] The antibody according to [1], wherein the antibody substantially does not bind to any of the constant regions of the naturally occurring human IgG and the chimeric constant region obtained from at least two constant regions selected from the constant regions of naturally occurring human IgG.

[0018] [3] The antibody according to [1] or [2], wherein the constant region of the naturally occurring human IgG is 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.

[0019] [4] The antibody according to any one of [1] to [3], wherein the modified heavy chain constant region is derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and IgG4.

[0020] [5] The antibody according to any one of [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 are numbered according to the EU numbering system).

[0021] [6] The antibody according to any one of [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 are numbered according to the EU numbering system).

[0022] [7] The antibody according to any one of [1] to [6], wherein the modified IgG heavy chain constant region comprises Arg at position 235, and any one or both of Arg at position 236 and Lys at position 239 (all positions are numbered according to the EU numbering system).

[0023] [8] The antibody according to any one of [1] to [7] binds to the 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).

[0024] [9] The antibody according to any one of [1] to [8] comprises any one of the following (a) to (f):

[0025] (a) Variable region, which includes

[0026] HVR-H1, which contains the amino acid sequence of SEQ ID NO:33,

[0027] HVR-H2, which contains the amino acid sequence of SEQ ID NO:45,

[0028] HVR-H3, which contains the amino acid sequence of SEQ ID NO:57,

[0029] HVR-L1, which contains the amino acid sequence of SEQ ID NO:69,

[0030] HVR-L2, which contains the amino acid sequence of SEQ ID NO:81, and

[0031] HVR-L3, which contains the amino acid sequence of SEQ ID NO:93;

[0032] (b) Variable region, which includes

[0033] HVR-H1, which contains the amino acid sequence of SEQ ID NO:34,

[0034] HVR-H2, which contains the amino acid sequence of SEQ ID NO:46,

[0035] HVR-H3, which contains the amino acid sequence of SEQ ID NO:58,

[0036] HVR-L1, which contains the amino acid sequence of SEQ ID NO:70,

[0037] HVR-L2, which contains the amino acid sequence of SEQ ID NO:82, and

[0038] HVR-L3, which contains the amino acid sequence of SEQ ID NO:94;

[0039] (c) Variable region, which includes

[0040] HVR-H1, which contains the amino acid sequence of SEQ ID NO:37,

[0041] HVR-H2, which contains the amino acid sequence of SEQ ID NO:49,

[0042] HVR-H3, which contains the amino acid sequence of SEQ ID NO:61,

[0043] HVR-L1, which contains the amino acid sequence of SEQ ID NO:73,

[0044] HVR-L2, which contains the amino acid sequence of SEQ ID NO:85, and

[0045] HVR-L3, which contains the amino acid sequence of SEQ ID NO:97;

[0046] (d) Variable region, which includes

[0047] HVR-H1, which contains the amino acid sequence of SEQ ID NO:38,

[0048] HVR-H2, which contains the amino acid sequence of SEQ ID NO:50,

[0049] HVR-H3, which contains the amino acid sequence of SEQ ID NO:62,

[0050] HVR-L1, which contains the amino acid sequence of SEQ ID NO:74,

[0051] HVR-L2, which contains the amino acid sequence of SEQ ID NO:86, and

[0052] HVR-L3, which contains the amino acid sequence of SEQ ID NO:98;

[0053] (e) Variable region, which includes

[0054] HVR-H1, which contains the amino acid sequence of SEQ ID NO:39,

[0055] HVR-H2, which contains the amino acid sequence of SEQ ID NO:51,

[0056] HVR-H3, which contains the amino acid sequence of SEQ ID NO:63,

[0057] HVR-L1, which contains the amino acid sequence of SEQ ID NO:75,

[0058] HVR-L2, which contains the amino acid sequence of SEQ ID NO:87, and

[0059] HVR-L3, which contains the amino acid sequence of SEQ ID NO:99; and

[0060] (f) Variable region, which includes

[0061] HVR-H1, which contains the amino acid sequence of SEQ ID NO:41,

[0062] HVR-H2, which contains the amino acid sequence of SEQ ID NO:53,

[0063] HVR-H3, which contains the amino acid sequence of SEQ ID NO:65,

[0064] HVR-L1, which contains the amino acid sequence of SEQ ID NO:77,

[0065] HVR-L2, which contains the amino acid sequence of SEQ ID NO:89, and

[0066] HVR-L3 contains the amino acid sequence of SEQ ID NO:101.

[0067]

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

[0068]

[11] The antibody according to 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 are numbered according to the EU numbering system).

[0069]

[12] The antibody according to any one of [1] to [5],

[10] and

[11] binds to the 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).

[0070]

[13] The antibody according to any one of [1] to [5] and

[10] to

[12] comprises any one of the following (g) to (l):

[0071] (g) Variable region, which includes

[0072] HVR-H1, which contains the amino acid sequence of SEQ ID NO:32,

[0073] HVR-H2, which contains the amino acid sequence of SEQ ID NO:44,

[0074] HVR-H3, which contains the amino acid sequence of SEQ ID NO:56,

[0075] HVR-L1, which contains the amino acid sequence of SEQ ID NO:68,

[0076] HVR-L2, which contains the amino acid sequence of SEQ ID NO:80, and

[0077] HVR-L3, which contains the amino acid sequence of SEQ ID NO:92;

[0078] (h) Variable region, which includes

[0079] HVR-H1, which contains the amino acid sequence of SEQ ID NO:35,

[0080] HVR-H2, which contains the amino acid sequence of SEQ ID NO:47,

[0081] HVR-H3, which contains the amino acid sequence of SEQ ID NO:59,

[0082] HVR-L1, which contains the amino acid sequence of SEQ ID NO:71,

[0083] HVR-L2, which contains the amino acid sequence of SEQ ID NO:83, and

[0084] HVR-L3, which contains the amino acid sequence of SEQ ID NO:95;

[0085] (i) Variable region, which includes

[0086] HVR-H1, which contains the amino acid sequence of SEQ ID NO:36,

[0087] HVR-H2, which contains the amino acid sequence of SEQ ID NO:48,

[0088] HVR-H3, which contains the amino acid sequence of SEQ ID NO:60,

[0089] HVR-L1, which contains the amino acid sequence of SEQ ID NO:72,

[0090] HVR-L2, which contains the amino acid sequence of SEQ ID NO:84, and

[0091] HVR-L3, which contains the amino acid sequence of SEQ ID NO:96;

[0092] (j) Variable region, which includes

[0093] HVR-H1, which contains the amino acid sequence of SEQ ID NO:40,

[0094] HVR-H2, which contains the amino acid sequence of SEQ ID NO:52,

[0095] HVR-H3, which contains the amino acid sequence of SEQ ID NO:64,

[0096] HVR-L1, which contains the amino acid sequence of SEQ ID NO:76,

[0097] HVR-L2, which contains the amino acid sequence of SEQ ID NO:88, and

[0098] HVR-L3, which contains the amino acid sequence of SEQ ID NO:100;

[0099] (k) Variable region, which includes

[0100] HVR-H1, which contains the amino acid sequence of SEQ ID NO:42,

[0101] HVR-H2, which contains the amino acid sequence of SEQ ID NO:54,

[0102] HVR-H3, which contains the amino acid sequence of SEQ ID NO:66,

[0103] HVR-L1, which contains the amino acid sequence of SEQ ID NO:78,

[0104] HVR-L2, which contains the amino acid sequence of SEQ ID NO:90, and

[0105] HVR-L3, which contains the amino acid sequence of SEQ ID NO:102; and

[0106] (l) Variable region, which includes

[0107] HVR-H1, which contains the amino acid sequence of SEQ ID NO:43,

[0108] HVR-H2, which contains the amino acid sequence of SEQ ID NO:55,

[0109] HVR-H3, which contains the amino acid sequence of SEQ ID NO:67,

[0110] HVR-L1, which contains the amino acid sequence of SEQ ID NO:79,

[0111] HVR-L2, which contains the amino acid sequence of SEQ ID NO:91, and

[0112] HVR-L3 contains the amino acid sequence of SEQ ID NO:103.

[0113]

[14] The isolated antibody binds to the same epitope as the antibody in any of [1] to

[13] .

[0114]

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

[14] for binding to the modified IgG heavy chain constant region, wherein the modified IgG heavy chain constant region is derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from 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 are numbered according to the EU numbering system).

[0115]

[16] A composition for detecting or capturing peptides in a sample, wherein the composition comprises any one of [1] to

[15] antibodies.

[0116]

[17] The composition according to

[16] , wherein the polypeptide comprises a modified IgG heavy chain constant region derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from 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 are numbered according to the EU numbering system).

[0117]

[18] The composition according to

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

[0118]

[19] The composition according to

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

[0119]

[20] A method for detecting or capturing polypeptides in a sample, wherein the method comprises contacting the sample with an antibody as described in any one of [1] to

[15] or with a composition as described in any one of

[16] to

[19] .

[0120]

[21] According to the method of

[20] , the polypeptide comprises a modified IgG heavy chain constant region derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from 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 are numbered according to the EU numbering system).

