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

Abstract

The present disclosure provides antibodies for detecting or capturing a polypeptide in a sample, compositions, and methods for detecting or capturing a polypeptide in a sample.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610323658.9 (22) Application Date 2020.04.17 (30) Priority Data 2019-079740 2019.04.19 JP (62) Divisional Application Data 202080019187.3 2020.04.17 (71) Applicant Chugai Pharmaceutical Co., Ltd. Address Japan (72) Inventors Eriko Matsuda, Mitsuko Shibuya, Masao Nishidana (74) Patent Agency Zhongke Patent & Trademark Agency Co., Ltd. 11021 Patent Attorney Gao Lina (51) Int.Cl. C07K 16 / 42 (2006.01) C12N 15 / 13 (2006.01) C12P 21 / 02(2006.01) G01N 33 / 68(2006.01) G01N 33 / 543(2006.01) (54) Title of Invention Antibodies and compositions for detecting or capturing peptides in a sample, and methods for detecting or capturing peptides in a sample (57) Abstract This disclosure provides antibodies, compositions, and methods for detecting or capturing peptides in a sample. Claims 3 pages, Description 48 pages, Sequence Listing (electronic publication), Drawings 8 pages, CN 122145637 A 2026.06.05 CN 1 22 14 56 37 A 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, wherein said isolated antibody comprises any one of the following: (1) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20; (2) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 21; (3) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22; (4) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 11, and the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 23; (5) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 12,(6) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 13 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 25; (7) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 14 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 26; (8) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 15 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 27; (9) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 16 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 28; (10) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 17 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 29; and (11) The heavy chain variable region composed of the amino acid sequence of SEQ ID NO: 19 and the light chain variable region composed of the amino acid sequence of SEQ ID NO: 28. 1. A light chain variable region consisting of an amino acid sequence of 31. 2. The antibody of claim 1, wherein the antibody substantially does not bind 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. 3. The antibody of claim 1, wherein the modified IgG heavy chain constant region is derived from a chimeric constant region obtained from the constant regions of naturally occurring human IgG1 and IgG4. 4. The antibody of claim 1, 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 numbered according to the EU numbering system. 5. The antibody of claim 4, wherein the modified IgG heavy chain constant region further comprises a threonine residue at position 436, the position being numbered according to the EU numbering system. 6. A composition for detecting or capturing a polypeptide in a sample, wherein the composition comprises the antibody of any one of claims 1 to 5. Claims 1 / 3 page 2 CN 122145637 A 7. The composition of claim 6, 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.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 being numbered according to the EU numbering system. 8. The composition according to claim 6, wherein the polypeptide comprises any one of the following: the amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, the amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, the amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and the amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO: 118. 9. The composition of claim 8, wherein the polypeptide comprises a modified IgG heavy chain constant region, said modified IgG heavy chain constant region comprising any one of the following: an amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, an amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO: 118. 10. An in vitro method for detecting or capturing a polypeptide in a sample, wherein the method comprises contacting the sample with an antibody as described in any one of claims 1 to 5 or a composition as described in any one of claims 6 to 9. 11. The method of claim 10, wherein the polypeptide comprises 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 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 being numbered according to the EU numbering system. 12. The method of claim 10, wherein the polypeptide comprises any one of the following: the amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, the amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, or the amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105.13. The method of claim 12, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region comprising any one of the following: an amino acid sequence comprising RRGPK as shown in SEQ ID NO: 104, an amino acid sequence comprising RRGPK as shown in SEQ ID NO: 117, an amino acid sequence comprising LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence comprising LHEALHAHTTRKE as shown in SEQ ID NO: 118. 14. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 5. 15. A host cell comprising the nucleic acid of claim 14. 16. A method of preparing an antibody comprising culturing the host cell of claim 15 to prepare the antibody. 17. A method for measuring the concentration of a first antibody in a sample, wherein the first antibody is capable of binding a first epitope of an antigen, wherein the sample comprises the first antibody and the antigen, and wherein the method comprises (A) contacting the sample with a plate or bead immobilized with a second antibody, (B) after (A) contacting a solution comprising the antigen but excluding the first antibody and the second antibody with the plate or bead, and (C) after (B) detecting the antigen captured on the plate or bead by the second antibody and the first antibody using a third antibody, wherein the first antibody 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. 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 being numbered according to the EU numbering system; wherein the second antibody is the antibody of any one of claims 1 to 5, and wherein 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 having an amino acid sequence different from that of the first antibody and the second antibody IgG heavy chain constant region. 18. A method for measuring 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: (D) contacting the sample with a plate or bead immobilized with a third antibody,(E) Following (D), a solution comprising the first antibody but excluding the antigen and the third antibody is contacted with the plate or the bead, and (F) the first antibody captured on the plate or bead by the third antibody and the antigen is detected using a second antibody, wherein the first antibody 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 being numbered according to the EU numbering system. Wherein, the second antibody is the antibody according to any one of claims 1 to 5, and has an IgG heavy chain constant region whose amino acid sequence is different from that of the first antibody and the third antibody, and wherein the third antibody is capable of binding a second epitope of the antigen that is different from the first epitope. Claims 3 / 3 Page 4 CN 122145637 A Antibody and composition for detecting or capturing polypeptides in a sample, and a method for detecting or capturing polypeptides in a sample

[0001] This application is a divisional application of the application filed on April 17, 2020, with international application number PCT / JP2020 / 016813, entering the Chinese national phase, application number 202080019187.3, entitled "Antibody and composition for detecting or capturing polypeptides in a sample, and a method for detecting or capturing polypeptides in a sample". Technical Field

[0002] This invention relates to antibodies and compositions for detecting or capturing peptides in samples, and methods for detecting or capturing peptides in samples. Background Art

[0003] Hybridoma technology has enabled the production of monoclonal antibodies, which have been widely used in many scientific fields (NPL1). Following this technological achievement, further efforts have been made in the field of therapeutic and diagnostic antibodies. It has been 30 years 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 the standard therapeutic molecule and are used in a variety of disease areas, such as cancer, autoimmune diseases, respiratory diseases, infectious diseases, and neurological diseases (NPL3).

[0004] To increase the value of therapeutic antibodies, many different types of engineered Fc modifications have been identified to improve function.For example, those Fc modifications used 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).

[0005] 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.

[0006] Citation List

[0007] Patent Documents

[0008] [PTL1] WO2017072210A1

[0009] Non-Patent Documents

[0010] [NPL1] Kohler, G. et al., Nature 256:495-497 (1975)

[0011] [NPL2] Reichert, J. M. et al., Curr. Pharm. Biotechnol. 9:423-430 (2008)

[0012] [NPL3] Lagasse HAD et al. F1000Research 2017, 6 (F1000 Faculty Rev): 113

[0013] [NPL4] Mimoto et al., Curr. Pharm. Biotechnol. 17:1298-1314 (2016)

[0014] [NPL5] Yu et al., Antimicrob Agents Chemother. 61 (2016) Summary of the Invention

[0015] We provide antibodies comprising modified IgG heavy chain constant regions, said modified IgG heavy chain constant regions being derived from any constant region of naturally occurring human IgG or from chimeric constant regions derived from at least two constant regions selected from naturally occurring human IgG. Antibodies include, for example, satralizumab, nemolizuma b, emicizuma b, SKY59 (crovalimab), AMY109, and GYM329. For example, the modified IgG heavy chain constant region in one of them contains at least one amino acid selected from Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, and Ser at position 330.The group consisting of 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 antibody, and methods of using the antibody.

