Antibodies and compositions for detecting or capturing polypeptides in a sample, and methods for detecting or capturing polypeptides in a sample
阅读说明：本技术 用于检测或捕获样品中的多肽的抗体和组合物，以及用于检测或捕获样品中的多肽的方法 (Antibodies and compositions for detecting or capturing polypeptides in a sample, and methods for detecting or capturing polypeptides in a sample ) 是由 松田绘里子 澁谷光子 西馆正修 于 2020-04-17 设计创作，主要内容包括：本公开提供了用于检测或捕获样品中的多肽的抗体、组合物,以及用于检测或捕获样品中的多肽的方法。(The present disclosure provides antibodies, compositions for detecting or capturing polypeptides in a sample, and methods for detecting or capturing polypeptides in a sample.)
1. An isolated antibody that specifically binds to a modified IgG heavy chain constant region derived from any one of the constant regions in a human naturally occurring IgG, or a chimeric constant region derived from at least two constant regions selected from the group consisting of the constant regions in a human naturally occurring 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 numbered according to the EU numbering system).
2. The antibody of claim 1, wherein the antibody does not substantially bind to any one of the constant regions in the human naturally occurring IgG and the chimeric constant regions obtained from at least two constant regions selected from the group consisting of constant regions in a human naturally occurring IgG.
3. The antibody of claim 1 or 2, wherein the constant region in the human naturally occurring IgG is an IgGl constant region consisting of the amino acid sequence of SEQ ID No. 106, an IgG2 constant region consisting of the amino acid sequence of SEQ ID No. 107, an IgG3 constant region consisting of the amino acid sequence of SEQ ID No. 108 and an IgG4 constant region consisting of the amino acid sequence of SEQ ID No. 109.
4. The antibody of any one of claims 1 to 3, wherein the modified IgG heavy chain constant region is derived from a chimeric constant region obtained from the constant regions of human naturally occurring IgG1 and IgG 4.
5. The antibody of any one of claims 1-4, 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 numbered according to the EU numbering system).
6. The antibody of any one of claims 1-5, wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions numbered according to the EU numbering system).
7. The antibody of any one of claims 1-6, wherein the modified IgG heavy chain constant region comprises either or both of Arg at position 235, and Arg at position 236 and Lys at position 239 (all positions numbered according to the EU numbering system).
8. The antibody of any one of claims 1 to 7, which 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).
9. The antibody according to any one of claims 1 to 8, comprising any one of the following (a) to (f):
(a) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:33,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:45,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:57,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:69,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 93;
(b) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:34,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:46,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:58,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:70,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:82, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 94;
(c) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:37,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:49,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:61,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:73,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:85, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 97;
(d) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:38,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:50,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:62,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:74,
HVR-L2 comprising the amino acid sequence of SEQ ID NO 86, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 98;
(e) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:39,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:51,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:63,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:75,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:87, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 99; and
(f) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:41,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:53,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:65,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:77,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:89, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 101.
10. The antibody of any one of claims 1-5, 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 numbered according to the EU numbering system).
11. The antibody of any one of claims 1-5 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 numbered according to the EU numbering system).
12. The antibody of any one of claims 1 to 5, 10 and 11, which binds to a portion of the modified IgG heavy chain constant region consisting of amino acid sequence LHEALHAHYTRKE (SEQ ID NO:105) or LHEALHAHTTRKE (SEQ ID NO: 118).
13. The antibody according to any one of claims 1 to 5 and 10 to 12, comprising any one of the following (g) to (l):
(g) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:32,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:44,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:56,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:68,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 92;
(h) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:35,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:47,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:59,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:71,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 95;
(i) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:36,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:48,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:60,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:72,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:84, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 96;
(j) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:40,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:52,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:64,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:76,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:88, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 100;
(k) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:42,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:54,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:66,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:78,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:90, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 102; and
(l) A variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:43,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:55,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:67,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:79,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:91, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 103.
14. An isolated antibody that binds to the same epitope as the antibody of any one of claims 1 to 13.
15. An isolated antibody that specifically binds to a modified IgG heavy chain constant region, wherein the antibody competes for binding to the modified IgG heavy chain constant region with the antibody of any one of claims 1 to 14, wherein the modified IgG heavy chain constant region is derived from any one of the constant regions in a human naturally occurring IgG or from a chimeric constant region obtained from at least two constant regions selected from the constant regions in a human naturally occurring 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 numbered according to the EU numbering system).
Technical Field
The present invention relates to antibodies and compositions for detecting or capturing polypeptides in a sample, and methods for detecting or capturing polypeptides in a sample
Background
The hybridoma technology has made possible the production of monoclonal antibodies, which have been widely used in many scientific fields (NPL 1). After this technical effort has been achieved, further efforts have been made in the field of therapeutic and diagnostic antibodies. Since the first approval of monoclonal antibody therapy in the united states, 30 years have passed (NPL 2). Over 30 antibodies have been FDA approved and a number of candidate antibodies are undergoing clinical and preclinical evaluation. To date, monoclonal antibodies remain standard therapeutic molecules and are used in various disease areas, such as cancer, autoimmune diseases, respiratory diseases, infectious diseases and neurological diseases (NPL 3).
To increase the value of therapeutic antibodies, many different types of engineered Fc modifications were identified to improve function, such as those used for antibody-dependent cell-mediated enhanced cytotoxicity, complement-dependent enhanced cytotoxicity, extended antibody half-life, regulation of antigen clearance, and promotion of heavy chain heterodimerization (NPL 4).
Antibodies that specifically bind to an engineered Fc region but do not bind to wild-type Fc have been reported (NPL5, PTL 1). Antibodies directed against engineered Fc regions have proven to be very useful for a variety of purposes.
Reference list
Patent document
[PTL1]WO2017072210A1
Non-patent document
[NPL1]Kohler,G.et al.,Nature 256:495-497(1975)
[NPL2]Reichert,J.M.et al.,Curr.Pharm.Biotechnol.9:423-430(2008)
[NPL3]Lagasse HAD et al.F1000Research 2017,6(F1000 Faculty Rev):113
[NPL4]Mimoto et al.,Curr.Pharm.Biotechnol.17:1298-1314(2016)
[NPL5]Yu et al.,Antimicrob Agents Chemother.61(2016)
Disclosure of Invention
We provide antibodies comprising a modified IgG heavy chain constant region derived from any one of the constant regions of a human naturally occurring IgG or from a chimeric constant region derived from at least two constant regions selected from the constant regions of a human naturally occurring IgG. Antibodies include, for example, satralizumab (satralizumab), nemulizumab (nemolizumab), eimericzumab (emilizumab), SKY59 (covalimab), AMY109, and GYM 329. For example, the modified IgG heavy chain constant region in one of them comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions numbered according to the EU numbering system). The invention provides antibodies that specifically bind, detect and/or capture polypeptides comprising modified IgG heavy chain constant regions or epitopes therein, compositions comprising the antibodies, and methods of using the antibodies.
Specifically, the present invention relates to the following [1] to [25 ].
[1] An isolated antibody that specifically binds to a modified IgG heavy chain constant region derived from any one of the constant regions in a human naturally occurring IgG, or a chimeric constant region derived from at least two constant regions selected from the group consisting of the constant regions in a human naturally occurring 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 numbered according to the EU numbering system).
[2] The antibody of [1], wherein the antibody does not substantially bind to any one constant region in the human naturally occurring IgG and the chimeric constant region obtained from at least two constant regions selected from the group consisting of constant regions in the human naturally occurring IgG.
[3] The antibody according to [1] or [2], wherein the constant region in the human naturally occurring 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.
[4] The antibody of 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 human naturally occurring IgG1 and IgG 4.
[5] The antibody of 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 numbered according to the EU numbering system).
[6] The antibody of any one of [1] to [5], wherein the modified IgG heavy chain constant region comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, and Lys at position 239 (all positions numbered according to the EU numbering system).
[7] The antibody of any one of [1] to [6], wherein the modified IgG heavy chain constant region comprises Arg at position 235, and either or both of Arg at position 236 and Lys at position 239 (all positions numbered according to the EU numbering system).
[8] The antibody according to any one of [1] to [7], which binds to the part of the modified IgG heavy chain constant region consisting of the amino acid sequence RRGPK (SEQ ID NO:104) or RRGPS (SEQ ID NO: 117).