[0121]

[22] According to the method of

[20] , the polypeptide comprises any one of the following: 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).

[0122]

[23] According to the method of

[22] , the polypeptide includes a modified IgG heavy chain constant region, the modified IgG heavy chain constant region including any one of the following amino acid sequences: RRGPK (SEQ ID NO: 104), RRGPS (SEQ ID NO: 117), LHEALHAHYTRKE (SEQ ID NO: 105), and LHEALHAHTTRKE (SEQ ID NO: 118).

[0123]

[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, wherein the sample comprises the first antibody and the antigen, and wherein the method comprises

[0124] (A) Contact the sample with a plate or beads immobilized with a second antibody.

[0125] (B) After (A), contact the plate or bead with a solution comprising the antigen but excluding the first antibody and the second antibody, and

[0126] (C) Following (B), a third antibody is used to detect the antigen captured on the plate or bead by the second antibody and the first antibody, wherein...

[0127] The first antibody includes a modified IgG heavy chain constant region derived from any constant region of naturally occurring human IgG, or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG, wherein the modified IgG heavy chain constant region includes 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 are numbered according to the EU numbering system), the second antibody is any one of [1] to

[15] , and the third antibody is capable of binding a second epitope of the antigen that is different from the first epitope and has an IgG heavy chain constant region whose amino acid sequence is different from that of the first antibody and the second antibody.

[0128]

[25] A method for determining the concentration of an antigen in a sample, wherein the sample comprises the antigen and a first antibody capable of binding a first epitope of the antigen, wherein the method comprises

[0129] (D) Contact the sample with a plate or beads immobilized with a third antibody.

[0130] (E) After (D), contact the solution comprising the first antibody but excluding the antigen and the third antibody with the plate or the bead, and

[0131] (F) Detection of the first antibody captured on the plate or bead by the third antibody and the antigen using a second antibody, wherein...

[0132] The first antibody includes a modified IgG heavy chain constant region, said modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG, or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG, wherein said modified IgG heavy chain constant region includes 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 are numbered according to the EU numbering system).

[0133] The second antibody is any one of [1] to

[15] , and has an IgG heavy chain constant region whose amino acid sequence is different from that of the first antibody and the third antibody.

[0134] The third antibody can bind to a second epitope of the antigen that is different from the first epitope. Attached Figure Description

[0135] [ Figure 1 ] Figure 1 The sequence alignments of the five modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) described in Example 2 were explained. Human IgG CH germline sequences, namely IGHG1_01 (J00228) and IGHG4_01 (K01316), were also aligned for comparison. Dots indicate amino acids identical to SG115 at that position.

[0136] [ Figure 2-1 ] Figure 2-1 and Figure 2-2 The binding of 12 anti-SG115 antibodies to 5 modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) in the ELISA was demonstrated. SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127 showed selective binding to SG115v1, while SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171 showed selective binding to SG115v2, as described in Example 2.

[0137] [ Figure 2-2 ] Figure 2-2 yes Figure 2-1 The continuation of.

[0138] [ Figure 3 ] Figure 3The protocol for the detection and assay of Fc mutant antibodies is explained.

[0139] [ Figure 4 ] Figure 4 The protocol for antigen detection assay is explained.

[0140] [ Figure 5 ] Figure 5 The method for Simoa (registered trademark) testing is explained.

[0141] [ Figure 6 ] Figure 6 A sensor map illustrating the dissociation of human C5 from anti-hC5 antibodies, which were captured by SKA0016 and SKA0117 at pH 7.4 and pH 6.0, is presented. Neither SKA0016 nor SKA0117 interferes with the pH-dependent interaction between the anti-hC5 antibody and human C5.

[0142] [ Figure 7 ] Figure 7 This diagram illustrates the sensor mapping for the binding analysis of human Fc receptors to anti-hC5 antibodies captured by SKA0016. SKA0016 did not interrupt the binding between hFcRn and the anti-hC5 antibody. Detailed Implementation

[0143] I. Definition

[0144] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" or "binding activity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is typically expressed as a dissociation constant (Kd). Affinity can be measured using methods commonly known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0145] The term "isolated antibody that specifically binds to a modified IgG heavy chain constant region, wherein the modified IgG heavy chain constant region is derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG" refers to an antibody capable of binding to a specific type of modified IgG heavy chain constant region with sufficient affinity, such that the antibody can be used as a detection, capture, or diagnostic reagent targeting the modified IgG heavy chain constant region. In one embodiment, for an antibody that specifically binds to a modified IgG heavy chain constant region, the degree of binding of the antibody to an unmodified human IgG heavy chain constant region is less than about 10% of the binding to the modified IgG heavy chain constant region, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the antibody binding to the modified IgG heavy chain constant region has a concentration 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 nM). -8 M or smaller, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M). In some embodiments, the antibody that binds to the modified IgG heavy chain constant region binds to the epitope in the modified IgG heavy chain constant region.

[0146] The term “antibody” is used in the broadest sense herein and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0147] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0148] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the preparation and production of monoclonal antibodies), which are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the invention can be prepared 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 preparing monoclonal antibodies are described herein.

[0149] The term "constant region" as used herein refers to the region in an antibody corresponding to any one of the following: 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 comprises the CH1 region (positions 118 to 215 according to the EU numbering system), the hinge region (positions 216 to 230 according to the EU numbering system), the CH2 region (positions 231 to 340 according to the EU numbering system), and the CH3 region (positions 341 to 446 according to the EU numbering system).

[0150] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0151] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, libraries of complementary VL or VH domains can be screened separately using VH or VL domains from antibodies binding to antigens, thereby isolating antibodies binding to specific antigens. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0152] "Frame" or "FR" refers to the variable domain residues outside the hypervariable region (HVR). The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences in VH (or VL) generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0153] As used herein, the term “hypervariant region” or “HVR” refers to each region of an antibody variable domain that is sequence-highly variable (“complementarity-determining region” or “CDR”) and / or forms a structurally defined loop (“hypervariant loop”) and / or contains antigen contact residues (“antigen contact”). Typically, an antibody contains six HVRs: three of the VHs (H1, H2, H3) and three of the VLs (L1, L2, L3). Exemplary HVRs in this document include:

[0154] (a) Hypervariable rings appearing 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));

[0155] (b) CDRs appearing 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));

[0156] (c) Antigen contacts appearing 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

[0157] (d) Combinations of (a), (b) and / or (c) including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).

[0158] The "percentage (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after the sequences have been aligned and, where necessary, vacancies have been introduced to obtain the maximum percentage sequence identity, and no conservative substitutions have been considered as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENTYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0159] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the U.S. Copyright Office (Washington DC, 20559) along with user documentation, under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change. When using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (or, in other words, a given amino acid sequence A has or contains a specific % amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:

[0160] 100 multiplied by the fraction X / Y

[0161] Where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and Y is the total number of amino acid residues in B. It should be understood that when 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 relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described above.

[0162] An antibody that "binds to the same epitope as the reference antibody" means an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay, and conversely, that the reference antibody blocks 50% or more of the binding of the reference antibody to its antigen in a competitive assay. Exemplary competitive assays are provided herein.

[0163] II. Antibodies

[0164] The antibody in this invention is a separation antibody that specifically binds to the constant region of the modified IgG heavy chain.

[0165] In one embodiment, the antibody substantially does not bind to constant regions of naturally occurring human IgG or chimeric constant regions obtained from at least two constant regions selected from naturally occurring human IgG. In this embodiment, the binding activity of the antibody to constant regions of naturally occurring human IgG and chimeric IgG composed of at least two IgGs selected from naturally occurring human IgG is below the detection limit in an enzyme-linked immunosorbent assay (ELISA). On the other hand, the binding activity of the antibody to modified IgG heavy chain constant regions is detectable in an ELISA.

[0166] In another aspect of the invention, the antibody is a monoclonal antibody, including chimeric antibodies, humanized antibodies, or human antibodies. In one embodiment, the antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1, IgG2, IgG3, and IgG4 antibody, or other antibody classes or isotypes as defined herein.

[0167] A. Modified IgG heavy chain constant region

[0168] In one embodiment, the modified IgG heavy chain constant region is derived from any constant region of naturally occurring human IgG, or from a chimeric constant region obtained from at least two constant regions selected from naturally occurring human IgG. The naturally occurring human IgG constant regions are the IgG1 constant region composed of the amino acid sequence of SEQ ID NO:106, the IgG2 constant region composed of the amino acid sequence of SEQ ID NO:107, the IgG3 constant region composed of the amino acid sequence of SEQ ID NO:108, and the IgG4 constant region composed of the amino acid sequence of SEQ ID NO:109.

[0169] In one embodiment, the modified IgG heavy chain constant region includes 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 are numbered according to the EU numbering system).

[0170] 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 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 IgG4.

[0171] In a preferred embodiment, the modified IgG heavy chain constant region includes 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 are numbered according to the EU numbering system).

[0172] In one embodiment, the modified IgG heavy chain constant region can form a dimer, such as the heavy chain constant region in naturally occurring IgG, or it can form a hemimer, such as the heavy chain constant region in monomeric Fc reported by Ishino T. et al., J. Biol. Chem. 288:16259-37 (2013).