[0016] Specifically, the present invention relates to the following [1] through

[25] .

[0017] [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 natural 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).

[0018] [2] An antibody according to [1], wherein the antibody substantially does not bind to any constant region of naturally occurring human IgG or the chimeric constant region obtained from at least two constant regions selected from natural human IgG.

[0019] [3] The antibody according to [1] or [2], wherein the constant region in the naturally occurring human IgG is 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.

[0020] [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 in the naturally occurring human IgG1 and IgG4.

[0021] [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).

[0022] [6] The antibody according to any one of [1] to [5],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).

[0023] [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).

[0024] [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).

[0025] [9] The antibody according to any one of [1] to [8] comprises any one of the following (a) to (f):

[0026] (a) a variable region comprising

[0027] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 33, specification 2 / 48 page 6 CN 122145637 A

[0028] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 45,

[0029] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 57,

[0030] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 69,

[0031] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 81, and

[0032] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 93;

[0033] (b) a variable region comprising

[0034] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 34,

[0035] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 46,

[0036] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 58,

[0037] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 70,

[0038] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 82, and

[0039] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 94;

[0040] (c) a variable region comprising

[0041] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 37,

[0042] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 49,

[0043] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 61,

[0044] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 73,

[0045] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 85, and

[0046] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 97;

[0047] (d) a variable region comprising

[0048] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 38,

[0049] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 50,

[0050] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 62,

[0051] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 74,

[0052] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 86. The amino acid sequence, and

[0053] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 98;

[0054] (e) a variable region, which includes

[0055] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 39,

[0056] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 51,

[0057] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63,

[0058] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 75,

[0059] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 87, and

[0060] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 99; and

[0061] (f) a variable region, which includes

[0062] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 41,

[0063] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 53,

[0064] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 65,

[0065] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 77,

[0066] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 89, and specification page 3 / 48, CN 122145637 A

[0067] HVR-L3,It contains the amino acid sequence of SEQ ID NO: 101.

[0068]

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

[0069]

[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).

[0070]

[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).

[0071]

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

[10] to

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

[0072] (g) a variable region comprising

[0073] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 32,

[0074] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 44,

[0075] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 56,

[0076] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 68,

[0077] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 80, and

[0078] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 92;

[0079] (h) a variable region comprising

[0080] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 92 ... The amino acid sequence of SEQ ID NO: 35,

[0081] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 47,

[0082] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 59,

[0083] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 71,

[0084] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 83, and

[0085] HVR-L3,It contains the amino acid sequence of SEQ ID NO: 95;

[0086] (i) a variable region comprising

[0087] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 36,

[0088] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 48,

[0089] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 60,

[0090] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 72,

[0091] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 84, and

[0092] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 96;

[0093] (j) a variable region comprising

[0094] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40,

[0095] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 96,

[0096] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 96,

[0097] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 40,

[0098] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 96,

[0099] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 96,

[0090] HVR-H1 ... The amino acid sequence of SEQ ID NO: 52,

[0096] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 64,

[0097] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 76,

[0098] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 88, and specification page 4 / 48 8 CN 122145637 A

[0099] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 100;

[0100] (k) Variable region, which includes

[0101] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 42,

[0102] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 54,

[0103] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 66,

[0104] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 78 The amino acid sequence,

[0105] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 90, and

[0106] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 102; and

[0107] (l) a variable region, which includes

[0108] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 43,

[0109] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 55,

[0110] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 67,

[0111] HVR-L1,It contains the amino acid sequence of SEQ ID NO: 79,

[0112] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 91, and

[0113] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 103.

[0114]

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

[13] .

[0115]

[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 natural 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).

[0116]

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

[15] .

[0117]

[17] According to the composition of

[16] , 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).

[0118]

[18] The composition according to

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

[0119]

[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 amino acid sequences consisting of RRGPK (SEQ ID NO: 104), RRGPS (SEQ ID NO: 117), LHEALHAHYTRKE (SEQ ID NO: 105), and LHEALHAHTTRKE (SEQ ID NO: 118). Specification 5 / 48 pages 9 CN 122145637 A

[0120]

[20] A method for detecting or capturing a polypeptide 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] .

[0121]

[21] According to the method of

[20] , 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 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).

[0122]

[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).

[0123]

[23] According to the method of

[22] , the polypeptide comprises a modified IgG heavy chain constant region.The modified IgG heavy chain constant region includes 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).

[0124]

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

[0125] (A) contacting the sample with a plate or bead immobilized with a second antibody,

[0126] (B) after (A) contacting the plate or bead with a solution comprising the antigen but excluding the first antibody and the second antibody, and

[0127] (C) after (B) detecting the antigen captured on the plate or bead by the second antibody and the first antibody using a third antibody, wherein

[0128] the first antibody 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 The 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 second antibody being any one of [1] to

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

[0129]

[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

[0130] (D) contacting the sample with a plate or bead immobilized with a third antibody,

[0131] (E) after (D) contacting the plate or bead with a solution comprising the first antibody but excluding the antigen and the third antibody, and

[0132] (F) detecting the first antibody captured on the plate or bead by the third antibody and the antigen using a second antibody, wherein

[0133] the first antibody comprises a modified IgG heavy chain constant region.The modified IgG heavy chain constant region is derived from any constant region of naturally occurring 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),

[0134] 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, and

[0135] the third antibody is capable of binding a second epitope of the antigen that is different from the first epitope.

[0136] [Figure 1] Figure 1 illustrates the sequence alignment of the five modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) described in Example 2. 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.

[0137] [Figure 2-1] Figures 2-1 and 2-2 illustrate the binding of 12 anti-SG115 antibodies to the five modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m, and G4d) in ELISA. 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.

[0138] [Figure 2-2] Figure 2-2 is a continuation of Figure 2-1.

[0139] [Figure 3] Figure 3 illustrates the assay protocol for Fc mutant antibodies.

[0140] [Figure 4] Figure 4 illustrates the assay protocol for antigen detection.

[0141] [Figure 5] Figure 5 illustrates the assay protocol for Simoa (registered trademark).

[0142] [Figure 6] Figure 6 illustrates a sensory map showing the dissociation of human C5 from anti-hC5 antibodies.The anti-hC5 antibody was captured by SKA0016 and SKA0117 at pH 7.4 and pH 6.0. Neither SKA0016 nor SKA0117 interfered with the pH-dependent interaction between the anti-hC5 antibody and human C5.

[0143] [Figure 7] Figure 7 illustrates the sensor map of the binding assay of human Fc receptor to anti-hC5 antibody captured by SKA0016. SKA0016 did not interrupt the binding between hFcRn and anti-hC5 antibody. Detailed Description

[0144] I. Definitions

[0145] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise stated, as used herein, “binding affinity” or “binding activity” refers to the intrinsic binding affinity that reflects the 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 by methods commonly known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0146] 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 with sufficient affinity to a specific type of modified IgG heavy chain constant region, 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 dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10⁻⁸ M or less, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M). In some embodiments, the antibody binding to the modified IgG heavy chain constant region binds to an epitope in the modified IgG heavy chain constant region.