[9] The antibody according to any one of [1] to [8], which comprises any one of the following (a) to (f):
(a) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:33,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:45,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:57,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:69,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 93;
(b) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:34,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:46,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:58,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:70,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:82, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 94;
(c) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:37,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:49,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:61,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:73,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:85, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 97;
(d) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:38,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:50,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:62,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:74,
HVR-L2 comprising the amino acid sequence of SEQ ID NO 86, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 98;
(e) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:39,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:51,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:63,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:75,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:87, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 99; and
(f) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:41,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:53,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:65,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:77,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:89, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 101.
[10] The antibody of 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 numbered according to the EU numbering system).
[11] The antibody of any one of [1] to [5] and [10], wherein the modified IgG heavy chain constant region comprises Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, and optionally threonine at position 436 (all positions numbered according to the EU numbering system).
[12] The antibody according to any one of [1] to [5], [10] and [11], which binds to a portion of the modified IgG heavy chain constant region consisting of amino acid sequence LHEALHAHYTRKE (SEQ ID NO:105) or LHEALHAHTTRKE (SEQ ID NO: 118).
[13] The antibody according to any one of [1] to [5] and [10] to [12], which comprises any one of the following (g) to (l):
(g) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:32,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:44,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:56,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:68,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 92;
(h) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:35,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:47,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:59,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:71,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 95;
(i) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:36,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:48,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:60,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:72,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:84, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 96;
(j) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:40,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:52,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:64,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:76,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:88, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 100;
(k) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:42,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:54,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:66,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:78,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:90, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 102; and
(l) A variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:43,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:55,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:67,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:79,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:91, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 103.
[14] An isolated antibody that binds to the same epitope as the antibody of any one of [1] to [13 ].
[15] An isolated antibody that specifically binds to a modified IgG heavy chain constant region, wherein the antibody competes for binding to the modified IgG heavy chain constant region with the antibody of any one of [1] to [14], wherein the modified IgG heavy chain constant region is derived from any one of the constant regions in a human naturally occurring IgG or from a chimeric constant region obtained from at least two constant regions selected from the constant regions in a human naturally occurring 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 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).
[16] A composition for detecting or capturing a polypeptide in a sample, wherein the composition comprises the antibody of any one of [1] to [15 ].
[17] The composition of [16], wherein the polypeptide comprises a modified IgG heavy chain constant region derived from any one of the constant regions in a human naturally occurring IgG or from a chimeric constant region obtained from at least two constant regions selected from the group consisting of constant regions in a human naturally occurring 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, 327, Gly 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).
[18] The composition of [16], wherein the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO:104), an amino acid sequence consisting of RRGPS (SEQ ID NO:117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).
[19] The composition of [18], wherein the polypeptide comprises a modified IgG heavy chain constant region comprising any one of an amino acid sequence consisting of RRGPK (SEQ ID NO:104), an amino acid sequence consisting of RRGPS (SEQ ID NO:117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).
[20] A method of detecting or capturing a polypeptide in a sample, wherein the method comprises contacting the sample with an antibody of any one of [1] to [15] or with a composition of any one of [16] to [19 ].
[21] The method of [20], wherein the polypeptide comprises a modified IgG heavy chain constant region derived from any one of the constant regions in a human naturally occurring IgG or from a chimeric constant region obtained from at least two constant regions selected from the group consisting of constant regions in a human naturally occurring 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, 327, Gly 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).
[22] The method according to [20], wherein the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO:104), an amino acid sequence consisting of RRGPS (SEQ ID NO:117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).
[23] The method of [22], wherein the polypeptide comprises a modified IgG heavy chain constant region comprising any one of the amino acid sequence consisting of RRGPK (SEQ ID NO:104), the amino acid sequence consisting of RRGPS (SEQ ID NO:117), the amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and the amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118).
[24] A method of measuring the concentration of a first antibody in a sample, wherein said first antibody is capable of binding a first epitope of an antigen, wherein said sample comprises said first antibody and said antigen, and wherein said method comprises
(A) Contacting the sample with a plate or bead having a second antibody immobilized thereon,
(B) contacting a solution comprising the antigen and not the first and second antibodies with the plate or beads after (A), and
(C) detecting an antigen captured on the plate or bead by the second antibody and the first antibody using a third antibody after (B), wherein
The first antibody comprises a modified IgG heavy chain constant region derived from any constant region in a human naturally occurring IgG, or a chimeric constant region derived from at least two constant regions selected from the group consisting of constant regions in human naturally occurring 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 numbered according to the EU numbering system), the second antibody is the antibody of any one of [1] to [15], and the third antibody is capable of binding a second epitope of the antigen different from the first epitope, and has an IgG heavy chain constant region with an amino acid sequence different from that of the first and second antibodies.
[25] A method for determining the concentration of an antigen in a sample, wherein said sample comprises said antigen and a first antibody capable of binding to a first epitope of said antigen, wherein said method comprises
(D) Contacting the sample with a plate or bead having a third antibody immobilized thereon,
(E) contacting a solution comprising the first antibody and not comprising the antigen and the third antibody with the plate or the beads after (D), and
(F) detecting the first antibody captured on the plate or bead by the third antibody and the antigen using a second antibody, wherein
The first antibody comprises a modified IgG heavy chain constant region derived from any one of the constant regions in a human naturally occurring IgG or a chimeric constant region derived from at least two constant regions selected from the group consisting of the constant regions in a human naturally occurring 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 numbered according to the EU numbering system),
the second antibody is the antibody of 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
the third antibody is capable of binding a second epitope of the antigen different from the first epitope.
Drawings
FIG. 1 illustrates the sequence alignment of the 5 modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m and G4d) described in example 2. Human IgG CH germline sequences, IGHG1_01(J00228) and IGHG4_01(K01316), were also aligned for comparison. Dots indicate the same amino acid as SG115 at this position.
FIG. 2-1 FIGS. 2-1 and 2-2 illustrate the binding of 12 anti-SG 115 antibodies to 5 modified IgG heavy chain constant regions (SG115, SG115v1, SG115v2, G1m and G4d) in an ELISA. SKA0009, SKA0016, SKA0046, SKA0052, SKA0054 and SKA0127 show selective binding to SG115v1, while SKA0001, SKA0027, SKA0028, SKA0117, SKA0141 and SKA0171 show selective binding to SG115v2, as described in example 2.
Fig. 2-2 is a continuation of fig. 2-1.
FIG. 3 illustrates a protocol for an Fc mutant antibody detection assay.
FIG. 4 illustrates a protocol for an antigen detection assay.
FIG. 5A scheme of the measurement of Simoa (registered trademark) is shown in FIG. 5.
FIG. 6 illustrates a sensorgram for dissociation of human C5 from anti-hC 5 antibody captured by SKA0016 and SKA0117 at pH7.4 and pH 6.0. Neither SKA0016 nor SKA0117 interfered with pH-dependent interactions between anti-hC 5 antibodies and human C5.
FIG. 7 illustrates sensorgrams for the binding assay of human Fc receptor to anti-hC 5 antibody captured by SKA 0016. SKA0016 did not interrupt the binding between hFcRn and anti-hC 5 antibody.
Detailed Description
I. Definition of
"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" or "binding activity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.
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 one of the constant regions in human naturally occurring IgG or from a chimeric constant region obtained from at least two constant regions selected from the constant regions in human naturally occurring IgG" refers to an antibody that is capable of binding a particular type of modified IgG heavy chain constant region with sufficient affinity such that the antibody is useful as a detection, capture, or diagnostic reagent that targets 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 the 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, a Radioimmunoassay (RIA). In certain embodiments, an antibody that binds to a modified IgG heavy chain constant region has 1 μ M or less, 100nM or less, 10nM or less, 1nM or less, 0.1nM or less, 0.01nM or less, or 0.001nM or less (e.g., 10 nM)-8M or less, e.g. 10-8M to 10-13M, e.g. 10-9M to 10-13M) dissociation constant (Kd). In certain embodiments, an antibody that binds to a modified IgG heavy chain constant region binds to an epitope in the modified IgG heavy chain constant region.
The term "antibody" is used herein in the broadest sense and includes a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during the production of the monoclonal antibody). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.
The term "constant region" herein is a region of an antibody corresponding to any one of the IgG1 constant region consisting of the amino acid sequence of SEQ ID NO 106, the IgG2 constant region consisting of the amino acid sequence of SEQ ID NO 107, the IgG3 constant region consisting of the amino acid sequence of SEQ ID NO 108, and the IgG4 constant region consisting of the amino acid sequence of SEQ ID NO 109. The constant region consists of 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).
