Peptides analyzable and / or assessable by mass spectrometry and uses thereof

Abstract

The inventors have devised a method for designing unique sequences by modifying a part of the sequence of an antigen binding molecule and for analyzing and / or evaluating said sequences with a mass spectrometer. Based on the devised idea, the present application provides, for example: antigen binding molecules; methods for detecting and / or quantifying antigen binding molecules in a biological sample; methods for designing, selecting or producing modified antigen binding molecules; modified antigen binding molecules designed, selected or produced by said methods; peptides derived from antigen binding molecules and capable of being analyzed and / or evaluated by mass spectrometry; and collections comprising two or more types of said peptides.

Description

Technical Field

[0001] This application relates to peptides that can be analyzed and / or evaluated by mass spectrometry and their uses. Background Technology

[0002] One technique for detecting, quantifying, or studying the in vivo kinetics and distribution of antigen-binding molecules (such as antibodies) is mass spectrometry of peptides containing specific sequences contained in antigen-binding molecules (Patent Document (PTL) 1). Examples of measurements using the unique sequences contained in each antibody are described in Non-Patent Document (NPL) 1. There is also a method for identifying antibodies and antigen-binding molecules by adding an amino acid sequence used as a barcode to the antigen-binding molecule (such as an antibody) and measuring the barcode portion.

[0003] Cartridge dosing studies (mixed administration studies) are known as a technique that simultaneously administers multiple drugs or samples to an animal and simultaneously evaluates their pharmacokinetics and distribution in the same animal. Cartridge dosing is advantageous because it allows for the simultaneous evaluation of multiple samples in a single animal, facilitating comparisons with standards and reducing the number of animals used. To date, the following four cartridge dosing studies have been conducted on antibodies:

[0004] (1) Mix three types of antibodies that recognize different antigens and measure them using the ECL method (NPL 1) with three different antigens.

[0005] (2) Method of labeling antibodies with different radioactive metals (NPL 2);

[0006] (3) A method for measuring a sequence containing an antibody-specific sequence present in the variable region of an antibody using a mass spectrometer (NPL 3); and

[0007] (4) A method for adding an amino acid sequence to the end of an antibody as a barcode and measuring the barcode site.

[0008] Citation List

[0009] Non-patent literature

[0010] [NPL 1] Pharmaceutical Research, Vol. 38, 583-592 (2021)

[0011] [NPL 2] Methods Mol Biol. 2014; 1141: 147-157

[0012] [NPL 3] Clin Vaccine Immunol. May 2017; 24(5): e00545-16

[0013] Patent documents

[0014] [PTL 1] WO2012 / 155019

[0015] [PTL 2] WO2008 / 093762 Summary of the Invention

[0016] Technical issues

[0017] The purpose of this application is to provide a new technique for analyzing and / or evaluating antigen-binding molecules using mass spectrometry.

[0018] Solution to the problem

[0019] The inventors have invented a technique for designing unique sequences by modifying a portion of the sequence of an antigen-binding molecule and analyzing and / or evaluating the sequence using mass spectrometry. Based on this invention, this application provides antigen-binding molecules, methods for detecting and / or quantifying antigen-binding molecules in biological samples, methods for designing, selecting, or generating modified antigen-binding molecules, and peptides derived from antigen-binding molecules that can be analyzed and / or evaluated by mass spectrometry. For example, this application provides the following:

[0020] [A1] An antigen-binding molecule comprising an antigen-binding domain, a CL domain, and a non-natural human IgG CH1 domain, wherein the amino acid sequence in the non-natural human IgG CH1 domain between positions 121 and 133 according to the EU index is different from the corresponding sequence in natural human IgG having the same isotype as the non-natural human IgG.

[0021] [A2] According to the antigen-binding molecule of [A1], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0022] (1) The amino acid at any one or more sites from position 122 to 132 is different from the amino acid at the corresponding site in the same type of natural human IgG, and the amino acid at said site is an amino acid other than Lys, Arg and Met (provided that the amino acid at position 122 is not Pro).

[0023] (2) An amino acid other than Lys, Arg and Met has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130, the site between positions 130 and 131, the site between positions 131 and 132, and the site between positions 132 and 133 (provided that the amino acid at the site between positions 121 and 122 is not Pro).

[0024] [A3] The antigen-binding molecule according to [A1] or [A2], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0025] (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala;

[0026] (2) The amino acid at any one or more sites from position 122 to 132 is Ile;

[0027] (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val;

[0028] (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu;

[0029] (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe;

[0030] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0031] [A4] The antigen-binding molecule according to [A1] or [A2], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0032] (1) The amino acid at any of the positions 122 to 128 and 130 to 132 is Ala;

[0033] (2) The amino acid at any of the positions 122 to 132 is Ile;

[0034] (3) The amino acid at any of the positions 122 to 124 and 126 to 132 is Val;

[0035] (4) The amino acid at any of the positions 122 to 127 and 129 to 132 is Leu;

[0036] (5) The amino acid at any of the positions 122 to 125 and 127 to 132 is Phe;

[0037] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, or the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0038] [A5] The antigen-binding molecule according to any one of [A1] to [A4], wherein the non-natural human IgG CH1 domain is a non-natural human IgG1 CH1 domain, a non-natural human IgG2 CH1 domain, a non-natural human IgG3 CH1 domain, or a non-natural human IgG4 CH1 domain.