[0147] The term “antibody” is used herein in the broadest sense and includes various antibody structures.This includes, but is 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.

[0148] The “class” of an antibody 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 which can be further subdivided 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 referred to as α, δ, ε, γ, and μ, respectively.

[0149] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical and / or bind the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during the preparation and production of monoclonal antibodies), which are typically present in small amounts. Unlike 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 interpreted 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.

[0150] The term “constant region” as used herein refers to a 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 consists of 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).

[0151] The term “Fc region” is used herein to define the C-terminal region of the immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes the native sequence Fc region and the variant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. Unless otherwise 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.

[0152] 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 Specification 8 / 48 pages 12 CN 122145637 A Immunology, 6th edition, WH Freeman and Co., page 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 using the VH or VL domains of antibodies that bind to antigens, thereby isolating antibodies that bind to specific antigens. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0153] “Frame” or “FR” refers to variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, the HVR and FR sequences in the VH (or VL) generally appear in the following order: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0154] 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 as described herein include:

[0155] (a) appearing at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), and 26–32 (H1).(a) Hypervariable rings at amino acid residues 53-55 (H2) and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0156] (b) CDRs 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));

[0157] (c) CDRs at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 Antigen contacts at (H2) and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0158] (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).

[0159] The “percentage (%) amino acid sequence identity” relative to the reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences are aligned and vacancies are introduced where necessary to obtain the maximum percentage sequence identity, and no conserved substitutions are considered as part of the sequence identity. The 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 the appropriate parameters for aligning the sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

[0160] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted to the US Copyright Office (Washington DC, 20559) along with user documentation.Its U.S. copyright registration number is TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from 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:

[0161] 100 multiplied by X / Y.

[0162] 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 where 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 are obtained using the ALIGN-2 computer program as described above.

[0163] "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 antibody to its antigen in a competitive assay. Exemplary competitive assays are provided herein.

[0164] II. Antibody

[0165] The antibody in this invention is a separate antibody that specifically binds to a modified IgG heavy chain constant region.

[0166] In one embodiment, the antibody substantially does not bind to constant regions in naturally occurring human IgG and 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 in 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. On the other hand, the binding activity of the antibody to the modified IgG heavy chain constant region is detectable in an enzyme-linked immunosorbent assay.

[0167] 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.For example, complete IgG1, IgG2, IgG3, and IgG4 antibodies, or other antibody classes or isotypes as defined herein.

[0168] A. Modified IgG Heavy Chain Constant Region

[0169] In one embodiment, 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. The constant regions of naturally occurring human IgG are 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.

[0170] In one embodiment, the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions are numbered according to the EU numbering system).

[0171] In a further embodiment, the modified IgG heavy chain constant region may be derived from a chimeric constant region obtained from constant regions in naturally occurring human IgG1 and IgG4. In a preferred embodiment, the constant region is derived from a chimeric constant region obtained from constant regions in naturally occurring human IgG1 and IgG4.

[0172] In a preferred embodiment, the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions are numbered according to the EU numbering system).

[0173] In one embodiment, the modified IgG heavy chain constant region may form a dimer, such as the heavy chain constant region in naturally occurring IgG, or may form a hemimer, such as the heavy chain constant region in monomer Fc reported in Ishino T. et al., J. Biol. Chem. 288: 16259-37 (2013).

[0174] In one embodiment, when the modified IgG heavy chain constant region is in the human modified IgG heavy chain, the human modified IgG heavy chain is selected from human modified IgG1, IgG2, IgG3 and IgG4 heavy chains, as specified in the specification 10 / 48 pages 14 CN 122145637 A.And the group consisting of their chimeric IgG heavy chains. In a preferred embodiment, the human IgG heavy chain is the human IgG1 heavy chain, the human IgG4 heavy chain, or a chimeric IgG heavy chain thereof.

[0175] B. Exemplary antibody with a specific modification that recognizes the CH2 region of the modified IgG heavy chain constant region

[0176] The Arg at position 235, the Arg at position 236, and the Lys at position 239 (all positions are numbered according to the EU numbering system) are those amino acids that are 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 of a chimeric constant region that corresponds to at least one of the constant regions of naturally occurring human IgG or is obtained from at least two constant regions selected from the constant regions of naturally occurring human IgG.

[0177] 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).

[0178] In a preferred embodiment, the modified IgG heavy chain constant region comprises 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).

[0179] 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 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,The amino acid sequences comprising: (ii) HVR-H2, comprising the amino acid sequences ...

[0180] 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 comprising 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,It contains the amino acid sequence of SEQ ID NO: 45, 46, 49, 50, 51 or 53; and (iii) HVR-H3, whose specification page 11 / 48, CN 122145637 A, contains the amino acid sequence of SEQ ID NO: 57, 58, 61, 62, 63 or 65.

[0181] In another aspect, the present invention provides an antibody containing at least one, at least two or all three VL HVR sequences 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. In one embodiment, the antibody comprises (iv) HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 69, 70, 73, 74, 75 or 77; (v) HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 93, 94, 97, 98, 99 or 101.

[0182] 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: 69, 70, 73, 74, 75, or 77. The amino acid sequence of 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.

[0183] In another aspect, the present invention provides an antibody,It 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; (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 selected from SEQ ID NO: 93, 94, 97, 98, 99 or 101.

[0184] In another aspect, the antibodies described herein include a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 comprises 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 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 the 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 by pyroglutamylation.

[0185] In another aspect,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 comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the antibody comprising 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.

[0186] 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 respectively in SEQ ID NO: 9, 10, 13, 14, 15 or 17 and SEQ ID NO: 21, 22, 25, 26, 27 or 29, 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 to pyroglutamic acid by pyroglutamylation.

[0187] In one aspect, an antibody is provided, wherein the antibody competes with an antibody comprising the following: (i) HVR-H1,(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.

[0188] 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, or 99. Or the amino acid sequence of 101.

[0189] 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 in the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO: 104) or RRGPS (SEQ ID NO: 117), the antibody comprising any one of the following (a) to (f):

[0190] (a) a variable region comprising

[0191] HVR-H1,It comprises the amino acid sequence of SEQ ID NO: 33,

[0192] HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 45,

[0193] HVR-H3, which comprises the amino acid sequence of SEQ ID NO: 57,

[0194] HVR-L1, which comprises the amino acid sequence of SEQ ID NO: 69,

[0195] HVR-L2, which comprises the amino acid sequence of SEQ ID NO: 81, and specification page 13 / 48 17 CN 122145637 A

[0196] HVR-L3, which comprises the amino acid sequence of SEQ ID NO: 93;

[0197] (b) a variable region comprising

[0198] HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 34,

[0199] HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 46,

[0200] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 58,

[0201] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 70,

[0202] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 82, and

[0203] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 94;

[0204] (c) a variable region comprising

[0205] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 37,

[0206] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 49,

[0207] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 61,

[0208] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 73,

[0209] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 85

[0210] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 97;

[0211] (d) Variable region, which includes

[0212] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 38,

[0213] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 50,

[0214] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 62,

[0215] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 74,

[0216] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 86, and

[0217] HVR-L3,It contains the amino acid sequence of SEQ ID NO: 98;

[0218] (e) a variable region comprising

[0219] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 39,

[0220] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 51,

[0221] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 63,

[0222] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 75,

[0223] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 87, and

[0224] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 99; and

[0225] (f) a variable region comprising

[0226] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 41,

[0227] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 98,

[0228] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 99,

[0228] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 99,

[0229] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 99,

[0228] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 99,

[0229] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 99, [0229 ... The amino acid sequence of 53,

[0228] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 65,

[0229] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 77,

[0230] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 89, and

[0231] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 101.