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is 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.
The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). (see, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antigen-binding antibodies can be used to screen libraries of complementary VL or VH domains, respectively, to isolate antibodies that bind a particular antigen. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature 352: 624-628(1991).
"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Accordingly, HVR and FR sequences typically occur in VH (or VL) in the following order: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.
As used herein, the term "hypervariable region" or "HVR" refers to each region which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or antibody variable domains containing antigen-contacting residues ("antigen-contacts"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:
(a) the hypervariable loops which occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196: 901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) antigen contacts occurring at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al, J.mol.biol.262: 732-745 (1996)); and
(d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).
"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (GENETYX co., Ltd.). One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.
The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the us copyright Office (u.s.copy Office, Washington d.c., 20559) with the user document, with us copyright registration accession No. TXU 510087. The ALIGN-2 program is publicly available from Genentech, Inc. of St.Francisco, Calif., or may 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 were set by the ALIGN-2 program and were unchanged. In the case of amino acid sequence comparisons using ALIGN-2, the% amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or so to say that a given amino acid sequence A has or contains a particular% amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the X/Y fraction
Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and wherein Y is the total number of amino acid residues in B. It will 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 specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.
An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that: blocking 50% or more of the binding of a reference antibody to its antigen in a competition assay, and conversely, blocking 50% or more of the binding of the reference antibody to its antigen in a competition assay. Exemplary competition assays are provided herein.
Antibodies
The antibodies of the invention are isolated antibodies that specifically bind to a modified IgG heavy chain constant region.
In one embodiment, the antibody does not substantially bind to a constant region in a human naturally occurring IgG and a chimeric constant region obtained from at least two constant regions selected from the group consisting of constant regions in a human naturally occurring IgG. In this embodiment, the binding activity of the antibody to the constant region of human naturally occurring IgG and chimeric IgG consisting of at least two IgG selected from the group consisting of human naturally occurring IgG is below the detection limit in enzyme-linked immunoassay. On the other hand, the binding activity of the antibody to the modified IgG heavy chain constant region is detectable in an enzyme-linked immunoassay.
In another aspect of the invention, the antibody is a monoclonal antibody, including a chimeric, humanized or human antibody. In one embodiment, the antibody is an antibody fragment, such as an Fv, Fab ', scFv, diabody, or F (ab')2 fragment. In another embodiment, the antibody is a full length antibody, such as the intact IgG1, IgG2, IgG3, and IgG4 antibodies, or other antibody classes or isotypes defined herein.
A. Modified IgG heavy chain constant region
In one embodiment, the modified IgG heavy chain constant region is derived from any one of the constant regions in a human naturally occurring IgG, or from a chimeric constant region obtained from at least two constant regions selected from the constant regions in a human naturally occurring IgG. The constant regions in human naturally occurring 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.
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 numbered according to the EU numbering system).
In further embodiments, the modified IgG heavy chain constant region may be derived from a chimeric constant region obtained from the constant regions in human naturally occurring IgG1 and IgG 4. In a preferred embodiment, the constant region is derived from a chimeric constant region obtained from the constant regions in human naturally occurring IgG1 and IgG 4.
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 numbered according to the EU numbering system).
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 a monomeric Fc reported in Ishino t, et al, j.biol.chem.288:16259-37 (2013).
In one embodiment, when the modified IgG heavy chain constant region is in a human modified IgG heavy chain, the human modified IgG heavy chain is selected from the group consisting of human modified IgG1, IgG2, IgG3, and IgG4 heavy chains, and chimeric IgG heavy chains thereof. In preferred embodiments, the human IgG heavy chain is a human IgG1 heavy chain, a human IgG4 heavy chain, or a chimeric IgG heavy chain thereof.
B. Exemplary antibodies specifically recognizing a modification characteristic of the CH2 region of the modified IgG heavy chain constant region
Arg at position 235, Arg at position 236 and Lys at position 239 (all positions 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. Thus, the modified IgG heavy chain constant region in the exemplary antibodies herein preferably comprises a region corresponding to at least the CH2 region of any one of the constant regions of human naturally occurring IgG or a chimeric constant region derived from at least two constant regions selected from the constant regions of human naturally occurring IgG.
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 numbered according to the EU numbering system). In this embodiment, the modified IgG heavy chain constant region comprises Arg at position 235, and either or both Arg at position 236 and Lys at position 239 (all positions numbered according to the EU numbering system).
In preferred embodiments, the modified IgG heavy chain constant region includes all three of these mutations, or Arg at position 235 and Arg at position 236 (all positions numbered according to the EU numbering system). In that case, the antibody binds to the part of the modified IgG heavy chain constant region that consists of the amino acid sequence RRGPK (SEQ ID NO:104) or RRGPS (SEQ ID NO: 117).
In aspects in which the antibody specifically binds to the following (1) to (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 numbered according to the EU numbering system), (2) a modified IgG heavy chain constant region comprising Arg at position 235, and either or both of Arg at position 236 and Lys at position 239 (all positions numbered according to the EU numbering system), or (3) a portion consisting of the amino acid sequence RRGPK (SEQ ID NO:104) or RRGPS (SEQ ID NO:117) in the modified IgG heavy chain constant region, the invention provides an antibody comprising at least one, two, three, four, five, or six HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO 33, 34, 37, 38, 39 or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 45, 46, 49, 50, 51 or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63 or 65; (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75 or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO 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, said HVR-H3 comprises the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63, or 65. In another embodiment, an antibody comprises HVR-H3 and HVR-L3, said HVR-H3 comprises the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63, or 65, and said HVR-L3 comprises the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99, or 101. In another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, said HVR-H3 comprises the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63, or 65, said HVR-L3 comprises the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99, or 101, and said HVR-H2 comprises the amino acid sequence of SEQ ID NO:45, 46, 49, 50, 51, or 53. In further embodiments, the antibody comprises (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 another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75, or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101. In one embodiment, the antibody comprises (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 69, 70, 73, 74, 75 or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101.
In another aspect, an antibody of the 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, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, 82, 85, 86, 87, or 89, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99, or 101.
In another aspect, the invention provides an antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:33, 34, 37, 38, 39, or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 45, 46, 49, 50, 51 or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63 or 65; (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75 or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs 93, 94, 97, 98, 99 or 101.
In another aspect, the antibodies described herein comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID No. 9, 10, 13, 14, 15, or 17. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an antibody described herein comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NOs 9, 10, 13, 14, 15, or 17 are substituted, inserted, and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the antibody comprises the VH sequence in SEQ ID NO 9, 10, 13, 14, 15 or 17, including post-translational modifications of that sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:33, 34, 37, 38, 39 or 41, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:45, 46, 49, 50, 51 or 53, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63 or 65. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In another aspect, antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:21, 22, 25, 26, 27 or 29. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an antibody comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO 21, 22, 25, 26, 27 or 29 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the antibody includes the VL sequence in SEQ ID NO 21, 22, 25, 26, 27 or 29, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75, or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In another aspect, there is provided an antibody, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO 9, 10, 13, 14, 15 or 17 and SEQ ID NO 21, 22, 25, 26, 27 or 29, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In one aspect, an antibody is provided, wherein the antibody competes for binding to a first modified IgG heavy chain constant region with an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO 33, 34, 37, 38, 39 or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 45, 46, 49, 50, 51 or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63 or 65; (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75 or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101.
In one aspect, antibodies are provided, wherein the antibodies bind to the same epitope as an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO 33, 34, 37, 38, 39 or 41; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO 45, 46, 49, 50, 51 or 53; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:57, 58, 61, 62, 63 or 65; (iv) HVR-L1, comprising the amino acid sequence of SEQ ID NO:69, 70, 73, 74, 75 or 77; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 81, 82, 85, 86, 87 or 89; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO:93, 94, 97, 98, 99 or 101.
In particular embodiments wherein the antibody specifically binds to (1) - (3) below: (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 numbered according to the EU numbering system), (2) a modified IgG heavy chain constant region comprising Arg at position 235, and either or both of Arg at position 236 and Lys at position 239 (all positions numbered according to the EU numbering system), or (3) a portion consisting of the amino acid sequence RRGPK (SEQ ID NO:104) or RRGPS (SEQ ID NO:117) in a modified IgG heavy chain constant region, the antibody comprising any one of the following (a) to (f):
(a) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:33,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:45,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:57,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:69,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:81, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 93;
(b) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:34,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:46,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:58,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:70,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:82, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 94;
(c) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:37,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:49,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:61,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:73,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:85, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 97;
(d) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:38,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:50,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:62,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:74,
HVR-L2 comprising the amino acid sequence of SEQ ID NO 86, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 98;
(e) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:39,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:51,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:63,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:75,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:87, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 99; and
(f) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:41,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:53,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:65,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:77,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:89, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 101.