[0039] [A6] The antigen-binding molecule according to any one of [A1] to [A5], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is the amino acid sequence of any one of SEQ ID NO: 6 to 294 and 1565 to 1833.

[0040] [A7] An antigen-binding molecule according to any one of [A1] to [A6], wherein the antigen-binding domain is a VH domain and a VL domain.

[0041] [A8] The antigen-binding molecule according to [A7], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain.

[0042] [A9] According to the antigen-binding molecule of [A7] or [A8], wherein the C-terminus of the VH domain is linked to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VL domain is linked to the N-terminus of the CL domain.

[0043] [A10] According to the antigen-binding molecule of [A7] or [A8], wherein the C-terminus of the VL domain is linked to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VH domain is linked to the N-terminus of the CL domain.

[0044] [A11] The antigen-binding molecule according to any one of [A1] to [A10] further comprises an antibody Fc region.

[0045] [A12] The antigen-binding molecule according to any one of [A1] to [A11] is a non-natural IgG antibody.

[0046] [A13] According to the antigen-binding molecule described in [A12], wherein the non-natural IgG antibody is a non-natural IgG1 antibody, a non-natural IgG2 antibody, a non-natural IgG3 antibody, or a non-natural IgG4 antibody.

[0047] [A14] An antigen-binding molecule according to any one of [A1] to [A13], which is partially conjugated to a drug.

[0048] [B1] A method for detecting peptides, the method comprising:

[0049] (a) Preparing a sample containing a peptide, said peptide consisting of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG.

[0050] The amino acid sequence between positions 121 and 133 of the CH1 domain of the non-natural human IgG, according to the EU index, differs from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG; and

[0051] (b) The sample prepared in (a) was analyzed by mass spectrometry to detect the peptide.

[0052] [B2] A method for quantifying peptides, the method comprising:

[0053] (a) Preparing a sample containing a peptide, said peptide consisting of an amino acid sequence in the CH1 domain of a non-natural human IgG cell between positions 121 and 133 according to the EU index.

[0054] The amino acid sequence between positions 121 and 133 of the CH1 domain of the non-natural human IgG, according to the EU index, is different from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG;

[0055] (b) Analyzing the sample prepared in (a) by mass spectrometry to detect the peptide; and

[0056] (c) Quantify the peptide based on the results of the analysis described in (b).

[0057] [B3] A method for detecting and / or quantifying antigen-binding molecules in a biological sample, the method comprising:

[0058] (a) Treating a biological sample containing an antigen-binding molecule with a digestive enzyme to produce a peptide from the antigen-binding molecule, wherein

[0059] The antigen-binding molecule comprises an antigen-binding domain, a CL domain, and a non-natural human IgG CH1 domain.

[0060] The amino acid sequence between positions 121 and 133 of the CH1 domain of the non-natural human IgG, according to the EU index, differs from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG, and

[0061] The peptide consists of the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index;

[0062] (b) Analyzing the biological sample treated in (a) by mass spectrometry to detect the peptide; and

[0063] (c) Detect and / or quantify the antigen-binding molecules based on the results of the analysis described in (b).

[0064] [B4] According to any one of [B1] to [B3], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0065] (1) The amino acid at any one or more sites from position 122 to 132 is different from the amino acid at the corresponding site in the same type of natural human IgG, and the amino acid at said site is an amino acid other than Lys, Arg and Met (provided that the amino acid at position 122 is not Pro).

[0066] (2) An amino acid other than Lys, Arg and Met has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130, the site between positions 130 and 131, the site between positions 131 and 132, and the site between positions 132 and 133 (provided that the amino acid at the site between positions 121 and 122 is not Pro).

[0067] [B5] According to any one of [B1] to [B3], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0068] (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala;

[0069] (2) The amino acid at any one or more sites from position 122 to 132 is Ile;

[0070] (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val;

[0071] (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu;

[0072] (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe;

[0073] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0074] [B6] According to any one of [B1] to [B3], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0075] (1) The amino acid at any of the positions 122 to 128 and 130 to 132 is Ala;

[0076] (2) The amino acid at any of the positions 122 to 132 is Ile;

[0077] (3) The amino acid at any of the positions 122 to 124 and 126 to 132 is Val;

[0078] (4) The amino acid at any of the positions 122 to 127 and 129 to 132 is Leu;

[0079] (5) The amino acid at any of the positions 122 to 125 and 127 to 132 is Phe;

[0080] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, or the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0081] [B7] The method according to any one of [B1] to [B6], wherein the non-natural human IgG CH1 domain is a non-natural human IgG1 CH1 domain, a non-natural human IgG2 CH1 domain, a non-natural human IgG3 CH1 domain, or a non-natural human IgG4 CH1 domain.