[0232] C. Exemplary antibodies that specifically recognize modifications specific to the CH3 region of the constant region of the modified IgG heavy chain

[0233] 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, on pages 14 / 48 of the specification, 18 CN 122145637 A, the modified IgG heavy chain constant region in the exemplary antibody herein preferably includes a region of the CH3 region of a chimeric constant region obtained from at least one constant region corresponding to any one of the constant regions of naturally occurring human IgG or from at least two constant regions selected from the constant regions of naturally occurring human IgG.

[0234] In one embodiment, 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). In this embodiment, the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440.And optionally, threonine at position 436 (all positions are numbered according to the EU numbering system).

[0235] 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).

[0236] 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 NO: (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.

[0237] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences, said VH HVR sequences being selected from (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2,It comprises (iii) HVR-H3, 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. In one embodiment, the antibody comprises HVR-H3, which comprises 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.

[0238] 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, page 15 / 48 of CN 122145637 A, the antibody comprises (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2,It 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.

[0239] 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; (v) HVR-L2, It 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.

[0240] In another aspect, the present invention provides 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 an amino acid sequence selected from SEQ ID NO: 92, 95, 96, 100, 102. Or an amino acid sequence of 103.

[0241] In another aspect, the antibodies described herein include heavy chain variable domain (VH) sequences,The amino acid sequence of SEQ ID NO: 8, 11, 12, 16, 18, or 19 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. 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 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: 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.

[0242] In another aspect, 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 comprises substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but the antibody comprising 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: 20, 23, 24, 28, 30, or 31 are substituted, inserted, and / or deleted. In some implementations, 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 by pyroglutamylation.

[0243] 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.

[0244] In one aspect, an antibody is provided, wherein the antibody competes with an antibody comprising: (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2, comprising the amino acid sequence of SEQ ID NO: 44, 47, 48, 52, 54, or 55; (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO: 56, 59, 60, 64, 66, or 67; (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO: 68, 71, 72, 76, 78, or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO: 80, 83, 84, 88, 90, or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO: The amino acid sequence is 92, 95, 96, 100, 102, or 103.

[0245] In one aspect, an antibody is provided, wherein the antibody binds to the same epitope as an antibody comprising: (i) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 32, 35, 36, 40, 42, or 43; (ii) HVR-H2,It 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.

[0246] 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 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 antibody comprising any one of the following (g) to (l):

[0247] (g) a variable region comprising

[0248] HVR-H1, which contains SEQ ID NO: The amino acid sequence of SEQ ID NO: 32,

[0249] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 44,

[0250] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 56,

[0251] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 68,

[0252] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 80, and

[0253] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 92;

[0254] (h) Variable region, which includes

[0255] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 35,

[0256] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 47,

[0257] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 59,

[0258] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 92,

[0259] SEQ ID NO: 92,

[0250] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 92,

[0250] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 92,

[0250] HVR-L1 ... The amino acid sequence of 71,Specification page 17 / 48 21 CN 122145637 A

[0259] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 83, and

[0260] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 95;

[0261] (i) a variable region comprising

[0262] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 36,

[0263] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 48,

[0264] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 60,

[0265] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 72,

[0266] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 84, and

[0267] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 96;

[0268] (j) A variable region comprising

[0269] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 40,

[0270] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 52,

[0271] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 64,

[0272] HVR-L1, comprising the amino acid sequence of SEQ ID NO: 76,

[0273] HVR-L2, comprising the amino acid sequence of SEQ ID NO: 88, and

[0274] HVR-L3, comprising the amino acid sequence of SEQ ID NO: 100;

[0275] (k) A variable region comprising

[0276] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 42,

[0277] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 54,

[0278] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 40,

[0279] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 42,

[0277] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 54,

[0278] HVR-H3, comprising the amino acid sequence of SEQ ID NO: 54,

[0279] HVR-H1, comprising the amino acid sequence of SEQ ID NO: 54,

[0279] HVR-H2, comprising the amino acid sequence of SEQ ID NO: 54, [0279 ...2, comprising the amino acid sequence of SEQ ID NO: 54, [02 The amino acid sequence of SEQ ID NO: 76,

[0279] HVR-L1, which contains the amino acid sequence of SEQ ID NO: 78,

[0280] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 90, and

[0281] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 102; and

[0282] (l) a variable region, which includes

[0283] HVR-H1, which contains the amino acid sequence of SEQ ID NO: 43,

[0284] HVR-H2, which contains the amino acid sequence of SEQ ID NO: 55,

[0285] HVR-H3, which contains the amino acid sequence of SEQ ID NO: 67,

[0286] HVR-L1,It contains the amino acid sequence of SEQ ID NO: 79,

[0287] HVR-L2, which contains the amino acid sequence of SEQ ID NO: 91, and

[0288] HVR-L3, which contains the amino acid sequence of SEQ ID NO: 103.

[0289] D. Other embodiments

[0290] In one embodiment, the antibody of the present invention comprises an antibody that binds to the same epitope as any of the antibodies mentioned in the foregoing section, wherein the foregoing section is “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”.

[0291] In one embodiment, the antibody of the present invention comprises an antibody that specifically binds to the modified IgG heavy chain constant region, wherein the binding of the antibody to the modified IgG heavy chain constant region competes with the antibodies mentioned in the foregoing section, wherein the foregoing section is “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”. 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 the above portion.

[0292] E. Recombinant methods and compositions

[0293] 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 the following): (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.A second vector containing nucleic acid, said nucleic acid encoding an amino acid sequence containing the VH of an 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 said method comprises culturing a host cell containing nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally recovering said antibody from said host cell (or host cell culture medium).

[0294] For recombinant production of the antibody described herein, nucleic acid encoding an antibody, for example as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody.

[0295] 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 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245–254, which describes the expression of antibody fragments in Escherichia coli). After expression, the antibody can be separated from the bacterial cell paste in soluble fractions and can be further purified.

[0296] In addition to 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).

[0297] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells.

[0298] 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 (describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0299] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; the human embryonic kidney line (293 or 293 cells, as described in Graham et al., J. Gen Virol. 36:59 (1977)); 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. The MRC 5 cells and FS4 cells described in NY Acad. Sci. 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / O. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BK.C. Lo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0300] F. Assays

[0301] The antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

[0302] G. Binding assay and other assays

[0303] On 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.

[0304] 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, 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, wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, and Gly at position 330. Ser at position 331, Ser at position 428, Leu at position 434, 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., a linear or conformational epitope) as any of the antibodies used in the examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA0171) that bind to the epitope. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology vol.66 (Humana Press, Totowa, NJ).