[0173] In one embodiment, when the constant region of the modified IgG heavy chain is in a human-modified IgG heavy chain, the human-modified IgG heavy chain is selected from the group consisting of human-modified IgG1, IgG2, IgG3, and IgG4 heavy chains, and their chimeric IgG heavy chains. In a preferred embodiment, the human IgG heavy chain is a human IgG1 heavy chain, a human IgG4 heavy chain, or their chimeric IgG heavy chains.

[0174] B. Exemplary antibodies that specifically recognize modifications unique to the CH2 region of the constant region of the IgG heavy chain.

[0175] Arg at position 235, Arg at position 236, and Lys at position 239 (all positions are numbered according to the EU numbering system) are those amino acids specifically present in the CH2 region of SG115 and SG115v1 used in the examples. Therefore, the modified IgG heavy chain constant region in the exemplary antibody herein preferably includes a region of the CH2 region corresponding to at least one constant region of naturally occurring human IgG or a chimeric constant region obtained from at least two constant regions selected from the constant regions of naturally occurring human IgG.

[0176] 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 are numbered according to the EU numbering system). In this embodiment, the modified IgG heavy chain constant region comprises Arg at position 235, and any one or both of Arg at position 236 and Lys at position 239 (all positions are numbered according to the EU numbering system).

[0177] In a preferred embodiment, the modified IgG heavy chain constant region includes all three mutations, or Arg at positions 235 and 236 (all positions are numbered according to the EU numbering system). In that case, the antibody binds to the 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).

[0178] In which the antibody specifically binds to the following aspects (1)-(3): (1) 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 are numbered according to the EU numbering system), (2) a modified IgG heavy chain constant region comprising Arg at position 235, and any one or both of Arg at position 236 and Lys at position 239 (all positions are numbered according to the EU numbering system), or (3) 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 present invention provides an antibody comprising at least one, two, three, four, five or six HVRs selected from: (i) HVR-H1, comprising SEQ ID NO: 104, SEQ ID NO: 117 ... (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39 or 41; (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63 or 65; (iv) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75 or 77; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99 or 101.

[0179] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three of the following VH HVR sequences: (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, which comprises the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. In another embodiment, the antibody comprises HVR-H3 and HVR-L3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101. In another embodiment, the antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101, and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53. In a further embodiment, the antibody comprises (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39 or 41; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51 or 53; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63 or 65.

[0180] In another aspect, the present invention provides an antibody 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, which contains the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75 or 77; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99 or 101.

[0181] In another aspect, the antibody of the present invention comprises (I) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 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) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; and (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65; and (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; and (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; and (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 69, 70, 73, 74 The amino acid sequences NO:81, 82, 85, 86, 87 or 89, and (vi)HVR-L3, which contains the amino acid sequences SEQ ID NO:93, 94, 97, 98, 99 or 101.

[0182] In another aspect, the present invention provides 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 selected from SEQ ID NO: 93, 94, 97, 98, 99 or 101.

[0183] On the other hand, the antibodies described herein include a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9, 10, 13, 14, 15, or 17. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the antibody described herein containing this sequence retains the ability to bind the first modified IgG heavy chain constant region. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 9, 10, 13, 14, 15, or 17 are substituted, inserted, and / or deleted. In some embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody includes a VH sequence in SEQ ID NO: 9, 10, 13, 14, 15, or 17, including post-translational modifications of that sequence. In a particular embodiment, the VH includes one, two, or three HVRs selected from: (i) HVR-H1, which contains the amino acid sequence of SEQ ID NO: 33, 34, 37, 38, 39, or 41; (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51, or 53; and (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63, or 65. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain with pyroglutamic acid via pyroglutamylation.

[0184] On the other hand, an antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21, 22, 25, 26, 27, or 29. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the antibody containing this sequence retains the ability to bind to the first modified IgG heavy chain constant region. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 21, 22, 25, 26, 27, or 29 are substituted, inserted, and / or deleted. In some embodiments, substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody includes a VL sequence in SEQ ID NO: 21, 22, 25, 26, 27, or 29, including post-translational modifications of that sequence. In a particular embodiment, the VL includes one, two, or three HVRs selected from (iv) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75, or 77; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87, or 89; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99, or 101. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain with pyroglutamic acid via pyroglutamylation, replacing glutamine or glutamate.

[0185] In another aspect, an antibody is provided, wherein the antibody comprises VH as in any of the embodiments provided above and VL as in any of the embodiments provided above. In one embodiment, the antibody comprises VH and VL sequences in SEQ ID NO: 9, 10, 13, 14, 15 or 17 and SEQ ID NO: 21, 22, 25, 26, 27 or 29, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain of glutamine or glutamate to pyroglutamic acid by pyroglutamylation.

[0186] In one aspect, an antibody is provided, wherein the antibody competitively binds to a first modified IgG heavy chain constant region 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.

[0187] In one aspect, an antibody is provided, wherein the antibody binds 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.

[0188] In a specific embodiment in which the antibody specifically binds to the following (1)-(3): (1) 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 are numbered according to the EU numbering system), (2) a modified IgG heavy chain constant region comprising Arg at position 235, and any one or both of Arg at position 236 and Lys at position 239 (all positions are numbered according to the EU numbering system), or (3) 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), wherein the antibody comprises any one of the following (a) to (f):

[0189] (a) Variable region, which includes

[0190] HVR-H1, which contains the amino acid sequence of SEQ ID NO:33,

[0191] HVR-H2, which contains the amino acid sequence of SEQ ID NO:45,

[0192] HVR-H3, which contains the amino acid sequence of SEQ ID NO:57,

[0193] HVR-L1, which contains the amino acid sequence of SEQ ID NO:69,

[0194] HVR-L2, which contains the amino acid sequence of SEQ ID NO:81, and

[0195] HVR-L3, which contains the amino acid sequence of SEQ ID NO:93;

[0196] (b) Variable region, which includes

[0197] HVR-H1, which contains the amino acid sequence of SEQ ID NO:34,

[0198] HVR-H2, which contains the amino acid sequence of SEQ ID NO:46,

[0199] HVR-H3, which contains the amino acid sequence of SEQ ID NO:58,

[0200] HVR-L1, which contains the amino acid sequence of SEQ ID NO:70,

[0201] HVR-L2, which contains the amino acid sequence of SEQ ID NO:82, and

[0202] HVR-L3, which contains the amino acid sequence of SEQ ID NO:94;

[0203] (c) Variable region, which includes

[0204] HVR-H1, which contains the amino acid sequence of SEQ ID NO:37,

[0205] HVR-H2, which contains the amino acid sequence of SEQ ID NO:49,

[0206] HVR-H3, which contains the amino acid sequence of SEQ ID NO:61,

[0207] HVR-L1, which contains the amino acid sequence of SEQ ID NO:73,

[0208] HVR-L2, which contains the amino acid sequence of SEQ ID NO:85, and

[0209] HVR-L3, which contains the amino acid sequence of SEQ ID NO:97;

[0210] (d) Variable region, which includes

[0211] HVR-H1, which contains the amino acid sequence of SEQ ID NO:38,

[0212] HVR-H2, which contains the amino acid sequence of SEQ ID NO:50,

[0213] HVR-H3, which contains the amino acid sequence of SEQ ID NO:62,

[0214] HVR-L1, which contains the amino acid sequence of SEQ ID NO:74,

[0215] HVR-L2, which contains the amino acid sequence of SEQ ID NO:86, and

[0216] HVR-L3, which contains the amino acid sequence of SEQ ID NO:98;

[0217] (e) Variable region, which includes

[0218] HVR-H1, which contains the amino acid sequence of SEQ ID NO:39,

[0219] HVR-H2, which contains the amino acid sequence of SEQ ID NO:51,

[0220] HVR-H3, which contains the amino acid sequence of SEQ ID NO:63,

[0221] HVR-L1, which contains the amino acid sequence of SEQ ID NO:75,

[0222] HVR-L2, which contains the amino acid sequence of SEQ ID NO:87, and

[0223] HVR-L3, which contains the amino acid sequence of SEQ ID NO:99; and

[0224] (f) Variable region, which includes

[0225] HVR-H1, which contains the amino acid sequence of SEQ ID NO:41,

[0226] HVR-H2, which contains the amino acid sequence of SEQ ID NO:53,

[0227] HVR-H3, which contains the amino acid sequence of SEQ ID NO:65,

[0228] HVR-L1, which contains the amino acid sequence of SEQ ID NO:77,

[0229] HVR-L2, which contains the amino acid sequence of SEQ ID NO:89, and

[0230] HVR-L3 contains the amino acid sequence of SEQ ID NO:101.

[0231] C. Exemplary antibodies that specifically recognize modifications specific to the CH3 region of the constant region of the IgG heavy chain.

[0232] Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions are numbered according to the EU numbering system) are those amino acids specifically present in the CH3 region of SG115 and SG115v2 used in the examples. Therefore, the modified IgG heavy chain constant region in the exemplary antibody herein preferably includes a region of the CH3 region corresponding to at least one constant region of naturally occurring human IgG or a chimeric constant region obtained from at least two constant regions selected from the constant regions of naturally occurring human IgG.