[0082] [B8] The method according to any one of [B1] to [B6], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is an amino acid sequence of any one of SEQ ID NO: 6 to 294 and 1565 to 1833.

[0083] [B9] The method according to any one of [B3] to [B8], wherein the digestive enzyme is a protease.

[0084] [B10] The method according to any one of [B3] to [B8], wherein the digestive enzyme is trypsin.

[0085] [B11] The method according to any one of [B3] to [B10], wherein step (b) comprises contacting the biological sample treated in step (a) with an affinity capture medium or a chromatographic adsorbent and eluting the biological sample rich in said peptide, and analyzing the enriched biological sample by mass spectrometry.

[0086] [B12] According to the method of [B1] or [B2], wherein step (b) includes contacting the sample prepared in step (a) with an affinity capture medium or a chromatographic adsorbent and eluting the sample rich in said peptide, and analyzing the enriched sample by mass spectrometry.

[0087] [B13] The method according to any one of [B3] to [B10], wherein step (a) comprises contacting the biological sample with an affinity capture medium or a chromatographic adsorbent and eluting the biological sample rich in the antigen-binding molecules, and treating the enriched biological sample with a digestive enzyme.

[0088] [B14] The method according to any one of [B11] to [B13], wherein the affinity capture medium is protein A or protein G.

[0089] [B15] The method according to any one of [B11] to [B13], wherein the chromatographic adsorbent is a solid phase extraction (SPE) adsorbent.

[0090] [B16] The method according to any one of [B1] to [B10], wherein step (b) includes analyzing the biological sample treated in step (a) or the sample prepared in step (a) by mass spectrometry combined with liquid chromatography.

[0091] [B17] The method according to any one of [B1] to [B10], wherein step (b) includes separating a peptide-containing fraction from the biological sample treated in step (a) or the sample prepared in step (a) by liquid chromatography, and analyzing the separated fraction by mass spectrometry.

[0092] [B18] The method according to any one of [B3] to [B10], wherein step (a) comprises separating fractions containing the antigen-binding molecules from the biological sample by liquid chromatography, and treating the separated fractions with a digestive enzyme.

[0093] [B19] The method according to any one of [B1] to [B18], wherein the mass spectrometry is performed by tandem mass spectrometry or multistage mass spectrometry.

[0094] [B20] The method according to any one of [B1] to [B18], wherein the mass spectrometry is performed by tandem mass spectrometry or multistage mass spectrometry in the selected reaction monitoring (SRM) mode.

[0095] [B21] The method according to any one of [B3] to [B20], wherein the biological sample is serum, plasma, tissue or cells of an animal treated with the antigen-binding molecule.

[0096] [B22] The method according to any one of [B3] to [B21], wherein the antigen-binding domain comprises a VH domain and a VL domain.

[0097] [B23] According to the method described in [B22], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain.

[0098] [B24] According to the method of [B22] or [B23], wherein the C-terminus of the VH domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VL domain is connected to the N-terminus of the CL domain.

[0099] [B25] According to the method of [B22] or [B23], wherein the C-terminus of the VL domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VH domain is connected to the N-terminus of the CL domain.

[0100] [B26] The method according to any one of [B3] to [B25], wherein the antigen-binding molecule further comprises an antibody Fc region.

[0101] [B27] According to the method of [B11] or [B13], wherein the antigen-binding molecule further comprises an antibody Fc region, and the affinity capture medium is an anti-Fc antibody.

[0102] [B28] The method according to any one of [B3] to [B27], wherein the antigen-binding molecule is a non-natural IgG antibody.

[0103] [B29] According to the method of [B28], the non-natural IgG antibody is a non-natural IgG1 antibody, a non-natural IgG2 antibody, a non-natural IgG3 antibody, or a non-natural IgG4 antibody.

[0104] [B30] The method according to any one of [B3] to [B29], wherein the antigen-binding molecule is partially conjugated with the drug.

[0105] [C1] A method for detecting multiple types of peptides in a sample, the method comprising:

[0106] (a) Preparation of a sample containing multiple types of peptides, wherein

[0107] The various types of peptides consist of amino acid sequences at positions 121 to 133 according to the EU index in the CH1 domain of natural or non-natural human IgG, and the amino acid sequences are different among the various types of peptides; and

[0108] (b) Analyze the sample prepared in (a) by mass spectrometry to detect each of the multiple types of peptides.