C. Exemplary antibodies specifically recognizing a modification characteristic of the CH3 region of the modified IgG heavy chain constant region
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) are those amino acids specifically present in the CH3 region of SG115 and SG115v2 used in the examples. Thus, the modified IgG heavy chain constant region in the exemplary antibodies herein preferably comprises a region corresponding to at least the CH3 region of any one of the constant regions of human naturally occurring IgG or a chimeric constant region derived from at least two constant regions selected from the constant regions of human naturally occurring IgG.
In one embodiment, the modified IgG heavy chain constant region comprises at least one selected from the group consisting of Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions 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 numbered according to the EU numbering system).
In a preferred embodiment, the modified IgG heavy chain constant region includes all of these mutations. In that case, the antibody binds to the part of the modified IgG heavy chain constant region consisting of amino acid sequence LHEALHAHYTRKE (SEQ ID NO:105) or LHEALHAHTTRKE (SEQ ID NO: 118).
In aspects in which the antibody specifically binds to the following (1) to (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 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 numbered according to the EU numbering system), or (3) a portion consisting of amino acid sequence LHEALHAHYTRKE (SEQ ID NO:105) or LHEALHAHTTRKE (SEQ ID NO:118) in a modified IgG heavy chain constant region, the invention provides an antibody comprising at least one, two, three, four, five, or six 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; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 56, 59, 60, 64, 66 or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 68, 71, 72, 76, 78 or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103.
In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from (i) HVR-H1, comprising the amino acid sequence of SEQ ID NO:32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54 or 55; and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:56, 59, 60, 64, 66 or 67. In one embodiment, the antibody comprises HVR-H3, said HVR-H3 comprises the amino acid sequence of SEQ ID NO 56, 59, 60, 64, 66 or 67. In another embodiment, an antibody comprises HVR-H3 and HVR-L3, said HVR-H3 comprises the amino acid sequence of SEQ ID NO:56, 59, 60, 64, 66 or 67 and said HVR-L3 comprises the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103. In another embodiment, an antibody comprises HVR-H3, HVR-L3, and HVR-H2, said HVR-H3 comprises the amino acid sequence of SEQ ID NO:56, 59, 60, 64, 66, or 67, said HVR-L3 comprises the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102, or 103, and said HVR-H2 comprises the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54, or 55. In further embodiments, the antibody comprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:32, 35, 36, 40, 42, or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54 or 55; and (iii) HVR-H3, comprising the amino acid sequence of SEQ ID NO:56, 59, 60, 64, 66 or 67.
In another aspect, the invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:68, 71, 72, 76, 78, or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103. In one embodiment, the antibody comprises (iv) HVR-L1 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 another aspect, an antibody of the 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, 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 another aspect, the 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 NOs: 92, 95, 96, 100, 102, or 103.
In another aspect, the antibodies described herein comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO 8, 11, 12, 16, 18, or 19. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an antibody described herein comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids of SEQ ID NO 8, 11, 12, 16, 18 or 19 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of 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 particular embodiments, the VH comprises one, two, or three HVRs selected from: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:32, 35, 36, 40, 42, or 43, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54, or 55, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:56, 59, 60, 64, 66, or 67. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In another aspect, antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:20, 23, 24, 28, 30 or 31. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution (e.g., a conservative substitution), insertion, or deletion relative to a reference sequence, but an antibody comprising that sequence retains the ability to bind to the first modified IgG heavy chain constant region. In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO 20, 23, 24, 28, 30 or 31 are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). Optionally, the antibody includes the VL sequence in SEQ ID NO 20, 23, 24, 28, 30 or 31, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 68, 71, 72, 76, 78, or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In another aspect, there is provided an antibody, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NOs 8, 11, 12, 16, 18 or 19 and 20, 23, 24, 28, 30 or 31, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamate acylation.
In one aspect, an antibody is provided, wherein the antibody competes for binding to a first modified IgG heavy chain constant region with an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:32, 35, 36, 40, 42 or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54 or 55; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 56, 59, 60, 64, 66 or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 68, 71, 72, 76, 78 or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103.
In one aspect, antibodies are provided, wherein the antibodies bind to the same epitope as an antibody comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:32, 35, 36, 40, 42 or 43; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:44, 47, 48, 52, 54 or 55; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO 56, 59, 60, 64, 66 or 67; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO 68, 71, 72, 76, 78 or 79; (v) HVR-L2, comprising the amino acid sequence of SEQ ID NO 80, 83, 84, 88, 90 or 91; and (vi) HVR-L3, comprising the amino acid sequence of SEQ ID NO:92, 95, 96, 100, 102 or 103.
In particular embodiments wherein the antibody specifically binds to (1) - (3) below: (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 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 numbered according to the EU numbering system), or (3) a portion of the modified IgG heavy chain constant region consisting of amino acid sequence LHEALHAHYTRKE (SEQ ID NO:105) or LHEALHAHTTRKE (SEQ ID NO:118), the antibody comprising any one of (g) to (l) below:
(g) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:32,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:44,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:56,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:68,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:80, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 92;
(h) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:35,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:47,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:59,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:71,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:83, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 95;
(i) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:36,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:48,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:60,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:72,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:84, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO. 96;
(j) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:40,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:52,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:64,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:76,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:88, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO 100;
(k) a variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:42,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:54,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:66,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:78,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:90, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 102; and
(l) A variable region comprising
HVR-H1, comprising the amino acid sequence of SEQ ID NO:43,
HVR-H2, comprising the amino acid sequence of SEQ ID NO:55,
HVR-H3, comprising the amino acid sequence of SEQ ID NO:67,
HVR-L1, comprising the amino acid sequence of SEQ ID NO:79,
HVR-L2 comprising the amino acid sequence of SEQ ID NO:91, and
HVR-L3, comprising the amino acid sequence of SEQ ID NO: 103.
D. Other embodiments
In one embodiment, the antibody of the present invention includes an antibody that binds to the same epitope as any of the antibodies mentioned in the above sections "a.
In one embodiment, the antibodies of the invention include antibodies that specifically bind to a modified IgG heavy chain constant region, wherein the binding of the antibody to the modified IgG heavy chain constant region competes with the antibodies mentioned in the section "a. modified IgG heavy chain constant region" to "c. modified exemplary antibodies specific for the CH3 region that specifically recognizes the modified IgG heavy chain constant region". In this embodiment, the modified IgG heavy chain constant region is derived from any one of the constant regions in a human naturally occurring IgG, or from a chimeric constant region obtained from at least two constant regions selected from the constant regions in a human naturally occurring 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 numbered according to the EU numbering system). In this embodiment, the specific antibodies referred to herein are the same as the moieties described above.
E. Recombinant methods and compositions
Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In one embodiment, isolated nucleic acids encoding the antibodies described herein are provided. Such nucleic acids can encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell includes (e.g., has been transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising the VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp2/0 cell). In one embodiment, a method of producing an antibody described herein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of the antibodies described herein, nucleic acids encoding the antibodies, e.g., as described above, are 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 (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).
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 expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also Charlton, Methods in Molecular Biology, vol.248(B.K.C.Lo, ed., Humana Press, Totowa, NJ,2003), page 245-. After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized", thereby producing antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22: 1409-.
Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures may also be used as hosts. See, for example, U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plantsTMA technique).
Vertebral motionSomatic cells may also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described by Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse trophoblasts (TM4 cells, as described in Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as Mather et al, Annals NY Acad.Sci.383: 44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, methods of molecular biology, Vol.248(B.K.C.Lo ed., Humana Press, Totowa, NJ), pp.255-268 (2003).
F. Measurement of
The antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
G. Binding assays and other assays
In one aspect, antibodies of the invention are tested for antigen binding activity, e.g., by known methods such as ELISA, Western blot, and the like.