[0233] 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 are numbered according to the EU numbering system). In another 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 threonine at position 436 (all positions are numbered according to the EU numbering system).

[0234] In a preferred embodiment, the modified IgG heavy chain constant region includes all of these mutations. In that case, the antibody binds to the 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).

[0235] In which the antibody specifically binds to the following aspects (1)-(3): (1) a modified IgG heavy chain constant region, 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 are numbered according to the EU numbering system), (2) 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 are numbered according to the EU numbering system), or (3) 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 present invention provides an antibody comprising at least one, two, three, four, five, or six HVRs, wherein the HVRs are selected from: (i) HVR-H1, which comprises SEQ ID (ii) HVR-H2, which contains the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54 or 55; (iii) HVR-H3, which contains the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66 or 67; (iv) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78 or 79; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102 or 103.

[0236] In one aspect, the present invention provides an antibody 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 and HVR-L3, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67, and HVR-L3 comprises the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103. In another embodiment, the antibody comprises HVR-H3, HVR-L3, and HVR-H2, wherein HVR-H3 comprises the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67, HVR-L3 comprises the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103, and HVR-H2 comprises the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55. In a further embodiment, the antibody comprises (i) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42 or 43; (ii) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54 or 55; and (iii) HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66 or 67.

[0237] In another aspect, the present invention provides an antibody 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, which contains the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78 or 79; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102 or 103.

[0238] In another aspect, the antibody of the present invention comprises (I) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 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; and (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43. The amino acid sequence NO:80, 83, 84, 88, 90 or 91; and (vi)HVR-L3, which contains the amino acid sequence SEQ ID NO:92, 95, 96, 100, 102 or 103.

[0239] In another aspect, the present invention provides antibodies comprising (i) HVR-H1, comprising an amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42 or 43; (ii) HVR-H2, comprising an amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54 or 55; (iii) HVR-H3, comprising an amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66 or 67; (iv) HVR-L1, comprising an amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78 or 79; (v) HVR-L2, comprising an amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising an amino acid sequence selected from SEQ ID NO: 92, 95, 96, 100, 102 or 103.

[0240] On the other hand, the antibodies described herein include a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequences of SEQ ID NO: 8, 11, 12, 16, 18, or 19. In some embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the antibodies described herein containing this sequence retain the ability to bind to the first modified IgG heavy chain constant region. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 8, 11, 12, 16, 18, or 19 are substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody comprises the VH sequence of SEQ ID NO: 8, 11, 12, 16, 18, or 19, including post-translational modifications of that sequence. In a particular embodiment, 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, modifying the N-terminus of the heavy or light chain with pyroglutamic acid via pyroglutamylation.

[0241] On the other hand, an antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 20, 23, 24, 28, 30, or 31. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the antibody containing this sequence retains the ability to bind to the first modified IgG heavy chain constant region. In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 20, 23, 24, 28, 30, or 31 are substituted, inserted, and / or deleted. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody includes a VL sequence in SEQ ID NO: 20, 23, 24, 28, 30, or 31, including post-translational modifications of that sequence. In a particular embodiment, the VL includes one, two, or three HVRs selected from (iv) HVR-L1, which contains the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2, which contains the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3, which contains the amino acid sequence of SEQ ID NO: 92, 95, 96, 100, 102, or 103. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy or light chain with pyroglutamic acid via pyroglutamylation, replacing glutamine or glutamate.

[0242] In another aspect, an antibody is provided, wherein the antibody comprises VH as in any of the embodiments provided above and VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH sequence and VL sequence in SEQ ID NO: 8, 11, 12, 16, 18 or 19 and SEQ ID NO: 20, 23, 24, 28, 30 or 31, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modifying the N-terminus of the heavy chain or light chain with pyroglutamic acid by pyroglutamylation to pyroglutamic acid.

[0243] In one aspect, an antibody is provided, wherein the antibody competitively binds to a first modified IgG heavy chain constant region 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.

[0244] In one aspect, an antibody is provided, wherein the antibody binds 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.

[0245] In a specific embodiment in which the antibody specifically binds to the following (1)-(3): (1) a modified IgG heavy chain constant region comprising at least one of the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions are numbered according to the EU numbering system), (2) 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 are numbered according to the EU numbering system), or (3) 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), wherein the antibody comprises any one of the following (g) to (l):

[0246] (g) Variable region, which includes

[0247] HVR-H1, which contains the amino acid sequence of SEQ ID NO:32,

[0248] HVR-H2, which contains the amino acid sequence of SEQ ID NO:44,

[0249] HVR-H3, which contains the amino acid sequence of SEQ ID NO:56,

[0250] HVR-L1, which contains the amino acid sequence of SEQ ID NO:68,

[0251] HVR-L2, which contains the amino acid sequence of SEQ ID NO:80, and

[0252] HVR-L3, which contains the amino acid sequence of SEQ ID NO:92;

[0253] (h) Variable region, which includes

[0254] HVR-H1, which contains the amino acid sequence of SEQ ID NO:35,

[0255] HVR-H2, which contains the amino acid sequence of SEQ ID NO:47,

[0256] HVR-H3, which contains the amino acid sequence of SEQ ID NO:59,

[0257] HVR-L1, which contains the amino acid sequence of SEQ ID NO:71,

[0258] HVR-L2, which contains the amino acid sequence of SEQ ID NO:83, and

[0259] HVR-L3, which contains the amino acid sequence of SEQ ID NO:95;

[0260] (i) Variable region, which includes

[0261] HVR-H1, which contains the amino acid sequence of SEQ ID NO:36,

[0262] HVR-H2, which contains the amino acid sequence of SEQ ID NO:48,

[0263] HVR-H3, which contains the amino acid sequence of SEQ ID NO:60,

[0264] HVR-L1, which contains the amino acid sequence of SEQ ID NO:72,

[0265] HVR-L2, which contains the amino acid sequence of SEQ ID NO:84, and

[0266] HVR-L3, which contains the amino acid sequence of SEQ ID NO:96;

[0267] (j) Variable region, which includes

[0268] HVR-H1, which contains the amino acid sequence of SEQ ID NO:40,

[0269] HVR-H2, which contains the amino acid sequence of SEQ ID NO:52,

[0270] HVR-H3, which contains the amino acid sequence of SEQ ID NO:64,

[0271] HVR-L1, which contains the amino acid sequence of SEQ ID NO:76,

[0272] HVR-L2, which contains the amino acid sequence of SEQ ID NO:88, and

[0273] HVR-L3, which contains the amino acid sequence of SEQ ID NO:100;

[0274] (k) Variable region, which includes

[0275] HVR-H1, which contains the amino acid sequence of SEQ ID NO:42,

[0276] HVR-H2, which contains the amino acid sequence of SEQ ID NO:54,

[0277] HVR-H3, which contains the amino acid sequence of SEQ ID NO:66,

[0278] HVR-L1, which contains the amino acid sequence of SEQ ID NO:78,

[0279] HVR-L2, which contains the amino acid sequence of SEQ ID NO:90, and

[0280] HVR-L3, which contains the amino acid sequence of SEQ ID NO:102; and

[0281] (l) Variable region, which includes

[0282] HVR-H1, which contains the amino acid sequence of SEQ ID NO:43,

[0283] HVR-H2, which contains the amino acid sequence of SEQ ID NO:55,

[0284] HVR-H3, which contains the amino acid sequence of SEQ ID NO:67,

[0285] HVR-L1, which contains the amino acid sequence of SEQ ID NO:79,

[0286] HVR-L2, which contains the amino acid sequence of SEQ ID NO:91, and

[0287] HVR-L3 contains the amino acid sequence of SEQ ID NO:103.

[0288] D. Other implementation plans

[0289] In one embodiment, the antibody of the present invention includes an antibody that binds to the same epitope as any of the antibodies mentioned in the foregoing sections, wherein the foregoing sections are “A. Modified IgG heavy chain constant region” to “C. Exemplary antibodies that specifically recognize the CH3 region of the modified IgG heavy chain constant region”.

[0290] In one embodiment, the antibody of the present invention comprises an 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 the antibody mentioned in the foregoing section, which is “A. Modified IgG Heavy Chain Constant Region” through “C. Exemplary Modified Antibodies Specifically Recognizing the CH3 Region of the Modified IgG Heavy Chain Constant Region”. In this embodiment, the modified IgG heavy chain constant region is derived from any constant region of naturally occurring human IgG, or from a chimeric constant region obtained from at least two constant regions selected from 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 are numbered according to the EU numbering system). In this embodiment, the specific antibody referred to herein is the same as that described in the foregoing section.

[0291] E. Recombination methods and compositions

[0292] Antibodies can be generated using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising a VL of the antibody and / or an amino acid sequence comprising a VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed with): (1) a vector comprising nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody, or (2) a first vector comprising nucleic acid encoding an amino acid sequence comprising a VL of the antibody, and a second vector comprising nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., YO, NSO, Sp2 / O cells). In one embodiment, a method for preparing the antibody described herein is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0293] For the recombinant production of the antibodies described herein, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0294] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, the antibody can be separated from the bacterial cell paste in soluble fractions and can be further purified.