[0109] [C2] A method for quantifying multiple types of peptides in a sample, the method comprising:

[0110] (a) Preparation of a sample containing multiple types of peptides, wherein

[0111] The various types of peptides consist of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural or non-natural human IgG according to the EU index, and the amino acid sequence is different among the various types of peptides;

[0112] (b) Analyzing the sample prepared in (a) by mass spectrometry to detect each of the multiple types of peptides; and

[0113] (c) Quantify each of the multiple types of peptides based on the results of the analysis described in (b).

[0114] [C3] A method for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample, the method comprising:

[0115] (a) Treating a biological sample containing multiple types of antigen-binding molecules with a digestive enzyme to produce multiple types of peptides from said multiple types of antigen-binding molecules, wherein

[0116] The various types of antigen-binding molecules include antigen-binding molecules containing an antigen-binding domain, a CL domain, and a natural or non-natural human IgG CH1 domain.

[0117] The amino acid sequence between positions 121 and 133 of the human IgG CH1 domain, according to the EU index, differs among the various types of antigen-binding molecules.

[0118] The various types of peptides consist of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural or non-natural human IgG according to the EU index, and the amino acid sequence is different among the various types of peptides;

[0119] (b) Analyzing the biological sample treated in (a) by mass spectrometry to detect each of the multiple types of peptides; and

[0120] (c) Based on the results of the analysis described in (b), detect and / or quantify each of the multiple types of antigen-binding molecules.

[0121] [C4] The method according to any one of [C1] to [C3], wherein the plurality of peptides comprises:

[0122] (I) Two or more types of peptides comprising an amino acid sequence in the CH1 domain of a non-natural human IgG, between positions 121 and 133 according to the EU index, wherein said amino acid sequence differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG, or

[0123] (II) A peptide consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural human IgG according to the EU index; and one or more types of peptides consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of non-natural human IgG according to the EU index, wherein the amino acid sequence is different from the corresponding sequence in natural human IgG having the same isotype as non-natural human IgG.

[0124] [C5] According to any one of [C1] to [C4], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0125] (1) The amino acid at any one or more sites from position 122 to 132 is different from the amino acid at the corresponding site in the same type of natural human IgG, and the amino acid at said site is an amino acid other than Lys, Arg and Met (provided that the amino acid at position 122 is not Pro).

[0126] (2) An amino acid other than Lys, Arg and Met has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130, the site between positions 130 and 131, the site between positions 131 and 132, and the site between positions 132 and 133 (provided that the amino acid at the site between positions 121 and 122 is not Pro).

[0127] [C6] According to any one of [C1] to [C4], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0128] (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala;

[0129] (2) The amino acid at any one or more sites from position 122 to 132 is Ile;

[0130] (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val;

[0131] (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu;

[0132] (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe;

[0133] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0134] [C7] According to any one of [C1] to [C4], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0135] (1) The amino acid at any of the positions 122 to 128 and 130 to 132 is Ala;

[0136] (2) The amino acid at any of the positions 122 to 132 is Ile;

[0137] (3) The amino acid at any of the positions 122 to 124 and 126 to 132 is Val;

[0138] (4) The amino acid at any of the positions 122 to 127 and 129 to 132 is Leu;

[0139] (5) The amino acid at any of the positions 122 to 125 and 127 to 132 is Phe;

[0140] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, or the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0141] [C8] The method according to any one of [C1] to [C7], wherein the human IgG CH1 domain is a human IgG1 CH1 domain, a human IgG2 CH1 domain, a human IgG3 CH1 domain or a human IgG4 CH1 domain.

[0142] [C9] The method according to any one of [C1] to [C7], wherein the plurality of peptides comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 6 to 294 and 1565 to 1833.

[0143] [C10] The method according to any one of [C1] to [C7], wherein the plurality of peptides comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 6 to 294.

[0144] [C11] The method according to any one of [C1] to [C7], wherein the plurality of peptides comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 1565 to 1833.

[0145] [C12] The method according to any one of [C1] to [C7], wherein the plurality of peptides comprises: a peptide consisting of an amino acid sequence comprising, according to EU index positions 121 and 133, in the CH1 domain of a natural human IgG; and one or more types of peptides consisting of an amino acid sequence comprising, according to EU index positions 121 and 133, in the CH1 domain of a non-natural human IgG, wherein

[0146] When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 6 to 294, and

[0147] When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 1565 to 1833.