In another aspect, the competition assay can be used to identify antibodies that compete for binding to a modified IgG heavy chain constant region derived from any one of the constant regions in human naturally occurring IgG or from a chimeric constant region derived from at least two constant regions selected from the constant regions in human naturally occurring 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 numbered according to the EU numbering system) with any of the antibodies used in the examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA 0171). In certain embodiments, such competitive antibodies can bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by any of the antibodies used in the examples (SKA0001, SKA0009, SKA0016, SKA0027, SKA0028, SKA0046, SKA0052, SKA0054, KA0117, SKA0127, SKA0141, and SKA 0171). 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).
In an exemplary competition assay, the immobilized modified IgG heavy chain constant region is incubated in a solution comprising a labeled antibody that binds to the modified IgG heavy chain constant region and an unlabeled antibody that is being tested for its ability to compete with the labeled antibody for binding to the immobilized modified IgG heavy chain constant region. Unlabeled antibodies may be present in the B cell or hybridoma supernatant. As a control, the immobilized modified IgG heavy chain constant region was incubated in a solution comprising labeled antibody but not unlabeled antibody. After incubation under conditions that allow binding of the labeled antibody to the immobilized modified IgG heavy chain constant region, excess unbound antibody is removed and the amount of label associated with the immobilized modified IgG heavy chain constant region is measured. If the amount of label associated with the immobilized modified IgG heavy chain constant region in the test sample is significantly reduced relative to the control sample, it indicates that the unlabeled antibody competes with the labeled antibody for binding to the immobilized modified IgG heavy chain constant region. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Composition III
In one aspect, the compositions of the invention are compositions for detecting or capturing a polypeptide in a sample. The composition comprises any of the antibodies described in "ii.
In another aspect, the compositions of the present invention are compositions for treating or preventing a disease. When the antibody is used to treat or prevent any disease, the composition may be or include a cell expressing any of the antibodies described in "antibody ii" or fragments thereof that specifically bind to a modified IgG heavy chain constant region.
In a preferred embodiment, the polypeptide in the sample comprises a modified IgG heavy chain constant region derived from any one of the constant regions of a human naturally occurring IgG or from a chimeric constant region of at least two constant regions selected from human naturally occurring 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 numbered according to the EU numbering system). The specific antibodies referred to herein are the same as those described in "ii.
In another preferred embodiment, the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO:104), an amino acid sequence consisting of RRGPS (SEQ ID NO:117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118). 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 comprises a modified IgG heavy chain constant region comprising any one or more of the amino acid sequences.
In one embodiment, the polypeptide detected or captured by the composition may be an antibody, such as a human IgG1, IgG2, IgG3, or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide comprising a modified IgG heavy chain constant region or epitope therein.
Where the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region may comprise other amino acid substitutions or modifications so long as it comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions numbered according to the EU numbering system).
Method IV
In one aspect, the methods of the invention are methods for detecting or capturing a polypeptide in a sample. The method comprises contacting the sample with any one of the antibodies described in "ii.
In a preferred embodiment, the polypeptide comprises a modified IgG heavy chain constant region. The modified IgG heavy chain constant region is derived from any one of the constant regions in a human naturally occurring IgG or a chimeric constant region derived from at least two constant regions selected from the constant regions in a human naturally occurring 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 numbered according to the EU numbering system).
In another preferred embodiment, the polypeptide comprises any one of an amino acid sequence consisting of RRGPK (SEQ ID NO:104), an amino acid sequence consisting of RRGPS (SEQ ID NO:117), an amino acid sequence consisting of LHEALHAHYTRKE (SEQ ID NO:105), and an amino acid sequence consisting of LHEALHAHTTRKE (SEQ ID NO: 118). 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. The polypeptide preferably comprises a modified IgG heavy chain constant region comprising one or more of these amino acid sequences.
In one embodiment, the polypeptide detected or captured by the method may be an antibody, such as a human IgG1, IgG2, IgG3, or IgG4 molecule, an antibody fragment, a fusion protein, or any other form of polypeptide comprising a modified IgG heavy chain constant region or epitope therein.
Where the polypeptide comprises a modified IgG heavy chain constant region, the modified IgG heavy chain constant region may comprise other amino acid substitutions or modifications as long as it comprises at least one amino acid selected from the group consisting of Arg at position 235, Arg at position 236, Lys at position 239, Gly at position 327, Ser at position 330, Ser at position 331, Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 (all positions numbered according to the EU numbering system).
The ELISA method is illustrated in fig. 3 as a specific embodiment of the method. A "rabbit anti-Fc mutant antibody" corresponds to one of the antibodies described in "ii. antibody", the "anti-hC 5 antibody" in fig. 3A and the "anti-IL-8 antibody" in fig. 3B correspond to polypeptides comprising a modified IgG heavy chain constant region. The rabbit anti-Fc-mutant antibody immobilized on the plate captured the anti-hC 5 antibody in FIG. 3A and the anti-IL-8 antibody in FIG. 3B in the sample. Then, hC5 (human complement 5) as an antigen of the anti-hC 5 antibody, a "mouse anti-hC 5" bound to an epitope different from the epitope bound by the anti-hC 5 antibody, and anti-mouse-POD were reacted in this order in fig. 3A. In FIG. 3B, IL-8 as an antigen of the anti-IL-8 antibody, "mouse anti-IL-8" binding to an epitope different from that bound by the anti-IL-8 antibody, and anti-mouse-POD were reacted in this order. Finally, a POD substrate was added to the plate and its luminescence was measured. In this embodiment, luminescence is detected photometrically when an amount of polypeptide comprising a modified IgG heavy chain constant region is present in the sample.
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, the "mouse anti-Fc-mutant antibody" and the "rabbit anti-Fc-mutant antibody" correspond to any of the antibodies described in "antibody ii", and the "anti-hC 5 antibody" in fig. 4A and the "anti-IL-8 antibody" in fig. 4B correspond to a polypeptide comprising a modified IgG heavy chain constant region. In fig. 4A, a rabbit anti-hC 5 antibody immobilized on a plate captured hC5 in the sample. In FIG. 4B, a mouse anti-IL-8 antibody immobilized on a plate captures IL-8 in the sample. Then, in fig. 4A, the anti-hC 5 antibody, the mouse anti-Fc-mutant antibody, and the anti-mouse-POD reacted in this order. In FIG. 4B, anti-IL-8 antibody, rabbit anti-Fc mutant antibody and anti-rabbit HRP reacted in this order. A POD (peroxidase) substrate such as HRP (horseradish peroxidase) is finally added to the plate and its luminescence is measured. In this embodiment, when an amount of hC5 or IL-8 is present in the sample, luminescence is detected by a luminometer.
The above-described embodiment relating to the ELISA method may be replaced with a Simoa (registered trade Mark) assay. In one embodiment of the assay, the antibodies described in "ii. antibodies" can be used to detect an antigen, such as IL-8, as shown in figure 5. In this embodiment, a "rabbit anti-Fc-mutant antibody" corresponds to one of the antibodies described in "antibody ii", and an "anti-IL-8 antibody" corresponds to a polypeptide comprising a modified IgG heavy chain constant region. The mouse anti-IL-8 antibody immobilized on the beads captures IL-8 in the sample. Then anti-IL-8 antibody, biotinylated anti-Fc-mutant antibody and streptavidin-beta-galactosidase (SBG (Quanterix corporation)) were reacted in this order. Finally, a substrate for β -galactosidase (RGB) was added to the reaction and its luminescence was measured.
Example 1
Production of antibodies comprising constant regions comprising multiple mutations in the Fc region for antibody expression and purification
Antibodies comprising constant regions comprising multiple mutations in the Fc region were expressed by the FreeStyle293 expression system. The constant region used (SEQ ID NO:1) is referred to as SG115 in WO2016098356A 1. The Harvested Cell Culture Fluid (HCCF) was purified with r protein a resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex200pg, GE). During SEC, we changed the buffer to 20mmol/L histidine, 150mmol/L arginine-aspartic acid, pH 6.0. Finally, the antibody was concentrated to 143mg/mL using UF (ultrafiltration).
Digestion with papain
For papain digestion, we used the Pierce Fab preparation kit (Pierce, cat 44985). The papain digestion process is as follows.
The antibody concentration was adjusted to 8.0mg/mL with digestion buffer.
Add 0.5mL of antibody solution to the centrifuge column containing equilibrated papain resin. The top and bottom plugs were placed on a spin column.
-incubating the digestion reaction solution at 37 ℃ for 15 hours on a rotator.
After incubation, the bottom cap is removed and the column is placed in a microcentrifuge tube. The column was centrifuged at 5000x g for one minute.
The resin was washed with 0.5mL of Dulbecco's PBS (-). The column was placed in a microcentrifuge tube. The column was centrifuged at 5000x g for 1 min.