[0305] 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, the fixed modified IgG heavy chain constant region is incubated in a solution containing a labeled antibody but not an unlabeled antibody. After incubation under conditions that allow 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,This indicates that the unlabeled antibody competes with the labeled antibody for binding to the fixed modified IgG heavy chain constant region. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Manual 20 / 48 pages 24 CN 122145637 A Harbor Laboratory, Cold Spring Harbor, NY).

[0306] III. Compositions

[0307] In one aspect, 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".

[0308] In another aspect, the compositions of the present invention are compositions for treating or preventing diseases. When the antibody is used to treat or prevent any disease, the composition may be or include cells expressing any of the antibodies described in "II. Antibodies" or fragments thereof that specifically bind to the modified IgG heavy chain constant region.

[0309] In a preferred embodiment, the polypeptide in the sample comprises a modified IgG heavy chain constant region derived from any one constant region of naturally occurring human IgG or a chimeric constant region derived from at least two constant regions selected from 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”.

[0310] 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.

[0311] In one embodiment, the polypeptide detected or captured by the composition may be an antibody.For example, human IgG1, IgG2, IgG3 or IgG4 molecules, antibody fragments, fusion proteins or any other form of polypeptide containing a modified IgG heavy chain constant region or an epitope therein.

[0312] 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).

[0313] IV. Method

[0314] In one aspect, the method of the present invention is a method for detecting or capturing polypeptides 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”.

[0315] 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).

[0316] 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 any one or more of these amino acid sequences. Preferably, the polypeptide includes a modified IgG heavy chain constant region comprising one or more of these amino acid sequences.

[0317] In one embodiment, the polypeptide detected or captured by the method may be an antibody.For example, human IgG1, IgG2, IgG3 or IgG4 molecules, antibody fragments, fusion proteins or any other form of polypeptide containing a modified IgG heavy chain constant region or an epitope therein.

[0318] 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).

[0319] Figure 3 illustrates an ELISA method as a specific embodiment of the method. The "rabbit anti-Fc mutant antibody" corresponds to an antibody described in "II. Antibodies," and the "anti-hC5 antibody" in Figure 3A and the "anti-IL-8 antibody" in Figure 3B correspond to peptides containing modified IgG heavy chain constant regions. The rabbit anti-Fc mutant antibody immobilized on the plate captures the anti-hC5 antibody in Figure 3A and the anti-IL-8 antibody in Figure 3B from the sample. Then, hC5 (human complement 5), serving as the antigen for the anti-hC5 antibody, binds to an epitope different from that bound by the anti-hC5 antibody, and anti-mouse-POD react in this order in Figure 3A. In Figure 3B, 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, 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 by a photometer when a certain amount of a polypeptide containing the modified IgG heavy chain constant region is present in the sample.

[0320] In another embodiment, the antibodies described in “II. Antibodies” can be used to detect antigens, such as hC5 and IL-8 shown in Figure 4. 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 “anti-hC5 antibody” in Figure 4A and “anti-IL-8 antibody” in Figure 4B correspond to polypeptides containing the modified IgG heavy chain constant region. In Figure 4A, rabbit anti-hC5 antibody immobilized on a plate captures hC5 in the sample. In Figure 4B, mouse anti-IL-8 antibody immobilized on a plate captures IL-8 in the sample. Then, in Figure 4A, anti-hC5 antibody, mouse anti-Fc-mutant antibody, and anti-mouse-POD react in this order. In Figure 4B,Anti-IL-8 antibody, rabbit anti-Fc mutant antibody, and anti-rabbit HRP react 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.

[0321] The above-described embodiments relating to the 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 an antigen, such as IL-8, as shown in Figure 5. In this embodiment, "rabbit anti-Fc mutant antibody" corresponds to one of the antibodies described in "II. Antibody", and "anti-IL-8 antibody" corresponds to a polypeptide containing a modified IgG heavy chain constant region. Mouse anti-IL-8 antibody immobilized on beads captures IL-8 in the sample. Then, anti-IL-8 antibody, biotinylated anti-Fc mutant antibody, and streptavidin-β-galactosidase (SBG (Quanterix Corporation)) react in this order. Finally, the substrate (RGB) of β-galactosidase was added to the reactants and its luminescence was measured.

[0322] Example 1

[0323] Preparation of antibody containing a constant region comprising multiple mutations in the Fc region

[0324] Antibody expression and purification

[0325] An antibody containing a constant region comprising multiple mutations in the Fc region was expressed by the FreeStyle293 expression system. The constant region used (SEQ ID NO: 1) is referred to as SG115 in WO2016098356A1. The harvested cell culture medium (HCCF) was purified by r-protein A resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex 200pg, GE). During the SEC process, we replaced the buffer with 20 mmol / L histidine, 150 mmol / L arginine-aspartic acid, pH 6.0. Finally, we concentrated the antibody to 143 mg / mL using UF (ultrafiltration).

[0326] Papain Digestion

[0327] For papain digestion, we used the Pierce Fab Preparation Kit (Pierce,Catalog number 44985). The papain digestion process is as follows.

[0328] - Adjust the antibody concentration to 8.0 mg / mL with digestion buffer.

[0329] - 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.

[0330] - Incubate the digestion solution at 37°C on a rotary oscillator for 15 hours.

[0331] - After incubation, remove the bottom cap and place the centrifuge column in a microcentrifuge tube. Centrifuge the column at 5000 xg for one minute.

[0332] - Wash the resin with 0.5 mL of Dulbecco's PBS (-). Place the centrifuge column in a microcentrifuge tube. Centrifuge the column at 5000 xg for one minute.

[0333] - Combine the solutions from steps 4 and 5 as the digestion fraction. The total volume of one column is 1.0 mL.

[0334] Purification of Fc Fragment

[0335] The papain-digested sample was purified using r-protein A resin (MabScent SuRe, GE) and size exclusion chromatography (SEC, Superdex 200pg, GE). During the SEC process, the whole IgG (undigested molecule) was removed and the buffer was replaced with Dulbecco's PBS (-).

[0336] Example 2

[0337] Generation of Antibody Recognizing Mutations in SG115

[0338] Antibodies recognizing mutations in SG115, referred to as "anti-SG115 antibodies", were prepared, selected, and determined as described below.

[0339] Ten-week-old NZW rabbits were intradermally immunized with the Fc fragment of SG115 (50-100 μg / dose / rabbit). The administration was repeated 5 times over 2 months, and blood was then collected from the immunized rabbits. According to the procedure described in WO2016098356A1, antigen-specific B cells were sorted using a cell sorter, then plated and cultured. After culture, the B cell culture supernatant was collected for further analysis, and the precipitate was frozen.

[0340] The binding ability of B cell culture supernatant to SG115 was assessed by ELISA. We tested binding to five modified IgG heavy chain constant regions to assess 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). The sequence alignment of these five constant regions is shown in Figure 1.