[0148] [C13] The method according to any one of [C1] to [C7], wherein the plurality of peptides are two or more types of peptides selected from the group consisting of (1) to (26):

[0149] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0150] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0151] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0152] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0153] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 9;

[0154] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0155] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0156] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0157] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0158] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0159] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0160] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0161] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0162] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 38;

[0163] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 40;

[0164] (16) A peptide consisting of the amino acid sequence of SEQ ID NO: 42;

[0165] (17) A peptide consisting of the amino acid sequence of SEQ ID NO: 45;

[0166] (18) A peptide consisting of the amino acid sequence of SEQ ID NO: 46;

[0167] (19) A peptide consisting of the amino acid sequence of SEQ ID NO: 47;

[0168] (20) A peptide consisting of the amino acid sequence of SEQ ID NO: 73;

[0169] (21) A peptide consisting of the amino acid sequence of SEQ ID NO: 75;

[0170] (22) A peptide consisting of the amino acid sequence of SEQ ID NO: 76;

[0171] (23) A peptide consisting of the amino acid sequence of SEQ ID NO: 78;

[0172] (24) A peptide consisting of the amino acid sequence of SEQ ID NO: 79;

[0173] (25) A peptide consisting of the amino acid sequence of SEQ ID NO: 80; and

[0174] (26) A peptide consisting of the amino acid sequence of SEQ ID NO: 82.

[0175] [C14] The method according to any one of [C1] to [C7], wherein the plurality of peptides are two or more types of peptides selected from the group consisting of (1) to (13):

[0176] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0177] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0178] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0179] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0180] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 9;

[0181] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0182] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0183] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0184] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0185] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0186] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0187] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0188] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0189] [C15] The method according to any one of [C1] to [C7], wherein the plurality of peptides are two types of peptides, and the two types of peptides are selected from the group consisting of the following (1) to (15):

[0190] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0191] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0192] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0193] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0194] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0195] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0196] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0197] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0198] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0199] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0200] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0201] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0202] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0203] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0204] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0205] [C16] The method according to any one of [C1] to [C7], wherein the plurality of peptides are three types of peptides, and the three types of peptides are selected from the group consisting of (1) to (20):

[0206] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0207] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0208] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0209] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0210] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0211] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0212] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0213] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0214] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0215] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0216] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0217] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0218] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0219] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0220] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0221] (16) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0222] (17) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0223] (18) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0224] (19) A peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0225] (20) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0226] [C17] The method according to any one of [C1] to [C7], wherein the plurality of peptides are four types of peptides, and the four types of peptides are selected from the group consisting of (1) to (15):

[0227] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0228] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0229] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0230] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0231] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0232] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0233] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0234] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0235] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0236] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0237] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0238] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0239] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0240] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0241] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0242] [C18] The method according to any one of [C1] to [C7], wherein the plurality of peptides are five types of peptides, and the five types of peptides are selected from the group consisting of (1) to (6):

[0243] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0244] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0245] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0246] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0247] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0248] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0249] [C19] The method according to any one of [C1] to [C7], wherein the plurality of peptides are of the following six types:

[0250] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0251] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0252] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0253] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0254] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 17; and

[0255] (6) A peptide consisting of SEQ ID NO: 27.

[0256] [C20] The method according to any one of [C1] to [C7], wherein the plurality of peptides are seven types of peptides, and the seven types of peptides are selected from the group consisting of (1) to (4):

[0257] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0258] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0259] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24; and

[0260] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0261] [C21] The method according to any one of [C1] to [C7], wherein the plurality of peptides are eight types of peptides, and the eight types of peptides are selected from the group consisting of (1) to (6):

[0262] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0263] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0264] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0265] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0266] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; and

[0267] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0268] [C22] The method according to any one of [C1] to [C7], wherein the plurality of peptides are nine types of peptides, and the nine types of peptides are selected from the group consisting of (1) to (4):

[0269] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0270] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0271] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; and

[0272] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0273] [C23] The method according to any one of [C1] to [C7], wherein the plurality of peptides are of the following ten types:

[0274] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0275] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0276] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0277] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0278] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0279] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0280] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0281] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0282] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 24; and

[0283] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0284] [C24] The method according to any one of [C1] to [C7], wherein the plurality of peptides are eleven types of peptides, and the eleven types of peptides are selected from the group consisting of (1) to (2):

[0285] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, a peptide consisting of the amino acid sequence of SEQ ID NO: 36, and a peptide consisting of the amino acid sequence of SEQ ID NO: 6; and

[0286] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, a peptide consisting of the amino acid sequence of SEQ ID NO: 36, and a peptide consisting of the amino acid sequence of SEQ ID NO: 8.

[0287] [C25] The method according to any one of [C1] to [C7], wherein the plurality of peptides are twelve types of peptides:

[0288] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0289] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0290] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0291] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0292] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0293] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0294] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0295] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0296] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0297] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0298] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0299] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0300] [C26] The method according to any one of [C3] to [C25], wherein the digestive enzyme is a protease.

[0301] [C27] The method according to any one of [C3] to [C25], wherein the digestive enzyme is trypsin.

[0302] [C28] The method according to any one of [C3] to [C27], wherein step (b) comprises contacting the biological sample treated in step (a) with an affinity capture medium or a chromatographic adsorbent and eluting the biological sample rich in said peptide, and analyzing the enriched biological sample by mass spectrometry.