-combining the solutions of step 4 and step 5 as a digestion fraction. The total volume of one column was 1.0 mL.
Purification of Fc fragments
The papain digested samples were purified with r protein a resin (MabSelect SuRe, GE) and size exclusion chromatography (SEC, Superdex200pg, GE). During SEC, we removed whole IgG (undigested molecules) and changed the buffer to Dulbecco's PBS (-).
Example 2
Generation of antibodies recognizing mutations in SG115
Antibodies that recognize mutations in SG115, referred to as "anti-SG 115 antibodies," were prepared, selected, and determined as described below.
Ten week old NZW rabbits were immunized intradermally with the Fc fragment of SG115 (50-100 μ g/dose/rabbit). The administration was repeated 5 times within 2 months, and then blood was collected from the immunized rabbits. Antigen-specific B cells were sorted with a cell sorter according to the procedure described in WO2016098356a1, then plated and cultured. After incubation, the B cell culture supernatant was collected for further analysis, and the pellet was cryopreserved.
The ability to bind SG115 was assessed by ELISA using B cell culture supernatants. We tested binding to 5 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). An alignment of the sequences of these 5 constant regions is shown in FIG. 1.
10,560B cell lines were co-screened for binding to the 5 modified IgG heavy chain constant regions, 186 cell lines were selected from them and designated SKA0001-SKA0186, which bound to SG115 but not to G1m and G4d, and also to SG115v1 and/or Sg115v 2. RNA from the selected cell lines was purified from the cryopreserved cell pellets using the ZR-96 Quick-RNA kit (ZYMO RESEARCH, cat # R1053). The DNA encoding the antibody heavy chain variable region in the selected cell line was amplified by reverse transcription PCR and recombined with the DNA encoding the heavy chain constant region of rbIgGv 2(SEQ ID NO: 6). The DNA encoding the antibody light chain variable region was amplified by reverse transcription PCR and recombined with the DNA encoding the rbIgk light chain constant region (SEQ ID NO: 7). Antibodies in FreeStyleTM293-F cells (Invitrogen) and purified from culture supernatant. By further evaluation, 12 clones were selected based on binding ability and specificity in ELISA, and the sequence diversity of the heavy chain CDR 3. Of these clones, 6 clones (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054 and SKA0127) showed selective binding to SG115v1 but not to SG115v2, while the other 6 clones (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, SKA0171) showed selective binding to SG115v2 but not to SG115v1 (fig. 2-1 and 2-2). The VH and VL sequences of these 12 antibodies are listed in table 1.
[ Table 1]
Example 3
Detection of SG115 in a sample by anti-SG 115 antibody
In order to detect an antibody (hereinafter also referred to as "Fc-mutant antibody" or "Fc-mutant antibodies") comprising an Fc region containing a mutation of all or part of the Fc region of SG115 in a biological sample, the effectiveness of the above 12 monoclonal antibodies (hereinafter also referred to as "anti-Fc-mutant antibody" or "anti-Fc-mutant antibodies") was evaluated. A specific anti-human C5 antibody containing SG115 was used as a model for the Fc-mutant antibody in examples 3-5, which is hereinafter referred to as "anti-hC 5 antibody".
Measurement procedure
Each well of the 96-well immunoplate was coated with rabbit anti-Fc-mutant antibody and blocked with blocking buffer. Diluted serum samples were added to each well of the plate. Recombinant human C5 was added to each well of the plate. A mouse anti-hC 5 antibody was added followed by an anti-mouse-POD (Jackson ImmunoResearch Inc.). Finally, a POD substrate was added to each well of the plate and the OD was measured. The plate was washed between steps.
Reactivity test for antibody selection
12 anti-Fc-mutant antibodies were tested. anti-hC 5 antibodies containing SG115 were diluted with pooled human serum and measured using 12 candidate rabbit anti-Fc mutant antibodies. And calculating the signal-to-noise ratio. The measured OD is shown in Table 2. Three candidates (6 total) were selected from each epitope type for selectivity testing (table 2). That is, SKA0009, SKA0052 and SKA0127 were selected as antibodies specifically binding to SG115v1, and SKA0117, SKA0141 and SKA0171 were selected as antibodies specifically binding to SG115v 2.
[ Table 2]
Selective testing of antibody selection
10 individual serum and calibration curve samples were measured with or without a spiked (spiked) anti-hC 5 antibody using 6 candidate antibodies as capture reagents. In the absence of the spiked anti-hC 5 antibody, the measured concentration of all individual samples was below the limit of quantitation (BLQ) in any given rabbit anti-Fc mutant antibody. In the case of the spiked anti-hC 5 antibody, the Relative Error (RE) in the measured concentration was within +/-20% for all individual samples in any given rabbit anti-Fc mutant antibody (Table 3).
[ Table 3]
BLQ below the limit of quantitation
Selected antibodies
According to the results of the reactivity test and the selectivity test, SKA0141 was selected, which had the highest signal-to-noise ratio.
Example 4
Method for evaluating measurement of anti-hC 5 antibody in human serum (Fc mutation antibody detection assay)
Measurement method
A 96-well immunoplate was coated with rabbit anti-Fc-mutant antibody (SKA0141) and blocked with blocking buffer. A diluted serum sample comprising anti-hC 5 antibody was added to each well of the plate. Recombinant human C5 was added to each well of the plate. A mouse anti-hC 5 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.
Evaluation of the method
Reproducibility was tested. The in-batch accuracy (RE) and precision (CV) were-16.3% to-5.1% and 1.6% to 4.4%, respectively (table 4). The batch to batch accuracy (RE) and precision (CV) were-10.1% to-4.0% and 2.7% to 6.9%, respectively (table 5).
[ Table 4]
[ Table 5]
The selectivity was tested. The concentration measured for all individual samples was BLQ without the addition of a standard anti-hC 5 antibody. The RE measured concentration for all individual samples was-15.2% to 2.2% with the use of a spiked anti-hC 5 antibody (table 6).
[ Table 6]
Dilution linearity was tested. 1 mg per ml of anti-hC 5 antibody could be measured at a 50,000-fold dilution factor and no prozone effect was observed. (Table 7)
[ Table 7]
ALQ higher than the limit of quantitation
Interference from C5 was tested. No interference from C5 was observed (table 8).
[ Table 8]
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 FIG. 3.
Example 5
We also attempted to establish an assay for detecting antigens recognized by Fc mutant antibodies in biological samples. anti-hC 5 antibody was also used as a model for Fc mutant antibodies in this evaluation.
Method for evaluating and measuring C5 in human serum (antigen detection assay)
Measurement method
The 96-well immune plates were coated with rabbit anti-Fc-monoclonal antibodies and blocked with blocking buffer. Diluted serum samples were added to each well of the plate. anti-hC 5 antibody was added to each well of the plate. A mouse anti-Fc-mutant antibody obtained by replacing the Fc region in SKA0141 with a mouse Fc region was added to each well of the plate, followed by addition of 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.
Evaluation of the method
Reproducibility was tested. The in-batch accuracy (RE) and precision (CV) were-8.2% to 4.2% and 2.3% to 6.2%, respectively (table 9). The batch to batch accuracy (RE) and precision (CV) were-6.8% to 1.3% and 3.5% to 6.2%, respectively (table 10).
[ Table 9]
Recombinant human C5 was spiked into C5 depleted serum.
Pooled human serum (endogenous human C5). Concentrations are the average measured concentration in the batch-to-batch reproducibility test.
Recombinant human C5 was spiked into pooled human serum. The concentrations were endogenous human C5 concentration (89.1. mu.g/mL) + spiked recombinant human C5 concentration (65.0. mu.g/mL).
[ Table 10]
Recombinant human C5 was spiked into C5 depleted serum.
Pooled human serum (endogenous human C5). Concentrations are the average measured concentration in the batch-to-batch reproducibility test.
Recombinant human C5 was spiked into pooled human serum. The concentrations were endogenous human C5 concentration (89.1. mu.g/mL) + spiked recombinant human C5 concentration (65.0. mu.g/mL).
Parallelism was tested. Ten individual sera were serially diluted from 325-fold to 2600-fold and measured. At any dilution factor, the measured concentration was recovered (table 11).
[ Table 11]
Dilution linearity was tested. 1130 mg C5 per ml could be measured at a 26,000 fold dilution factor and no prozone effect was observed. (watch 12)
[ Table 12]
ALQ higher than the limit of quantitation
Interference from anti-hC 5 antibody was tested. No interference from anti-hC 5 antibody was observed (table 13).