[0341] A total of 10,560 B cell lines were screened for binding to the five modified IgG heavy chain constant regions.186 cell lines were selected and named SKA0001–SKA0186. These cells 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 in 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 expressed in FreeStyle™ 293-F cells (Invitrogen) and purified from the culture supernatant. Through further evaluation, based on binding affinity and specificity in ELISA and sequence diversity of the heavy chain CDR3, 12 clones were selected. Of these clones, 6 clones (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054 and SKA0127 (page 27, CN 122145637 A, 23 / 48 of the specification) showed selective binding to SG115v1 but not to SG115v2, while the other 6 clones (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171) showed selective binding to SG115v2 but not to SG115v1 (Figures 2-1 and 2-2). The VH and VL sequences of these 12 antibodies are listed in Table 1.

[0342] [Table 1]

[0343]

[0344] Example 3

[0345] Detection of SG115 in samples by anti-SG115 antibody

[0346] To detect antibodies (hereinafter also referred to as "Fc-mutant antibodies" or "multiple Fc-mutant antibodies") containing all or part of the mutated Fc region of SG115 in biological samples, the effectiveness of the above 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 of the Fc-mutant antibody in Examples 3-5, which is referred to as "anti-hC5 antibody" below.

[0347] Assay procedure

[0348] 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.Then anti-mouse-POD (Jackson ImmunoResearch Inc.) was added. Finally, the POD substrate was added to each well of the plate and the OD was measured. The plate was washed between steps.

[0349] Reactivity test for antibody selection

[0350] Twelve anti-Fc mutant antibodies were tested. Anti-hC5 antibodies containing SG115 were diluted with pooled human serum and measured using 12 candidate rabbit anti-Fc mutant antibodies. The signal-to-noise ratio was calculated. The measured ODs are listed in Table 2. Three candidates (a total of 6 candidates) were selected from each epitope type for a selectivity test (Table 2). Specifically, SKA0009, SKA0052, and SKA0127 were selected as antibodies that specifically bind to SG115v1, and SKA0117, SKA0141, and SKA0171 were selected as antibodies that specifically bind to SG115v2.

[0351] [Table 2] Specification 24 / 48 pages 28 CN 122145637 A

[0352]

[0353] Selectivity test for antibody selection

[0354] Ten individual serum and calibration curve samples with or without spiked anti-hC5 antibody were measured using six candidate antibodies as capture reagents. Without spiked anti-hC5 antibody, the measured concentration of all individual samples was below the limit of quantitation (BLQ) for any given rabbit anti-Fc mutant antibody. With spiked anti-hC5 antibody, the relative error (RE) of the measured concentration of all individual samples was within + / - 20% for any given rabbit anti-Fc mutant antibody (Table 3).

[0355] [Table 3]

[0356]

[0357] BLQ: Below the limit of quantitation

[0358] Selected antibody

[0359] Based on the results of the reactivity test and the selectivity test, SKA0141, which had the highest signal-to-noise ratio, was selected.

[0360] Example 4 Specification 25 / 48 pages 29 CN 122145637 A

[0361] Evaluation of a method for measuring anti-hC5 antibodies in human serum (Fc mutant antibody detection assay)

[0362] Assay method

[0363] A 96-well immunoassay plate was coated with rabbit anti-Fc mutant antibody (SKA0141) and blocked with blocking buffer. A diluted serum sample including anti-hC5 antibody was 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.

[0364] Method Evaluation

[0365] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) were -16.3% to -5.1% and 1.6% to 4.4%, respectively (Table 4). Inter-batch accuracy (RE) and precision (CV) were -10.1% to -4.0% and 2.7% to 6.9%, respectively (Table 5).

[0366] [Table 4]

[0367]

[0368] [Table 5] Specification 26 / 48 pages 30 CN 122145637 A

[0369]

[0370] Selectivity was tested. The measured concentration of all individual samples was BLQ without the addition of anti-hC5 antibody. When using spiked anti-hC5 antibody, the RE for the measured concentration of all individual samples was -15.2% to 2.2% (Table 6).

[0371] [Table 6]

[0372] Specification 27 / 48 pages 31 CN 122145637 A

[0373] Dilution linearity was tested. 1 mg of anti-hC5 antibody per milliliter could be measured at a dilution factor of 50,000, and no proband effect was observed. (Table 7)

[0374] [Table 7]

[0375]

[0376] ALQ: Above the limit of quantitation

[0377] Interference from C5 was tested. No interference from C5 was observed (Table 8).

[0378] [Table 8]

[0379]

[0380] A method for measuring Fc mutant antibodies in human serum using anti-Fc mutant antibodies was established. The protocol for this assay is shown in Figure 3.

[0381] Example 5

[0382] We also attempted to establish an assay for detecting antigens in biological samples that are recognized by Fc mutant antibodies. Anti-hC5 antibody was also used as a model for Fc mutant antibodies in this evaluation.

[0383] Evaluation of the method for measuring C5 in human serum (antigen detection assay) Specification 28 / 48 pages 32 CN 122145637 A

[0384] Assay method

[0385] 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.

[0386] Method Evaluation

[0387] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) were -8.2% to 4.2% and 2.3% to 6.2%, respectively (Table 9). Inter-batch accuracy (RE) and precision (CV) were -6.8% to 1.3% and 3.5% to 6.2%, respectively (Table 10).

[0388] [Table 9]

[0389]

[0390] [Table 10] Specification 29 / 48 pages 33 CN 122145637 A

[0391]

[0392] 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).

[0393] [Table 11] Specification 30 / 48 pages 34 CN 122145637 A

[0394]

[0395] Dilution linearity was tested. 1130 mg C5 per mL could be measured at a dilution factor of 26,000, and no prozone effect was observed. (Table 12)

[0396] [Table 12]

[0397]

[0398] ALQ: Above the limit of quantitation

[0399] Interference from anti-hC5 antibody was tested. No interference from anti-hC5 antibody was observed (Table 13).

[0400] [Table 13] Specification 31 / 48 pages 35 CN 122145637 A

[0401]

[0402] A method for measuring antigens in human serum using anti-Fc mutant antibodies was established. The protocol for this assay is shown in Figure 4.

[0403] Example 6

[0404] Evaluation of a method for measuring anti-IL-8 antibodies in human plasma (Fc mutant antibody detection assay)

[0405] Assay method

[0406] A specific anti-human IL-8 antibody comprising 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 for the Fc mutant antibody in Examples 6-8, and it was referred to as "anti-IL-8 antibody".

[0407] 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, ...) was added.SEQ ID NO: 114; light chain constant region, SEQ ID NO: 115), and then 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.

[0408] Method Evaluation

[0409] Reproducibility was tested. Known concentrations of anti-IL-8 antibody (50.0 ng / mL (REP-LL), 100 ng / mL (REP-L), 400 ng / mL (REP-M), 2400 ng / mL (REP-H), and 3200 ng / mL (REP-UL)) were measured. Intra-assay accuracy (RE) and precision (CV) were -14.2% to -9.7% and 4.9% to 7.3%, respectively (Table 14).

[0410] [Table 14] Specification 32 / 48 pages 36 CN 122145637 A

[0411]

[0412] Inter-batch accuracy (RE) and precision (CV) were -10.3% to -6.6% and 7.0% to 10.1%, respectively (Table 15).

[0413] [Table 15] Specification 33 / 48 pages 37 CN 122145637 A

[0414]

[0415] Selectivity was tested. Without anti-IL-8 antibody (SEL-O or SEL-EM-O), the measured concentration of all individual samples was BLQ. With anti-IL-8 antibody (50.0 ng / mL (SEL-LL or SEL-EM-LL)), the RE of the measured concentration of all individual samples was -23.2% to -4.3% (Table 16).