[0303] [C29] According to the method of [C1] or [C2], wherein step (b) includes contacting the sample prepared in step (a) with an affinity capture medium or a chromatographic adsorbent and eluting the sample rich in said peptide, and analyzing the enriched sample by mass spectrometry.

[0304] [C30] The method according to any one of [C3] to [C27], wherein step (a) comprises contacting the biological sample with an affinity capture medium or a chromatographic adsorbent and eluting the biological sample rich in the antigen-binding molecules, and treating the enriched biological sample with a digestive enzyme.

[0305] [C31] The method according to any one of [C28] to [C30], wherein the affinity capture medium is protein A or protein G.

[0306] [C32] The method according to any one of [C28] to [C30], wherein the chromatographic adsorbent is a solid phase extraction (SPE) adsorbent.

[0307] [C33] The method according to any one of [C1] to [C27], wherein step (b) includes analyzing the biological sample treated in step (a) or the sample prepared in step (a) by mass spectrometry combined with liquid chromatography.

[0308] [C34] The method according to any one of [C1] to [C27], wherein step (b) includes separating a peptide-containing fraction from the biological sample treated in step (a) or the sample prepared in step (a) by liquid chromatography, and analyzing the separated fraction by mass spectrometry.

[0309] [C35] The method according to any one of [C3] to [C27], wherein step (a) comprises separating fractions containing the antigen-binding molecules from the biological sample by liquid chromatography, and treating the separated fractions with a digestive enzyme.

[0310] [C36] The method according to any one of [C1] to [C35], wherein the mass spectrometry is performed by tandem mass spectrometry or multistage mass spectrometry.

[0311] [C37] The method according to any one of [C1] to [C35], wherein the mass spectrometry is performed by tandem mass spectrometry or multistage mass spectrometry in the selected reaction monitoring (SRM) mode.

[0312] [C38] The method according to any one of [C3] to [C37], wherein the biological sample is serum, plasma, tissue or cells of an animal treated with the antigen-binding molecule.

[0313] [C39] The method according to any one of [C3] to [C38], wherein the antigen-binding domain comprises a VH domain and a VL domain.

[0314] [C40] According to the method of [C39], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain.

[0315] [C41] According to the method of [C39] or [C40], wherein the C-terminus of the VH domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VL domain is connected to the N-terminus of the CL domain.

[0316] [C42] According to the method of [C39] or [C40], wherein the C-terminus of the VL domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VH domain is connected to the N-terminus of the CL domain.

[0317] [C43] The method according to any one of [C3] to [C42], wherein the antigen-binding molecule further comprises an antibody Fc region.

[0318] [C44] According to the method of [C28] or [C30], wherein the antigen-binding molecule further comprises an antibody Fc region, and the affinity capture medium is an anti-Fc antibody.

[0319] [C45] The antigen-binding molecule according to any one of [C3] to [C44], wherein the antigen-binding molecule is a non-natural IgG antibody.

[0320] [C46] According to the method of [C45], the non-natural IgG antibody is a non-natural IgG1 antibody, a non-natural IgG2 antibody, a non-natural IgG3 antibody, or a non-natural IgG4 antibody.

[0321] [C47] The method according to any one of [C3] to [C46], wherein the antigen-binding molecule is partially conjugated with the drug.

[0322] [D1] A method for designing, selecting, or generating a modified antigen-binding molecule, said modified antigen-binding molecule comprising an artificially modified peptide moiety detectable by mass spectrometry, wherein said method comprises:

[0323] (I) Identification of peptides obtained by treating an antigen-binding molecule with a digestive enzyme and detectable by mass spectrometry;

[0324] (II) Design artificially modified peptides using any of the following methods:

[0325] (II-1) Delete one or more amino acid residues in the peptide identified in (I) or replace one or more amino acid residues in the peptide identified in (I) with other amino acid residues;

[0326] (II-2) Insert one or more amino acid residues into the peptide identified in (I);

[0327] (II-3) Add an amino acid residue to the N-terminus or C-terminus of the peptide identified in (I);

[0328] (II-4) Combinations of (II-1) and (II-2);

[0329] (II-5) Combinations of (II-1) and (II-3);

[0330] Combinations of (II-6), (II-2), and (II-3);

[0331] Combinations of (II-7), (II-1), (II-2), and (II-3);

[0332] (III) Modify the peptide portion identified in (I) of the antigen-binding molecule of (I) in (II) to produce a modified antigen-binding molecule;

[0333] (IV) The modified antigen-binding molecule produced in (III) is selected if it satisfies any of the following:

[0334] (IV-1) The modified antigen-binding molecule has a Tm value of 50°C or higher;

[0335] (IV-2) The antigen-binding activity of the modified antigen-binding molecule is 10% or higher of the antigen-binding activity of the antigen-binding molecule in (I);

[0336] (IV-3) Both (IV-1) and (IV-2) mentioned above;

[0337] (V) The modified antigen-binding molecule is selected if the portion of the modified antigen-binding molecule selected in (IV) that corresponds to the peptide designed in (II) can be detected by mass spectrometry after the biological sample containing the modified antigen-binding molecule is treated with the digestive enzyme of (I).