[ Table 13]
A method for measuring an antigen in human serum using an anti-Fc mutant antibody was established. The protocol for this assay is shown in FIG. 4.
Example 6
Evaluation of the method for measuring anti-IL-8 antibodies in human plasma (Fc mutant antibody detection assay)
Measurement method
A specific anti-human IL-8 antibody comprising a modified IgG heavy chain constant region (wherein the modified IgG heavy chain constant region comprises a partial mutation in the Fc region of SG 115) (SEQ ID NO:110) was used as a model for the Fc-mutated antibody of examples 6-8, which was referred to as an "anti-IL-8 antibody".
A 96-well immunoplate was coated with one of the rabbit anti-Fc-mutant antibodies (SKA0117) and blocked with blocking buffer. Diluted plasma samples were added to each well of the plate. Recombinant human IL-8(SEQ ID NO:111) was added to each well of the plate. A mouse anti-IL-8 monoclonal antibody (heavy chain variable region, SEQ ID NO: 112; light chain variable region, SEQ ID NO: 113; heavy chain constant region, SEQ ID NO: 114; light chain constant region, SEQ ID NO:115) was added, followed by anti-mouse-POD (Jackson ImmunoResearch Inc.). Finally, POD substrate was added to each well of the plate and OD was measured. The plate was washed between steps.
Evaluation of the method
Reproducibility was tested. Known concentrations of anti-IL-8 antibody (50.0ng/mL (REP-LL), 100ng/mL (REP-L), 400ng/mL (REP-M), 2400ng/mL (REP-H), and 3200ng/mL (REP-UL)) were measured. The in-batch accuracy (RE) and precision (CV) were-14.2% to-9.7% and 4.9% to 7.3%, respectively (table 14).
[ Table 14]
The batch to batch accuracy (RE) and precision (CV) were-10.3% to-6.6% and 7.0% to 10.1%, respectively (table 15).
[ Table 15]
The selectivity was tested. In the absence of a labeled anti-IL-8 antibody (SEL-O or SEL-EM-O), all individual samples were measured at BLQ. The measured concentrations of RE were-23.2% to-4.3% for all individual samples using a spiked anti-IL-8 antibody (50.0ng/mL (SEL-LL or SEL-EM-LL)) (Table 16).
[ Table 16]
Lower limit of quantitation
Dilution linearity was tested. 1.6 mg per ml of anti-IL-8 antibody could be measured at a10,000 fold dilution factor and no prozone effect was observed (Table 17).
[ Table 17]
Quantitative range: 1.00ng/mL to 64.0ng/mL in assay wells
Higher than upper limit of quantitation
Interference from IL-8 was tested. IL-8 at concentrations of anti-IL-8 of 2400ng/mL did not interfere with the assay and IL-8 at concentrations of anti-IL-8 of 1.00ng/mL did not interfere with the assay at 50.0ng/mL (Table 18).
[ Table 18]
The effectiveness of the method for measuring anti-IL-8 antibodies in plasma was demonstrated.
Example 7
Evaluation of the method for measuring IL-8 in human plasma Using ELISA (antigen detection assay)
Measurement method
After blocking with blocking buffer, a 96-well streptavidin immunoplate was 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.
Evaluation of the method
Reproducibility was tested. The in-batch accuracy (RE) and precision (CV) were-6.4% to-1.9% and 1.5% to 2.9%, respectively (table 19).
[ Table 19]
Conversion values outside the quantitative range (less than 100pg/mL in plasma and more than 3200pg/mL in plasma) were included in the calculations.
The results of the day time reproducibility were used as the results of the first measurement of day time reproducibility.
CV: coefficient of variation
RE: relative error
The batch to batch accuracy (RE) and precision (CV) were-8.0% to 2.1% and 3.0% to 5.2%, respectively (table 20).
[ Table 20]
Dilution linearity was tested. 1 mg IL-8 per ml (spiked) could be measured at 20,000 fold dilution factor and no prozone effect was observed (Table 21).
[ Table 21]
Spiked concentrations in pooled human plasma: 1000ng/mL
ALQ: higher than the upper limit of quantitation (> 640pg/mL)
CV: coefficient of variation
RE: relative error
Interference from anti-IL-8 antibodies was tested. No interference from anti-IL-8 antibody (100. mu.l/mL in plasma) was observed (Table 22).
[ Table 22]
Batch number 190702
Difference% (% conversion of sample with added IS-conversion of sample without IS)/conversion of sample without IS x100
IS: interfering substances
ND: not detected
Underlining: below the lower limit of quantitation (< 100pg/mL in plasma)
NA: not applicable to
The effectiveness of the method for measuring IL-8 in human plasma using ELISA was confirmed.
Example 8
Evaluation of method for measuring IL-8 in human plasma Using Simoa (registered trademark) (Simoa (registered trademark) assay)
Measurement procedure
The measurement was automatically performed using a Simoa (registered trademark) system (Quanterix Corporation). The diluted sample, anti-IL-8 antibody and the coated beads on the mouse anti IL-8 monoclonal antibody mixture. The beads are loaded into the microwells of the array tray. Biotinylated anti-Fc mutant antibody (SKA0028) was added to the disc followed by streptavidin- β -galactosidase, sbg (quantrix corporation). Finally, the substrate RGB for β -galactosidase was added, and the fluorescence intensity was measured.
Evaluation of the method
Reproducibility was tested. The precision (CV) in the batch was 1.3% to 14.3% (Table 23).
[ Table 23]
The batch-to-batch precision (CV) was 8.6% to 24.7%, respectively (Table 24).
[ Table 24-1]
[ Table 24-2]
Parallelism was tested. Three separate plasma were serially diluted from 20-fold to 40-fold and measured. At any dilution factor, the measured concentration was recovered (table 25).
[ Table 25]
Dilution linearity was tested. 3.48 mg IL-8 per ml can be measured at a dilution factor of 50,000 and no prozone effect is observed. (Table 26).
[ Table 26]
-: not applicable to
*: the theoretical concentration was calculated taking into account the endogenous IL-8 concentration in plasma (batch No. PLA022A100E001) as determined in run-to-run precision.
Interference from anti-IL-8 antibodies was tested. No interference from anti-IL-8 antibody (100. mu.g/mL in plasma) was observed (Table 27).
[ Table 27]
The effectiveness of the method for measuring IL-8 in human plasma using the Simoa (registered trade Mark) assay was demonstrated.
Example 9
anti-Fc-mutant antibodies shown to selectively bind SG115v1 were evaluated for affinity for anti-hC 5 antibody.
anti-Fc-mutant antibodies (SKA0009, SKA0016, SKA0046, SKA0052, SKA0054 and SKA0127) that showed selective binding to SG115v1 at pH7.4 were directed against K of anti-hC 5 antibodyDValues were determined at 25 ℃ using a Biacore T200 instrument (GE Healthcare).
Mouse anti-rabbit IgG (FC) antibody (hereinafter referred to as anti-rabbit IgG) (Abbexa) was immobilized to Flow Cells (FC)1 and 2 of a CM5 sensor chip using an amine coupling kit (GE Healthcare). For the immobilization of anti-rabbit IgG, HBS-EP +, pH7.4(GE Healthcare) buffer was used as the running buffer. After fixation, the running buffer was changed to phosphate pH7.4 buffer (50mM phosphate buffer containing 150mM NaCl and 0.05 w/v% P-20, pH 7.4). Each anti-Fc-mutant antibody was captured by anti-rabbit IgG onto Fc2 of the sensor chip. The amount of anti-Fc-mutant antibody to be captured was adjusted so that the number of Resonance Units (RU) was 100. anti-hC 5 antibody was injected at 0, 50, 100, 200, 400, and 800nM at 10 μ l/min. After each cycle, the sensor surface was regenerated with 10mM glycine-HCl, ph2.0, which was injected at a flow rate of 30 μ l/min. KDValues were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare). Association rate (ka), dissociation rate (kd), and dissociation constant (K)D) Shown in table 28.
[ Table 28]
*: data reliability may be low due to slow dissociation rate
Example 10
anti-Fc-mutant antibodies that showed selective binding to SG115v1 were evaluated for affinity for anti-IL-8 antibodies.