[0295] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains with "humanized" glycosylation pathways, thereby producing antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0296] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells.

[0297] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for producing antibodies in transgenic plants). TM technology).

[0298] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, as described in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse trophoblast cells (TM4 cells, as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells, as described in Mather et al., Annals NY. The MRC 5 cells and FS4 cells described in Acad. Sci. 383:44-68 (1982) are also mentioned. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR cells. -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255–268 (2003).

[0299] F. Measurement

[0300] The antibodies described herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using a variety of assays known in the art.

[0301] G. Combination assay with other assays

[0302] On the one hand, the antigen-binding activity of the antibody of the present invention is tested, for example by known methods such as ELISA, Western blotting, etc.

[0303] On the other hand, the competition assay can be used to identify antibodies that compete with any 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, said modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG or from a source selected from... A chimeric constant region obtained from at least two constant regions of the constant region 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 are numbered according to the EU numbering system). In some embodiments, such competitive antibodies may bind to the same epitope (e.g., linear or conformational epitope) as the epitope bound by any of the antibodies used in the examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA0171). Morris (1996) provides detailed exemplary methods for mapping epitopes bound by antibodies in “Epitope Mapping Protocols”, Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ).

[0304] In an exemplary competitive assay, a fixed 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 being tested for its ability to competitively bind to the fixed modified IgG heavy chain constant region against the labeled antibody. The unlabeled antibody may be present in B cell or hybridoma supernatant. As a control, a fixed modified IgG heavy chain constant region is incubated in a solution containing the labeled antibody but not the unlabeled antibody. After incubation under conditions allowing the labeled antibody to bind to the fixed modified IgG heavy chain constant region, excess unbound antibody is removed, and the amount of labeling associated with the fixed modified IgG heavy chain constant region is measured. If the amount of labeling associated with the fixed modified IgG heavy chain constant region in the test sample is significantly reduced compared to the control sample, it indicates that the unlabeled antibody competitively binds to the fixed modified IgG heavy chain constant region against the labeled antibody. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0305] III. Composition

[0306] On one hand, the compositions of the present invention are compositions for detecting or capturing polypeptides in a sample. The compositions include any of the antibodies described in "II. Antibodies".

[0307] On the other hand, the compositions of the present invention are compositions for treating or preventing diseases. When antibodies are used to treat or prevent any disease, the composition may be or include cells expressing or comprising any of the antibodies or fragments thereof described in "II. Antibodies" that specifically bind to the constant region of the modified IgG heavy chain.

[0308] 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 from a chimeric constant region derived from at least two of the naturally occurring human constant regions. 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 are numbered according to the EU numbering system). The specific antibodies referred to herein are the same as those described in “II. Antibodies”.

[0309] In another preferred embodiment, the polypeptide comprises any one of the following amino acid sequences: RRGPK (SEQ ID NO: 104), RRGPS (SEQ ID NO: 117), LHEALHAHYTRKE (SEQ ID NO: 105), and LHEALHAHTTRKE (SEQ ID NO: 118). The polypeptide detected or captured by the composition is not particularly limited in its structure, as long as the polypeptide includes any one or more of these amino acid sequences. The polypeptide preferably includes a modified IgG heavy chain constant region comprising any one or more of the amino acid sequences.

[0310] In one embodiment, the polypeptide detected or captured by the composition may be an antibody, such as a human IgG1, IgG2, IgG3 or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide containing a modified IgG heavy chain constant region or an epitope therein.

[0311] In the case where the polypeptide includes a modified IgG heavy chain constant region, the modified IgG heavy chain constant region may include other amino acid substitutions or modifications, provided that it includes 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 are numbered according to the EU numbering system).

[0312] IV. Methods

[0313] On one hand, the method of the present invention is a method for detecting or capturing peptides in a sample. The method includes contacting the sample with any of the antibodies described in "II. Antibodies" or any of the compositions described in "III. Compositions".

[0314] In a preferred embodiment, the polypeptide includes a modified IgG heavy chain constant region. The modified IgG heavy chain constant region is derived from any constant region of naturally occurring human IgG, or from a chimeric constant region of at least two constant regions selected from naturally occurring human IgG. The modified IgG heavy chain constant region includes 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 are numbered according to the EU numbering system).

[0315] In another preferred embodiment, the polypeptide comprises any one of the following amino acid sequences: RRGPK (SEQ ID NO: 104), RRGPS (SEQ ID NO: 117), LHEALHAHYTRKE (SEQ ID NO: 105), and LHEALHAHTTRKE (SEQ ID NO: 118). The polypeptide detected or captured by the method is not particularly limited in its structure, as long as the polypeptide includes one or more of these amino acid sequences. The polypeptide preferably includes a modified IgG heavy chain constant region comprising one or more of these amino acid sequences.

[0316] In one embodiment, the polypeptide detected or captured by the method may be an antibody, such as a human IgG1, IgG2, IgG3 or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide containing a modified IgG heavy chain constant region or an epitope therein.

[0317] In the case where the polypeptide includes a modified IgG heavy chain constant region, the modified IgG heavy chain constant region may include other amino acid substitutions or modifications, provided that it includes 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 are numbered according to the EU numbering system).

[0318] Figure 3 The example illustrates the ELISA method as a specific implementation scheme for this method. "Rabbit anti-Fc mutant antibody" corresponds to an antibody described in "II. Antibodies". Figure 3 The "anti-hC5 antibody" in A and Figure 3 The "anti-IL-8 antibody" in B corresponds to a polypeptide containing the modified IgG heavy chain constant region. Rabbit anti-Fc-mutant antibody immobilized on the plate captures samples... Figure 3 Anti-hC5 antibody in A and Figure 3 Anti-IL-8 antibody in B. Then, hC5 (human complement 5), which serves as the antigen for anti-hC5 antibody, binds to an epitope different from the epitope bound by the anti-hC5 antibody, namely "mouse anti-hC5" and anti-mouse-POD. Figure 3 In A, the reactions occur in this order. Figure 3In step B, IL-8, serving as the antigen for the anti-IL-8 antibody, binds to an epitope different from that bound by the anti-IL-8 antibody, namely "mouse anti-IL-8," and anti-mouse-POD react in this order. Finally, the POD substrate is added to the plate, and its luminescence is measured. In this embodiment, luminescence is detected using a photometer when a certain amount of a polypeptide containing a modified IgG heavy chain constant region is present in the sample.

[0319] In another implementation, the antibodies described in "II. Antibodies" can be used to detect antigens, such as... Figure 4 The hC5 and IL-8 shown. In this embodiment, "mouse anti-Fc-mutant antibody" and "rabbit anti-Fc-mutant antibody" correspond to either of the antibodies described in "II. Antibodies", and Figure 4 The "anti-hC5 antibody" in A and Figure 4 The "anti-IL-8 antibody" in B corresponds to a polypeptide containing the modified IgG heavy chain constant region. Figure 4 In step A, rabbit anti-hC5 antibody immobilized on a plate captures hC5 in the sample. Figure 4 In step B, mouse anti-IL-8 antibody immobilized on the plate captures IL-8 in the sample. Then, in Figure 4 In A, anti-hC5 antibody, mouse anti-Fc-mutant antibody, and anti-mouse-POD react in this order. Figure 4 In step B, the anti-IL-8 antibody, rabbit anti-Fc mutant antibody, and anti-rabbit HRP are reacted in this order. Finally, a POD (peroxidase) substrate, such as HRP (horseradish peroxidase), is added to the plate and its luminescence is measured. In this embodiment, luminescence is detected by a photometer when a certain amount of hC5 or IL-8 is present in the sample.

[0320] The above-described ELISA method can be replaced by the Simoa (registered trademark) assay. In one embodiment of this assay, the antibody described in "II. Antibody" can be used to detect antigens, such as IL-8, as... Figure 5 As shown. 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. The anti-IL-8 antibody, biotinylated anti-Fc-mutant antibody, and streptavidin-β-galactosidase (SBG (Quanterix Corporation)) then react in this order. Finally, the substrate (RGB) of β-galactosidase is added to the reactants, and its luminescence is measured.

[0321] Example 1

[0322] Preparation of antibodies containing a constant region, wherein the constant region is contained in the Fc region, and expression and purification of multiple mutant antibodies.

[0323] Antibodies containing a constant region, which includes multiple mutations in the Fc region, were expressed using the FreeStyle293 expression system. The constant region used (SEQ ID NO:1) is referred to as SG115 in WO2016098356A1. Harvested cell culture medium (HCCF) was purified using rprotein A resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex 200 pg, GE). During SEC, the buffer was replaced with 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).

[0324] Papain digestion

[0325] For papain digestion, we used the Pierce Fab preparation kit (Pierce, catalog number 44985). The papain digestion process is described below.

[0326] - Adjust the antibody concentration to 8.0 mg / mL using digestion buffer.