[0338] [D2] According to the method described in [D1], the method further includes:

[0339] (VI) Treat biological samples containing antigen-binding molecules of (I) with the digestive enzyme of (I) and then perform pharmacokinetic tests by mass spectrometry to obtain pharmacokinetic spectra or pharmacokinetic parameters.

[0340] (VII) Treat a biological sample containing the modified antigen-binding molecule selected in (V) with the digestive enzyme of (I), and then perform pharmacokinetic testing by mass spectrometry to obtain a pharmacokinetic spectrum or pharmacokinetic parameters; and

[0341] (VIII) If the pharmacokinetic spectrum or pharmacokinetic parameters obtained in (VI) are comparable to those obtained in (VII), then the modified antigen-binding molecule is selected.

[0342] [D3] According to the method of [D1] or [D2], wherein, in step (I), the antigen-binding molecule comprises an antigen-binding domain, a CL domain and a human IgG CH1 domain, and the peptide that can be detected by mass spectrometry is derived from the human IgG CH1 domain.

[0343] [D4] According to the method of [D3], the peptide that can be detected by mass spectrometry consists of an amino acid sequence at positions 120 to 133 according to the EU index in the CH1 domain of human IgG.

[0344] [D5] According to the method described in [D3], the peptide that can be detected by mass spectrometry consists of an amino acid sequence between positions 121 and 133 of the human IgG CH1 domain according to the EU index.

[0345] [D6] The method according to any one of [D1] to [D5], wherein the other amino acid residues in (II-1) are Ala, Ile, Val, Leu or Phe.

[0346] [D7] The method according to any one of [D1] to [D6], wherein in (II-2), the inserted amino acid residue is one, two or three residues.

[0347] [D8] The method according to any one of [D1] to [D7], wherein, in (II-2), the inserted amino acid residue is Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe or Ala-Ala-Ala.

[0348] [D9] The method according to any one of [D1] to [D7], wherein, in (II-2), the amino acid residue is inserted at one, two or three sites of the peptide identified in (I).

[0349] [D10] The method according to any one of [D1] to [D9], wherein in (II-3), the added amino acid residue is one, two or three residues.

[0350] [D11] The method according to any one of [D1] to [D10], wherein the peptide designed in (II) does not contain a site cleaved by the digestive enzyme of (I).

[0351] [D12] The method according to any one of [D1] to [D11], wherein the digestive enzyme is a protease.

[0352] [D13] The method according to any one of [D1] to [D11], wherein the digestive enzyme is trypsin.

[0353] [D14] The method according to any one of [D1] to [D13], wherein the mass spectrometry is a combination of mass spectrometry and liquid chromatography.

[0354] [D15] The method according to any one of [D1] to [D14], wherein the mass spectrometry is a mass spectrometry by tandem mass spectrometry.

[0355] [D16] The method according to any one of [D1] to [D15], wherein the biological sample in steps (V) and (VII) is serum, plasma, tissue or cells of an animal treated with the modified antigen-binding molecule, and the biological sample in step (VI) is serum, plasma, tissue or cells of an animal treated with the antigen-binding molecule.

[0356] [D17] The method according to any one of [D1] to [D16], wherein the antigen-binding molecule in step (I) comprises an antigen-binding domain, a CL domain and a human IgG CH1 domain, and the antigen-binding domain comprises a VH domain and a VL domain.

[0357] [D18] According to the method described in [D17], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain.

[0358] [D19] According to the method of [D17] or [D18], wherein the C-terminus of the VH domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VL domain is connected to the N-terminus of the CL domain.

[0359] [D20] According to the method of [D17] or [D18], wherein the C-terminus of the VL domain is connected to the N-terminus of the non-natural human IgG CH1 domain, and the C-terminus of the VH domain is connected to the N-terminus of the CL domain.

[0360] [D21] The method according to any one of [D1] to [D20], wherein the antigen-binding molecule in step (I) further comprises an antibody Fc region.

[0361] [D22] The method according to any one of [D1] to [D21], wherein the antigen-binding molecule in step (I) is an IgG antibody.

[0362] [D23] According to the method of [D22], wherein the IgG antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody.

[0363] [D24] The method according to any one of [D1] to [D23], wherein the antigen-binding molecule in step (I) is partially conjugated with the drug.

[0364] [E1] A modified antigen-binding molecule designed, selected or generated by any one of [D1] to [D24].

[0365] [E2] peptide, which is designed by step (II) of the method according to any one of [D1] to [D24].