To confirm the binding ability of the anti-Fc-mutant antibody exhibiting selective binding to SG115v1, the anti-Fc-mutant antibody was directed against the K of the anti-IL-8 antibody at pH7.4DValues were determined at 25 ℃ using a Biacore T200 instrument (GE Healthcare). The sequence of the Fc region of the anti-IL-8 antibody was highly similar to that of the anti-hC 5 antibody.
Using amine coupling reagentsThe cassette (GE Healthcare) immobilizes anti-rabbit igg (abbexa) onto FC1 and 2 of a CM5 sensor chip. For the immobilization of anti-rabbit IgG, HBS-EP +, pH7.4(GE Healthcare) buffer was used as the running buffer. After fixation, the running buffer was changed to phosphate pH7.4 buffer (50mM phosphate buffer containing 150mM NaCl and 0.05 w/v% P-20, pH 7.4). Each anti-Fc-mutant antibody was captured by anti-rabbit IgG onto Fc2 of the sensor chip. The amount of anti-Fc-mutant antibody to be captured was adjusted so that the number of Resonance Units (RU) was 100. anti-IL-8 antibody was injected at 10 μ l/min at 0, 100, 400 and 800 nM. After each cycle, the sensor surface was regenerated with 10mM glycine-HCl, ph2.0, which was injected at a flow rate of 30 μ l/min. KDValues were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare).
ka. kd and KDAre listed in Table 29. Although the amino acid at position 239 was mutated from Ser to Lys in the anti-hC 5 antibody according to the EU numbering system, the amino acid at the corresponding position in the anti-IL-8 antibody was not mutated. In this case, the anti-Fc-mutant antibody can be combined with an anti-IL-8 antibody. This means that two mutations common to anti-hC 5 antibody and anti-IL-8 antibody in SG115v1, i.e. L235R and G236R (both positions numbered according to the EU numbering system), are essential for the selective binding of anti-Fc-mutant antibodies to SG115v 1.
[ Table 29]
*: data reliability may be low due to slow dissociation rate
Example 11
anti-Fc-mutant antibodies that showed selective binding to SG115v2 were directed against DENV E protein antibody affinity assessment.
anti-Fc-mutant antibodies (SKA0001, SKA0027, SKA0028, SKA0117, SKA0141, and SKA0171) that showed selective binding to SG115v2 at pH7.4 were directed against K of anti-DENV E protein antibodies that included a modified IgG heavy chain constant region (SEQ ID NO:116) as the Fc mutant antibodyDThe values were measured at 25 ℃ using a Biacore T200 instrument(GE Healthcare). Anti-rabbit IgG was immobilized to FC3 and 4 of CM5 sensor chip using amine coupling kit (GE Healthcare). For the immobilization of anti-rabbit IgG, HBS-EP +, pH7.4(GE Healthcare) buffer was used as the running buffer. After fixation, the running buffer was changed to phosphate ph7.4 buffer. Each antibody was captured by anti-rabbit IgG to FC4 on the sensor chip. The amount of anti-Fc-mutant antibody to be captured was adjusted so that the number of Resonance Units (RU) was 100. anti-DENV E protein antibodies were injected at 0, 12.5, 50 and 400nM at 10 μ l/min. After each cycle, the sensor surface was regenerated with 10mM glycine-HCl, ph2.0, which was injected at a flow rate of 30 μ l/min. KDValues were obtained using Biacore T200 evaluation software version 2.0 (GE Healthcare). ka. kd and KDShown in table 30.
[ Table 30]
*: data reliability may be low due to slow dissociation rate
Example 12
The affinity of anti-hC 5 antibodies for human C5 was evaluated using SKA0016 and SKA0117 as capture molecules.
K of anti-hC 5 antibody at pH7.4 to human C5 was determined at 37 ℃ using a Biacore T200 instrument (GE Healthcare)DThe value is obtained. SKA0016 was fixed to FC1 and 2 of CM5 sensor chip and SKA0117 was fixed to FC3 and 4 of CM5 sensor chip using amine coupling kit (GE Healthcare). For the immobilization of SKA0016 and SKA0117, HBS-EP +, pH7.4(GE Healthcare) buffer was used as the running buffer. After fixation, the running buffer was changed to phosphate ph7.4 buffer. The anti-hC 5 antibody was captured by SKA0016 and SKA0117 onto FC2 and FC4 of the sensor chip. The amount of anti-hC 5 antibody to be captured was adjusted so that the number of Resonance Units (RU) was 35. Human C5 was injected at 0, 2, 4,8, 16, and 32nM at 10 μ l/min. In each cycle, the sensor surface was regenerated with 100mM glycine-HCl, pH2.0, followed by 25mM NaOH, both injected at a flow rate of 30 μ l/min. KDValues were obtained using Biacore T200 evaluation software, version 2.0 (GE Healthcare). ka. kd and KDAre listed in Table 31.
[ Table 31]
Capture molecules
ka(1/Ms)
kd(1/s)
KD(M)
SKA0016
6.33×105
1.13×10-4
1.78×10-10
SKA0117
6.51×105
1.23×10-4
1.88×10-10
Example 13
pH-dependent interaction of anti-hC 5 antibodies with human C5 using SKA0016 and SKA0117 as immobilized molecules Qualitative analysis was performed.
The pH-dependent interaction of anti-hC 5 antibody with human C5 at pH7.4 and pH6.0 was evaluated at 37 ℃ using a Biacore T200 instrument (GE Healthcare). anti-hC 5 antibody was captured on FC2 and FC4 of the CM5 chip prepared in example 12. The amount of anti-hC 5 antibody to be captured was adjusted so that the number of Resonance Units (RU) was 35. To confirm the association between the anti-hC 5 antibody and human C5 at pH7.4, 32nM of human C5 was injected into all FCs in phosphate pH7.4 buffer. The dissociation phase was then monitored in phosphate pH7.4 buffer or phosphate pH6.0 buffer (50mM phosphate buffer containing 150mM NaCl and 0.05 w/v% P-20, pH 6.0) as the running buffer. After monitoring the dissociation phase, the sensor chip was regenerated by injecting 100mM Gly-HCl, pH2.0 and then 25mM NaOH, both injected at a flow rate of 30. mu.l/min. The pH-dependent interaction of anti-hC 5 antibody with human C5 was analyzed by comparing the dissociation phases of the sensorgrams at pH7.4 and pH6.0 using Biacore T200 evaluation software version 2.0. The sensorgram at FC2 was subtracted FC1, the sensorgram at FC4 was subtracted FC3, and each sensorgram was normalized by adjusting the human C5 binding response (5 seconds before the end of the human C5 injection) to the value "100".
Regardless of the capture molecule, the rate of dissociation of human C5 from anti-hC 5 antibody was faster than that at pH7.4 at pH6.0 (FIG. 6; anti-hC 5 antibody was immobilized by (a) SKA0016 and (b) SKA 0117). Thus, both SKA0016 and SKA0117 are believed to be effective in monitoring pH-dependent interactions between anti-hC 5 antibodies and human C5.
Example 14
Binding between human Fc receptor (hFcRn) and anti-hC 5 antibody captured by SKA0016 was assessed.
A Biacore T200 instrument (GE Healthcare) was used to assess whether human FcRn could bind to anti-hC 5 antibody captured by SKA0016 at pH 6.0. The anti-hC 5 antibody was captured onto FC2 by SKA0016, wherein said SKA0016 was immobilized onto a CM5 chip by the same procedure as in example 9. Phosphate ph6.0 buffer was used as running buffer. The amount of anti-hC 5 antibody to be captured was adjusted so that the number of Resonance Units (RU) was 400. hFcRn was injected with single cycle kinetics at 0, 26.3, 52.5, 105, 210 and 420nM at 10 μ l/min. The sensor surface was regenerated with 100mM glycine-HCl, pH2.0, followed by 25mM NaOH, both injected at a flow rate of 30 microliters/min. The increase in hFcRn binding response was confirmed using Biacore T200 evaluation software version 2.0 (GE Healthcare).
FIG. 7 shows a sensorgram for FC1 (dashed line) and FC2 (solid line). From the FC2 sensorgram (solid line) it can be seen that the binding response of hFcRn increases in a concentration-dependent manner. SKA0016 did not interrupt the binding between hFcRn and anti-hC 5 antibody. However, hFcRn appears to bind to the Fc region of SKA0016, as sensorgrams for Fc1 also show an increase in binding response (dashed line). This undesired binding of human FcRn to the capture molecule can be addressed by introducing amino acid substitutions into SKA0016 that abrogate binding to human FcRn.
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