[0327] Add 0.5 mL of antibody solution to a centrifuge column containing balanced papain resin. Place the top cap and bottom stopper on the centrifuge column.

[0328] - Incubate the digestion reaction solution at 37°C on a rotary instrument for 15 hours.

[0329] - After incubation, remove the bottom cap and place the centrifuge column into a microcentrifuge tube. Centrifuge the column at 5000 x g for one minute.

[0330] Wash the resin with 0.5 mL of Dulbecco's PBS (-). Place the column in a microcentrifuge tube. Centrifuge the column at 5000 x g for 1 minute.

[0331] - Combine the solutions from steps 4 and 5 to form the digestion fraction. The total volume of one column is 1.0 mL.

[0332] Purification of Fc fragments

[0333] The papain-digested sample was purified using r-protein A resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex 200 pg, GE). During the SEC process, whole IgG (undigested molecules) was removed and the buffer was replaced with Dulbecco's PBS (-).

[0334] Example 2

[0335] Generate antibodies that recognize mutations in SG115

[0336] Antibodies that recognize mutations in SG115 are called "anti-SG115 antibodies" and are prepared, selected, and determined as described below.

[0337] Ten-week-old NZW rabbits were intradermally immunized with the Fc fragment of SG115 (50-100 μg / dose / rabbit). The administration was repeated five times over two months, after which blood was collected from the immunized rabbits. Antigen-specific B cells were sorted using a cell sorter according to the procedure described in WO2016098356A1, then plated and cultured. Following culture, the B cell culture supernatant was collected for further analysis, and the precipitate was cryopreserved.

[0338] The ability to bind SG115 was assessed by ELISA using B cell culture supernatant. We tested binding to five modified IgG heavy chain constant regions to evaluate binding specificity: 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). Sequence alignments of these five constant regions are shown below. Figure 1 As shown.

[0339] A total of 10,560 B cell lines were screened for binding to five modified IgG heavy chain constant regions. From these, 186 cell lines were selected and named SKA0001-SKA0186. These lines bound SG115 but not G1m or G4d, and also bound SG115v1 and / or Sg115v2. RNA from the selected cell lines was purified from cryopreserved cell pellets using the ZR-96 Quick-RNA Kit (ZYMO RESEARCH, catalog number R1053). DNA encoding the antibody heavy chain variable region from the selected cell 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 antibody was analyzed in FreeStyle... TMThe gene was expressed in 293-F cells (Invitrogen) and purified from the culture supernatant. Twelve clones were selected through further evaluation based on binding ability and specificity in ELISA, and 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. Figure 2-1 and 2-2 The VH and VL sequences of these 12 antibodies are listed in Table 1.

[0340] [Table 1]

[0341]

[0342] Example 3

[0343] Detection of SG115 in samples using anti-SG115 antibody

[0344] To detect antibodies containing all or part of the mutated Fc region of SG115 in biological samples (hereinafter also referred to as "Fc-mutant antibodies" or "multiple Fc-mutant antibodies"), the effectiveness of the aforementioned 12 monoclonal antibodies (hereinafter also referred to as "anti-Fc-mutant antibodies" or "multiple anti-Fc-mutant antibodies") was evaluated. A specific anti-human C5 antibody containing SG115 was used as a model for the Fc-mutant antibodies in Examples 3-5, and is referred to below as "anti-hC5 antibody".

[0345] Measurement Procedure

[0346] Each well of a 96-well immunoassay plate 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 OD was measured. The plate was washed between steps.

[0347] Antibody selection reactivity test

[0348] Twelve anti-Fc mutant antibodies were tested. Anti-hC5 antibodies containing SG115 were diluted with pooled human serum and measured using twelve candidate rabbit anti-Fc mutant antibodies. The signal-to-noise ratio (SNR) was calculated. The measured OD values ​​are listed in Table 2. Three candidates (a total of six candidates) were selected from each epitope type for selective testing (Table 2). Specifically, SKA0009, SKA0052, and SKA0127 were selected as antibodies specifically binding to SG115v1, and SKA0117, SKA0141, and SKA0171 were selected as antibodies specifically binding to SG115v2.

[0349] [Table 2]

[0350]

[0351] Selectivity test for antibody selection

[0352] Ten individual serum and calibration curve samples, with and without spiked anti-hC5 antibodies, were measured using six candidate antibodies as capture reagents. Without spiked anti-hC5 antibodies, the measured concentrations of all individual samples were below the limit of quantitation (BLQ) for any given rabbit anti-Fc mutant antibody. With spiked anti-hC5 antibodies, the relative error (RE) of the measured concentrations of all individual samples was within + / - 20% for any given rabbit anti-Fc mutant antibody (Table 3).

[0353] [Table 3]

[0354]

[0355] BLQ: Below the limit of quantitation

[0356] Selected antibody

[0357] Based on the results of the reactivity and selectivity tests, SKA0141, which has the highest signal-to-noise ratio, was selected.

[0358] Example 4

[0359] Methods for assessing and measuring anti-hC5 antibodies in human serum (Fc mutant antibody detection assay)

[0360] Determination methods

[0361] A 96-well immunoassay plate was coated with rabbit anti-Fc mutant antibody (SKA0141) and blocked with blocking buffer. Diluted serum samples, including 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 OD was measured. The plate was washed between steps.

[0362] Method Evaluation

[0363] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) ranged from -16.3% to -5.1% and 1.6% to 4.4%, respectively (Table 4). Inter-batch accuracy (RE) and precision (CV) ranged from -10.1% to -4.0% and 2.7% to 6.9%, respectively (Table 5).

[0364] [Table 4]

[0365]

[0366] [Table 5]

[0367]

[0368] Selectivity was tested. Without spiked anti-hC5 antibody, the measured concentrations of all individual samples were BLQ. With spiked anti-hC5 antibody, the REs of the measured concentrations of all individual samples ranged from -15.2% to 2.2% (Table 6).

[0369] [Table 6]

[0370]

[0371] 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)

[0372] [Table 7]

[0373]

[0374] ALQ: Above the limit of quantitation

[0375] Interference from C5 was tested. No interference from C5 was observed (Table 8).

[0376] [Table 8]

[0377]

[0378] A method for measuring Fc mutant antibodies in human serum using anti-Fc mutant antibodies has been established. The assay protocol is as follows: Figure 3 As shown.

[0379] Example 5

[0380] We also attempted to develop an assay for detecting antigens in biological samples that are recognized by Fc mutant antibodies. Anti-hC5 antibodies were also used as a model for Fc mutant antibodies in this evaluation.

[0381] Evaluation of methods for measuring C5 in human serum (antigen detection assay)

[0382] Determination methods

[0383] A 96-well immunoassay plate was coated with rabbit anti-Fc monoclonal antibody and blocked with blocking buffer. Diluted serum samples were added to each well of the plate. Anti-hC5 antibody was added to each well of the plate. Mouse anti-Fc mutant antibody, obtained by replacing the Fc region in SKA0141 with the mouse Fc region, was added to each well of the plate, followed by anti-mouse-POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate, and OD was measured. The plate was washed between steps.

[0384] Method Evaluation

[0385] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) ranged from -8.2% to 4.2% and 2.3% to 6.2%, respectively (Table 9). Inter-batch accuracy (RE) and precision (CV) ranged from -6.8% to 1.3% and 3.5% to 6.2%, respectively (Table 10).

[0386] [Table 9]

[0387]

[0388] * Recombinant human C5 was spiked into serum that had been depleted of C5.

[0389] Combined human serum (endogenous human C5). Concentrations are the average measured concentrations in inter-batch reproducibility tests.

[0390] Recombinant human C5 was spiked into the combined human serum. The concentration was the sum of endogenous human C5 concentration (89.1 μg / mL) and spiked recombinant human C5 concentration (65.0 μg / mL).

[0391] [Table 10]

[0392]

[0393] * Recombinant human C5 was spiked into serum that had been depleted of C5.

[0394] Combined human serum (endogenous human C5). Concentrations are the average measured concentrations in inter-batch reproducibility tests.

[0395] Recombinant human C5 was spiked into the combined human serum. The concentration was the sum of endogenous human C5 concentration (89.1 μg / mL) and spiked recombinant human C5 concentration (65.0 μg / mL).

[0396] Parallelism was tested. Ten individual serum samples were serially diluted from 325 to 2600 times and measured. The measured concentrations were recovered at any dilution factor (Table 11).

[0397] [Table 11]

[0398]

[0399] Dilution linearity was tested. C5 was measured at a dilution factor of 26,000 to 1130 mg / mL, and no pre-band effect was observed. (Table 12)

[0400] [Table 12]

[0401]

[0402] ALQ: Above the limit of quantitation

[0403] Interference from the anti-hC5 antibody was tested. No interference from the anti-hC5 antibody was observed (Table 13).

[0404] [Table 13]

[0405]

[0406] A method for measuring antigens in human serum using anti-Fc mutant antibodies has been established. The protocol for this assay is as follows: Figure 4 As shown.