[0416] [Table 16] Specification 34 / 48 pages 38 CN 122145637 A

[0417]

[0418] Dilution linearity was tested. Anti-IL-8 antibody at a dilution factor of 10,000 was measurable at 1.6 mg / mL, and no proband effect was observed (Table 17).

[0419] [Table 17]

[0420]

[0421] 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.Furthermore, IL-8 concentrations up to 1.00 ng / mL did not interfere with the assay at an anti-IL-8 antibody concentration of 50.0 ng / mL (Table 18).

[0422] [Table 18] Specification 35 / 48 pages 39 CN 122145637 A

[0423]

[0424] The effectiveness of the method for measuring anti-IL-8 antibodies in plasma was confirmed.

[0425] Example 7

[0426] Evaluation of a method for measuring IL-8 in human plasma using ELISA (antigen detection assay)

[0427] Assay method

[0428] After blocking with blocking buffer, 96-well streptavidin immunoassay plates were coated with biotinylated mouse anti-IL-8 monoclonal antibody. Anti-IL-8 antibody was added to diluted plasma samples (reaction solution) in 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 plate was washed between steps.

[0429] Method Evaluation

[0430] Reproducibility was tested. Intra-batch accuracy (RE) and precision (CV) were -6.4% to -1.9% and 1.5% to 2.9%, respectively (Table 19).

[0431] [Table 19] Specification 36 / 48 pages 40 CN 122145637 A

[0432]

[0433] Inter-batch accuracy (RE) and precision (CV) were -8.0% to 2.1% and 3.0% to 5.2%, respectively (Table 20).

[0434] [Table 20] Specification 37 / 48 pages 41 CN 122145637 A

[0435]

[0436] Dilution linearity was tested. 1 mg IL-8 (spiked) per milliliter could be measured at a dilution factor of 20,000.And no prozone effect was observed (Table 21).

[0437] [Table 21]

[0438] Spiked concentration in combined human plasma: 1000 ng / mL Instructions for use 38 / 48 pages 42 CN 122145637 A

[0439]

[0440] Interference from anti-IL-8 antibody was tested. No interference from anti-IL-8 antibody (100 μ / mL in plasma) was observed (Table 22).

[0441] [Table 22] Instructions for use 39 / 48 pages 43 CN 122145637 A

[0442]

[0443] The effectiveness of the method for measuring IL-8 in human plasma using ELISA was confirmed.

[0444] Example 8

[0445] Evaluation of a method for measuring IL-8 in human plasma using Simoa (registered trademark) (Simoa (registered trademark) assay)

[0446] Assay Procedure

[0447] 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 substrate RGB of β-galactosidase was added, and fluorescence intensity was measured.

[0448] Method Evaluation

[0449] Reproducibility was tested. Intra-assay precision (CV) ranged from 1.3% to 14.3% (Table 23).

[0450] [Table 23] Specification 40 / 48 pages 44 CN 122145637 A

[0451]

[0452] Inter-batch precision (CV) ranged from 8.6% to 24.7% (Table 24).

[0453] [Table 24-1] Specification 41 / 48 pages 45 CN 122145637 A

[0454]

[0455] [Table 24-2] Specification 42 / 48 pages 46 CN 122145637 A

[0456]

[0457] 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).

[0458] [Table 25] Specification 43 / 48 pages 47 CN 122145637 A

[0459]

[0460] Dilution linearity was tested. 3.48 mg IL-8 per milliliter could be measured at a dilution factor of 50,000.And no prozone effect was observed. (Table 26).

[0461] [Table 26]

[0462]

[0463] Interference from anti-IL-8 antibody was tested. No interference from anti-IL-8 antibody (100 μg / mL in plasma) was observed (Table 27).

[0464] [Table 27] Specification 44 / 48 pages 48 CN 122145637 A

[0465]

[0466] The effectiveness of the method for measuring IL-8 in human plasma using Simoa (registered trademark) was confirmed.

[0467] Example 9

[0468] Affinity assessment of anti-Fc-mutant antibody selectively binding to SG115v1 against anti-hC5 antibody was shown.

[0469] The KD values ​​of the anti-Fc mutant antibodies (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054, and SKA0127) exhibiting selective binding to SG115v1 at pH 7.4 against the anti-hC5 antibody were determined using a Biacore T200 instrument (GE Healthcare) at 25°C.

[0470] 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 the immobilization of anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After immobilization, the running buffer was changed to phosphate pH 7.4 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) 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. KD values ​​were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare). The association rate (ka), dissociation rate (kd), and dissociation constant (KD) are shown in Table 28.

[0471] [Table 28]

[0472]

[0473] *: Due to the slow dissociation rate,Data reliability may be low

[0474] Example 10

[0475] Affinity assessment of anti-IL-8 antibody against an anti-Fc-mutant antibody showing selective binding to SG115v1.

[0476] To confirm the binding ability of the anti-Fc-mutant antibody that exhibits selective binding to SG115v1, the KD value of the anti-Fc-mutant antibody against the anti-IL-8 antibody at pH 7.4 was determined at 25°C using a Biacore T200 instrument (GE manual 45 / 48 pages 49 CN 122145637 A Healthcare). The Fc region sequence of the anti-IL-8 antibody is highly similar to the Fc region sequence of the anti-hC5 antibody.

[0477] Anti-rabbit IgG (Abbexa) was immobilized onto FC1 and 2 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare). For the immobilization of anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After fixation, the running buffer was changed to phosphate buffer pH 7.4 (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, which was injected at a flow rate of 30 μL / min. KD values ​​were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare).

[0478] ka, kd, and KD are listed in Table 29. Although the amino acid at position 239 of the anti-hC5 antibody is mutated from Ser to Lys according to the EU numbering system, the corresponding amino acid in the anti-IL-8 antibody is not mutated. 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 antibody and the anti-IL-8 antibody 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.

[0479] [Table 29]

[0480]

[0481] *: Due to the slow dissociation rate,Data reliability may be low

[0482] Example 11

[0483] Affinity assessment of anti-Fc-mutant antibodies selectively binding to SG115v2 against anti-DENV E protein antibodies.

[0484] The KD values ​​of anti-Fc-mutant antibodies (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141 and SKA0171) selectively binding to SG115v2 against anti-DENV E protein antibodies (which include a modified IgG heavy chain constant region (SEQ ID NO: 116) as an Fc-mutant antibody) were determined at pH 7.4 using a Biacore T200 instrument (GE Healthcare) at 25°C. Anti-rabbit IgG was immobilized onto FC3 and 4 of a CM5 sensor chip using an amine conjugation kit (GE Healthcare). For the immobilization of anti-rabbit IgG, HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the running buffer. After fixation, the run buffer was changed to phosphate buffer at pH 7.4. Each antibody was captured onto FC4 of the sensor chip using 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-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. KD values ​​were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare). ka, kd, and KD are shown in Table 30, page 46 / 48 of the instruction manual, CN 122145637 A.

[0485] [Table 30]

[0486]

[0487] *: Data reliability may be low due to slow dissociation rate

[0488] Example 12

[0489] The affinity of anti-hC5 antibody for human C5 was evaluated using SKA0016 and SKA0117 as capture molecules.