[0407] Example 6

[0408] Methods for evaluating and measuring anti-IL-8 antibodies in human plasma (Fc mutant antibody detection assay)

[0409] Determination methods

[0410] A specific anti-human IL-8 antibody, including a modified IgG heavy chain constant region (wherein the modified IgG heavy chain constant region contains a partial mutation in the SG115 Fc region) (SEQ ID NO: 110), was used as a model of the Fc-mutant antibody in Examples 6-8 and is referred to as "anti-IL-8 antibody".

[0411] A 96-well immunoassay plate 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 Immuno Research Inc.). Finally, POD substrate was added to each well of the plate and OD was measured. The plate was washed between steps.

[0412] Method Evaluation

[0413] Reproducibility was tested. Known concentrations of anti-IL-8 antibody were measured (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)). Intra-assay accuracy (RE) and precision (CV) ranged from -14.2% to -9.7% and 4.9% to 7.3%, respectively (Table 14).

[0414] [Table 14]

[0415]

[0416] Inter-batch accuracy (RE) and precision (CV) ranged from -10.3% to -6.6% and from 7.0% to 10.1%, respectively (Table 15).

[0417] [Table 15]

[0418]

[0419] Selectivity was tested. Without anti-IL-8 antibody spikes (SEL-O or SEL-EM-O), the measured concentrations for all individual samples were BLQ. With anti-IL-8 antibody spikes (50.0 ng / mL (SEL-LL or SEL-EM-LL)), the RE values ​​for the measured concentrations for all individual samples ranged from -23.2% to -4.3% (Table 16).

[0420] [Table 16]

[0421]

[0422] *Below the lower limit of quantification

[0423] Dilution linearity was tested. Anti-IL-8 antibody at a dilution factor of 10,000 was measured at 1.6 mg per mL, and no prozone effect was observed (Table 17).

[0424] [Table 17]

[0425]

[0426] Quantitative range: 1.00 ng / mL to 64.0 ng / mL in the assay wells.

[0427] *Higher than the upper limit of quantification

[0428] Interference from IL-8 was tested. Up to 50.0 ng / mL of IL-8 did not interfere with the assay at an anti-IL-8 antibody concentration of 2400 ng / mL, and up to 1.00 ng / mL of IL-8 did not interfere with the assay at an anti-IL-8 antibody concentration of 50.0 ng / mL (Table 18).

[0429] [Table 18]

[0430]

[0431] The effectiveness of the method for measuring anti-IL-8 antibodies in plasma has been confirmed.

[0432] Example 7

[0433] Evaluation of methods for measuring IL-8 in human plasma using ELISA (antigen detection assay)

[0434] Determination methods

[0435] After blocking with blocking buffer, 96-well streptavidin immunoassay plates were coated with biotinylated mouse anti-IL-8 monoclonal antibody. The anti-IL-8 antibody was added to diluted plasma samples (reaction solution) in the 96-well polypropylene plates. After incubation, the reaction solution was transferred to each well of the streptavidin plate. A rabbit anti-Fc mutant antibody (SKA0001) was added, followed by anti-rabbit HRP (Southern Biotechnology Associates Inc.). Finally, POD substrate was added to each well of the plate, and OD was measured. The streptavidin plates were washed between steps.

[0436] Method Evaluation

[0437] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) ranged from -6.4% to -1.9% and from 1.5% to 2.9%, respectively (Table 19).

[0438] [Table 19]

[0439]

[0440] Conversion values ​​outside the quantitative range (below 100 pg / mL in plasma, above 3200 pg / mL in plasma) are included in the calculation.

[0441] The results of intraday reproducibility were used as the results of the first determination of interday reproducibility.

[0442] CV: Coefficient of Variation

[0443] RE: Relative Error

[0444] Inter-batch accuracy (RE) and precision (CV) ranged from -8.0% to 2.1% and 3.0% to 5.2%, respectively (Table 20).

[0445] [Table 20]

[0446]

[0447] Dilution linearity was tested. 1 mg IL-8 (spiked) per mL could be measured at a dilution factor of 20,000, and no preband effect was observed (Table 21).

[0448] [Table 21]

[0449] Spiked concentration in combined human plasma: 1000 ng / mL

[0450]

[0451] ALQ: Above the upper limit of quantitation (>640 pg / mL)

[0452] CV: Coefficient of Variation

[0453] RE: Relative Error

[0454] Interference from anti-IL-8 antibody was tested. No interference from anti-IL-8 antibody (100 μ / mL in plasma) was observed (Table 22).

[0455] [Table 22]

[0456] Batch number 190702

[0457]

[0458] Difference % = (Transformation value of sample with IS added - Transformation value of sample without IS) / Transformation value of sample without IS x 100

[0459] IS: Interfering substance

[0460] ND: Not detected

[0461] Underline: Below the lower limit of quantification (<100 pg / mL in plasma)

[0462] NA: Not applicable

[0463] The effectiveness of the method for measuring IL-8 in human plasma using ELISA has been confirmed.

[0464] Example 8

[0465] Evaluation of the method for measuring IL-8 in human plasma using Simoa (registered trademark) (Simoa (registered trademark) assay)

[0466] Measurement Procedure

[0467] The assay was performed automatically using a Simoa (registered trademark) system (Quanterix Corporation). Diluted sample, anti-IL-8 antibody, and mouse anti-IL-8 monoclonal antibody coated on beads were mixed. Beads were loaded into the microwells of the array disk. Biotinylated anti-Fc mutant antibody (SKA0028) was added to the disk, followed by streptavidin-β-galactosidase, SBG (Quanterix Corporation). Finally, the β-galactosidase substrate RGB was added, and fluorescence intensity was measured.

[0468] Method Evaluation

[0469] Reproducibility was tested. Intra-batch precision (CV) ranged from 1.3% to 14.3% (Table 23).

[0470] [Table 23]

[0471]

[0472] Inter-batch precision (CV) ranged from 8.6% to 24.7% (Table 24).

[0473] [Table 24-1]

[0474]

[0475] [Table 24-2]

[0476]

[0477] Parallelism was tested. Three separate plasma samples were serially diluted from 20 to 40 times and measured. The measured concentrations were recovered at any dilution factor (Table 25).

[0478] [Table 25]

[0479]

[0480] Dilution linearity was tested. IL-8 of 3.48 mg / mL could be measured at a dilution factor of 50,000, and no prozone effect was observed (Table 26).

[0481] [Table 26]

[0482]

[0483] -:not applicable

[0484] *: The theoretical concentration is calculated by considering the endogenous IL-8 concentration in plasma (batch number PLA022A100E001) determined in the inter-operation precision test.

[0485] Interference from anti-IL-8 antibody was tested. No interference from anti-IL-8 antibody (100 μg / mL in plasma) was observed (Table 27).

[0486] [Table 27]

[0487]

[0488] The effectiveness of the Simoa (registered trademark) method for measuring IL-8 in human plasma has been confirmed.

[0489] Example 9

[0490] Affinity assessment of the anti-Fc-mutant antibody that selectively binds to SG115v1 against the anti-hC5 antibody.

[0491] Anti-Fc-mutant antibodies (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127) exhibiting selective binding to SG115v1 at pH 7.4 target the K-type anti-hC5 antibody. D The values ​​were determined at 25°C using a Biacore T200 instrument (GE Healthcare).

[0492] Mouse anti-rabbit IgG (Fc) antibody (hereinafter referred to as anti-rabbit IgG) (Abbexa) was immobilized onto flow cells (FC) 1 and 2 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare). For immobilization of the anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the run buffer. After immobilization, the run buffer was changed to phosphate buffer (50 mM phosphate buffer containing 150 mM NaCl and 0.05 w / v % P-20, pH 7.4). Each anti-Fc-mutant antibody was captured onto FC2 of the sensor chip by the anti-rabbit IgG. The amount of anti-Fc-mutant antibody to be captured was adjusted to achieve a resonance unit (RU) number of 100. Anti-hC5 antibody was injected at 0, 50, 100, 200, 400, and 800 nM at a flow rate of 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. K D The values ​​were obtained using Biacore T200 assessment software version 2.0 (GE Healthcare). Association rate (ka), dissociation rate (kd), and dissociation constant (K) were also measured. D The results are shown in Table 28.

[0493] [Table 28]

[0494]

[0495] *: Due to the slow dissociation speed, data reliability may be low.

[0496] Example 10

[0497] Affinity assessment of the anti-Fc-mutant antibody that selectively binds to SG115v1 against the anti-IL-8 antibody.

[0498] To confirm the binding affinity of the anti-Fc-mutant antibody, which exhibits selective binding to SG115v1, the anti-Fc-mutant antibody was tested at pH 7.4 against the K+ of the anti-IL-8 antibody. D The values ​​were determined at 25°C using a Biacore T200 instrument (GE Healthcare). The Fc region sequence of the anti-IL-8 antibody is highly similar to the Fc region sequence of the anti-hC5 antibody.

[0499] Anti-rabbit IgG (Abbexa) was immobilized onto FC1 and FC2 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare). For immobilization of the anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the run buffer. After immobilization, the run buffer was changed to phosphate buffer (50 mM phosphate buffer containing 150 mM NaCl and 0.05 w / v % P-20, pH 7.4). Each anti-Fc-mutant antibody was captured onto FC2 of the sensor chip by anti-rabbit IgG. The amount of anti-Fc-mutant antibody to be captured was adjusted to achieve a resonance unit (RU) count of 100. Anti-IL-8 antibody was injected at 0, 100, 400, and 800 nM at a flow rate of 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. D The values ​​were obtained using Biacore T200 assessment software version 2.0 (GE Healthcare).

[0500] ka, kd and K D Listed in Table 29. Although the amino acid at position 239 in the anti-hC5 antibody is mutated from Ser to Lys according to the EU numbering system, the corresponding amino acid at the same position in the anti-IL-8 antibody remains unchanged. In this case, the anti-Fc-mutant antibody can bind to the anti-IL-8 antibody. This means that the two mutations common to both the anti-hC5 and anti-IL-8 antibodies in SG115v1, namely L235R and G236R (both positions are numbered according to the EU numbering system), are essential for the selective binding of the anti-Fc-mutant antibody to SG115v1.

[0501] [Table 29]

[0502]

[0503] *: Due to the slow dissociation speed, data reliability may be low.

[0504] Example 11

[0505] The anti-Fc-mutant antibody, which selectively binds to SG115v2, targets anti-DENV. Affinity assessment of E protein antibody.

[0506] Anti-Fc mutant antibodies (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171) that selectively bind to SG115v2 at pH 7.4 target anti-DENV E protein antibodies (which include a modified IgG heavy chain constant region (SEQ ID NO: 116) as an Fc mutant antibody). DThe values ​​were determined at 25°C using a Biacore T200 instrument (GE Healthcare). Anti-rabbit IgG was immobilized onto FC3 and FC4 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare). For anti-rabbit IgG immobilization, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the run buffer. After immobilization, the run buffer was changed to phosphate-pH 7.4 buffer. Each antibody was captured by anti-rabbit IgG onto FC4 of the sensor chip. The amount of anti-Fc-mutant antibody to be captured was adjusted to achieve a resonance unit (RU) number of 100. Anti-DENV E protein antibody was injected at 0, 12.5, 50, and 400 nM at a flow rate of 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. D The values ​​were obtained using Biacore T200 assessment software version 2.0 (GE Healthcare). ka, kd, and K D As shown in Table 30.

[0507] [Table 30]

[0508]

[0509] *: Due to the slow dissociation speed, data reliability may be low.

[0510] Example 12

[0511] SKA0016 and SKA0117 were used as capture molecules to evaluate the affinity of anti-hC5 antibodies for human C5.

[0512] The potassium content of anti-hC5 antibody against human C5 at pH 7.4 was determined using a Biacore T200 instrument (GE Healthcare) at 37°C. D Values. SKA0016 was immobilized onto FC1 and 2 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare), and SKA0117 was immobilized onto FC3 and 4 of the CM5 sensor chip. For immobilization of SKA0016 and SKA0117, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the run buffer. After immobilization, the run buffer was changed to phosphate pH 7.4 buffer. Anti-hC5 antibodies were captured onto FC2 and FC4 of the sensor chip by SKA0016 and SKA0117. The amount of anti-hC5 antibody to be captured was adjusted to achieve a resonance unit (RU) number of 35. Human C5 was injected at 0, 2, 4, 8, 16, and 32 nM at a flow rate of 10 μL / min. 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.D The values ​​were obtained using Biacore T200 assessment software, version 2.0 (GE Healthcare). ka, kd, and K D Listed in Table 31.

[0513] [Table 31]

[0514] Capture molecules ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> SKA0016 <![CDATA[6.33×10 5 ]]> <![CDATA[1.13×10 -4 ]]> <![CDATA[1.78×10 -10 ]]> SKA0117 <![CDATA[6.51×10 5 ]]> <![CDATA[1.23×10 -4 ]]> <![CDATA[1.88×10 -10 ]]>

[0515] Example 13

[0516] Using SKA0016 and SKA0117 as immobilization molecules, the pH-dependent interaction of hC5 antibody with human C5 was antagonized. Conduct a qualitative analysis.

[0517] The pH-dependent interaction between anti-hC5 antibody and human C5 at pH 7.4 and pH 6.0 was evaluated using a Biacore T200 instrument (GE Healthcare) at 37°C. The anti-hC5 antibody was captured onto FC2 and FC4 of the CM5 chip prepared in Example 12. The amount of anti-hC5 antibody to be captured was adjusted to achieve a resonance unit (RU) number of 35. To confirm the association between the anti-hC5 antibody and human C5 at pH 7.4, 32 nM of human C5 was injected into all FCs in a phosphate pH 7.4 buffer. The dissociation phase was then monitored in either a phosphate pH 7.4 buffer or a phosphate pH 6.0 buffer (50 mM phosphate buffer containing 150 mM NaCl and 0.05 w / v % P-20, pH 6.0) as a running buffer. Following the dissociation phase, the sensor chip was regenerated by injecting 100 mM Gly-HCl, pH 2.0, followed by 25 mM NaOH, both at a flow rate of 30 μL / min. The pH-dependent interaction between the anti-hC5 antibody and human C5 was analyzed using Biacore T200 evaluation software version 2.0 by comparing the dissociation phases of the sensor maps at pH 7.4 and pH 6.0. The sensor map at FC2 was subtracted from FC1, and the sensor map at FC4 was subtracted from FC3. Each sensor map was normalized by adjusting the human C5 binding response (5 seconds before the end of human C5 injection) to a value of "100".

[0518] Regardless of the captured molecules, human C5 dissociates from anti-hC5 antibodies at pH 6.0 faster than at pH 7.4. Figure 6 (The anti-hC5 antibody was immobilized by (a)SKA0016 and (b)SKA0117). Therefore, it is believed that both SKA0016 and SKA0117 can effectively monitor the pH-dependent interaction between the anti-hC5 antibody and human C5.

[0519] Example 14

[0520] The binding between the human Fc receptor (hFcRn) and the anti-hC5 antibody captured by SKA0016 was evaluated.

[0521] The binding of human FcRn to anti-hC5 antibody captured by SKA0016 at pH 6.0 was assessed using a Biacore T200 instrument (GE Healthcare). The anti-hC5 antibody was captured onto FC2 by SKA0016, which was immobilized onto the CM5 chip using the same procedure as in Example 9. Phosphate-pH 6.0 buffer was used as the run buffer. The amount of anti-hC5 antibody to be captured was adjusted so that the number of resonance units (RUs) was 400. hFcRn was injected in a single-cycle kinetic manner at 0, 26.3, 52.5, 105, 210, and 420 nM at a rate of 10 μL / min. 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 increased hFcRn binding response was confirmed using Biacore T200 software version 2.0 (GE Healthcare).

[0522] Figure 7 Sensing plots for FC1 (dashed line) and FC2 (solid line) are shown. The FC2 sensing plot (solid line) shows that the binding response of hFcRn increases in a concentration-dependent manner. SKA0016 does not interrupt the binding between hFcRn and the anti-hC5 antibody. However, hFcRn appears to bind to the Fc region of SKA0016, as the FC1 sensing plot also shows an increased binding response (dashed line). This undesirable binding of human FcRn to the capture molecule can be resolved by introducing an amino acid substitution that disables binding to human FcRn into SKA0016.

Claims

1. An isolated antibody that specifically binds to a modified IgG heavy chain constant region, said modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG, or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG. The constant region of naturally occurring human IgG is any one of the following: the IgG1 constant region composed of the amino acid sequence of SEQ ID NO: 106, the IgG2 constant region composed of the amino acid sequence of SEQ ID NO: 107, the IgG3 constant region composed of the amino acid sequence of SEQ ID NO: 108, and the IgG4 constant region composed of the amino acid sequence of SEQ ID NO:

109. The modified IgG heavy chain constant region contains Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, all positions being numbered according to the EU numbering system. The isolated antibody comprises: The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO:

30.

2. The antibody according to 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 IgG4.

3. The antibody according to claim 1, 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, wherein all positions are numbered according to the EU numbering system.

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

5. A composition for detecting or capturing polypeptides in a sample, wherein the composition comprises the antibody according to any one of claims 1 to 4.

6. The composition of claim 5, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG, 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, all positions according to the EU numbering system, and wherein the constant region of naturally occurring human IgG is any one of the following: 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.

7. The composition according to claim 5, wherein the polypeptide comprises an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO:

105.

8. The composition of claim 7, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region comprising an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO:

105.

9. An in vitro method for detecting or capturing peptides in a sample, wherein the method comprises contacting the sample with an antibody according to any one of claims 1 to 4 or with a composition according to any one of claims 5 to 8.

10. The method of claim 9, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region being derived from any constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from naturally occurring human IgG, 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, all positions according to the EU numbering system, and wherein the constant region of naturally occurring human IgG is any one of the following: 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.

11. The method of claim 9, wherein the polypeptide comprises an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO:

105.

12. The method of claim 11, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region comprising an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO:

105.

13. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 4.

14. A host cell comprising the nucleic acid of claim 13.

15. A method for producing antibodies, comprising culturing the host cells of claim 14 to produce antibodies.

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