[0490] The KD value of anti-hC5 antibody against human C5 at pH 7.4 was determined using a Biacore T200 instrument (GE Healthcare) at 37°C. SKA0016 was immobilized onto FC1 and 2 of the CM5 sensor chip, and SKA0117 was immobilized onto FC3 and 4 of the CM5 sensor chip using an amine conjugation kit (GE Healthcare). For the immobilization of SKA0016 and SKA0117,HBS-EP+, pH 7.4 (GE Healthcare) buffer was used as the run buffer. After fixation, the run buffer was changed to phosphate pH 7.4 buffer. Anti-hC5 antibody was 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. KD values ​​were obtained using Biacore T200 evaluation software, version 2.0 (GE Healthcare). ka, kd, and KD are listed in Table 31.

[0491] [Table 31]

[0492]

[0493] Example 13

[0494] Qualitative analysis of the pH-dependent interaction between antihC5 antibody and human C5 was performed using SKA0016 and SKA0117 as immobilizing molecules.

[0495] The pH-dependent interaction between antihC5 antibody and human C5 at pH 7.4 and pH 6.0 was evaluated using a Biacore T200 instrument (GE Healthcare) at 37°C. AntihC5 antibody was captured onto FC2 and FC4 of the CM5 chip prepared in Example 12. The amount of antihC5 antibody to be captured was adjusted so that the resonance unit (RU) number was 35. To confirm the association between antihC5 antibody and human C5 at pH 7.4, 32 nM of human C5 was injected into all FCs of phosphate pH 7.4 buffer. The dissociation phase was then monitored in either phosphate buffer (pH 7.4) or phosphate buffer (pH 6.0, 50 mM phosphate buffer containing 150 mM NaCl and 0.05 w / v% P-20, pH 6.0) as the running buffer (pages 47 / 48, CN 122145637 A, CN 122145637 A, 51). After monitoring the dissociation phase, the sensor chip was regenerated by injecting 100 mM Gly-HCl, pH 2.0, followed by 25 mM NaOH, both at a flow rate of 30 μL / min. The pH-dependent interaction between the anti-hC5 antibody and human C5 was analyzed by comparing the dissociation phases of the sensor maps at pH 7.4 and pH 6.0 using Biacore T200 evaluation software version 2.0. The sensor map on FC2 was subtracted from FC1, and the sensor map on FC4 was subtracted from FC3.And 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”.

[0496] Regardless of the captured molecule, human C5 dissociates from the anti-hC5 antibody faster at pH 6.0 than at pH 7.4 (Fig. 6; anti-hC5 antibody was immobilized by (a) SKA0016 and (b) SKA0117). Therefore, both SKA0016 and SKA0117 are considered to be effective in monitoring the pH-dependent interaction between the anti-hC5 antibody and human C5.

[0497] Example 14

[0498] The binding between the human Fc receptor (hFcRn) and the anti-hC5 antibody captured by SKA0016 was evaluated.

[0499] The ability of human FcRn to bind to anti-hC5 antibodies 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 assessment software version 2.0 (GE Healthcare).

[0500] Figure 7 shows the sensor maps of FC1 (dashed line) and FC2 (solid line). The hFcRn binding response increases in a concentration-dependent manner as seen in the FC2 sensor map (solid line). SKA0016 did not interrupt the binding between hFcRn and the anti-hC5 antibody. However, hFcRn appears to bind to the Fc region of SKA0016.Because the FC1 sensor plot also shows an increase in binding response (dashed line). This undesirable binding of human FcRn to the capture molecule can be resolved by introducing an amino acid substitution that cancels the binding with human FcRn into SKA0016. Instruction manual, page 48 / 48, 52 CN 122145637 A, Figure 1; Instruction manual, Figure 1 / 8, page 53 CN 122145637 A, Figure 2-1; Instruction manual, Figure 2 / 8, page 54 CN 122145637 A, Figure 2-2; Instruction manual, Figure 3 / 8, page 55 CN 122145637 A, Figure 3; Instruction manual, Figure 4 / 8, page 56 CN 122145637 A, Figure 4; Instruction manual, Figure 5 / 8, page 57 CN 122145637 A, Figure 5; Instruction manual, Figure 6 / 8, page 58 CN 122145637 A, Figure 6; Instruction manual, Figure 7 / 8, page 59 CN 122145637 A, Figure 7; Instruction manual, Figure 8 / 8, page 60 CN 122145637 A. This disclosure provides antibodies, compositions, and methods for detecting or capturing peptides in a sample. Abstract,

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. in, The isolated antibody comprises any of the following: (1) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 8, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20; (2) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 9, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 21; (3) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 22; (4) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 11, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 23; (5) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 12, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 24; (6) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 25; (7) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26; (8) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 27; (9) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28; (10) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO: 29; and (11) The heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 19, and The light chain variable region consisting of the amino acid sequence of SEQ ID NO:

31.

2. The antibody of claim 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.

3. 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.

4. The antibody according to claim 1, wherein, The modified IgG heavy chain constant region contains 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 being numbered according to the EU numbering system.

5. The antibody according to claim 4, wherein, The modified IgG heavy chain constant region also contains threonine at position 436, which is numbered according to the EU numbering system.

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

7. The composition of claim 6, 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 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 being numbered according to the EU numbering system.

8. The composition according to claim 6, wherein the polypeptide comprises any one of the following: an amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, an amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO:

118.

9. The composition of claim 8, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region comprising any one of the following: an amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, an amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO:

118.

10. 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 5 or a composition according to any one of claims 6 to 9.

11. The method of claim 10, 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 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 being numbered according to the EU numbering system.

12. The method of claim 10, wherein the polypeptide comprises any one of the following: an amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, an amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO:

118.

13. The method of claim 12, wherein the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region comprising any one of the following: an amino acid sequence consisting of RRGPK as shown in SEQ ID NO: 104, an amino acid sequence consisting of RRGPS as shown in SEQ ID NO: 117, an amino acid sequence consisting of LHEALHAHYTRKE as shown in SEQ ID NO: 105, and an amino acid sequence consisting of LHEALHAHTTRKE as shown in SEQ ID NO:

118.

14. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 5.

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

16. A method for preparing an antibody, comprising culturing the host cells of claim 15 to prepare the antibody.

17. 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 (A) Contact the sample with a plate or beads immobilized with a second antibody. (B) After (A), contact the plate or bead with a solution comprising the antigen but excluding the first antibody and the second antibody, and (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. in, 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. 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 being numbered according to the EU numbering system. Wherein, the second antibody is the antibody according to any one of claims 1 to 5, and The third antibody is capable of binding to 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.

18. A method for measuring 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: (D) Contact the sample with a plate or beads immobilized with a third antibody. (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 (F) Detect the first antibody captured on the plate or bead by the third antibody and the antigen using the second antibody. The first antibody includes 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 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 being numbered according to the EU numbering system. Wherein, the second antibody is the antibody according to any one of claims 1 to 5, and has an IgG heavy chain constant region whose amino acid sequence is different from that of the first antibody and the third antibody, and The third antibody is capable of binding to a second epitope of the antigen that is different from the first epitope.

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