[0366] [F1] peptide, which consists of an amino acid sequence in the CH1 domain of non-natural human IgG between positions 121 and 133 according to the EU index, wherein the amino acid sequence is different from the corresponding sequence in natural human IgG that has the same isotype as non-natural human IgG.

[0367] [F2] The peptide according to [F1], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0368] (1) The amino acid at any one or more sites from position 122 to 132 is different from the amino acid at the corresponding site in the same type of natural human IgG, and the amino acid at said site is an amino acid other than Lys, Arg and Met (provided that the amino acid at position 122 is not Pro).

[0369] (2) An amino acid other than Lys, Arg and Met has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130, the site between positions 130 and 131, the site between positions 131 and 132, and the site between positions 132 and 133 (provided that the amino acid at the site between positions 121 and 122 is not Pro).

[0370] [F3] The peptide according to [F1] or [F2], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0371] (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala;

[0372] (2) The amino acid at any one or more sites from position 122 to 132 is Ile;

[0373] (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val;

[0374] (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu;

[0375] (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe;

[0376] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0377] [F4] The peptide according to [F1] or [F2], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0378] (1) The amino acid at any of the positions 122 to 128 and 130 to 132 is Ala;

[0379] (2) The amino acid at any of the positions 122 to 132 is Ile;

[0380] (3) The amino acid at any of the positions 122 to 124 and 126 to 132 is Val;

[0381] (4) The amino acid at any of the positions 122 to 127 and 129 to 132 is Leu;

[0382] (5) The amino acid at any of the positions 122 to 125 and 127 to 132 is Phe;

[0383] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, or the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0384] [F5] The peptide according to any one of [F1] to [F4], wherein the non-natural human IgG CH1 domain is a non-natural human IgG1 CH1 domain, a non-natural human IgG2 CH1 domain, a non-natural human IgG3 CH1 domain, or a non-natural human IgG4 CH1 domain.

[0385] [F6] The peptide according to any one of [F1] to [F5], wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is the amino acid sequence of any one of SEQ ID NO: 6 to 294 and 1565 to 1833.

[0386] [G1] A collection comprising two or more types of peptides, the two or more types of peptides consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural or non-natural human IgG according to the EU index, wherein the amino acid sequence is distinct between the two or more types of peptides.

[0387] [G2] The set described in [G1] comprises:

[0388] (I) Two or more types of peptides comprising an amino acid sequence in the CH1 domain of a non-natural human IgG, between positions 121 and 133 according to the EU index, wherein said amino acid sequence differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG, or

[0389] (II) A peptide consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural human IgG according to the EU index; and one or more types of peptides consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of non-natural human IgG according to the EU index, wherein the amino acid sequence is different from the corresponding sequence in natural human IgG having the same isotype as non-natural human IgG.

[0390] [G3] According to the set described in [G1] or [G2], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0391] (1) The amino acid at any one or more sites from position 122 to 132 is different from the amino acid at the corresponding site in the same type of natural human IgG, and the amino acid at said site is an amino acid other than Lys, Arg and Met (provided that the amino acid at position 122 is not Pro).

[0392] (2) An amino acid other than Lys, Arg and Met has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130, the site between positions 130 and 131, the site between positions 131 and 132, and the site between positions 132 and 133 (provided that the amino acid at the site between positions 121 and 122 is not Pro).

[0393] [G4] According to the set described in [G1] or [G2], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0394] (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala;

[0395] (2) The amino acid at any one or more sites from position 122 to 132 is Ile;

[0396] (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val;

[0397] (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu;

[0398] (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe;

[0399] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0400] [G5] According to the set described in [G1] or [G2], the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics:

[0401] (1) The amino acid at any of the positions 122 to 128 and 130 to 132 is Ala;

[0402] (2) The amino acid at any of the positions 122 to 132 is Ile;

[0403] (3) The amino acid at any of the positions 122 to 124 and 126 to 132 is Val;

[0404] (4) The amino acid at any of the positions 122 to 127 and 129 to 132 is Leu;

[0405] (5) The amino acid at any of the positions 122 to 125 and 127 to 132 is Phe;

[0406] (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, or the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

[0407] [G6] The set according to any one of [G1] to [G5], wherein the human IgG CH1 domain is a human IgG1 CH1 domain, a human IgG2 CH1 domain, a human IgG3 CH1 domain or a human IgG4 CH1 domain.

[0408] [G7] The set according to any one of [G1] to [G5], wherein the set comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 6 to 294 and 1565 to 1833.

[0409] [G8] The set according to any one of [G1] to [G5], wherein the plurality of peptide types comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 6 to 294.

[0410] [G9] The set according to any one of [G1] to [G5], wherein the plurality of peptide types comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 1565 to 1833.

[0411] [G10] The set according to any one of [G1] to [G5], wherein the set comprises: a peptide consisting of an amino acid sequence in the CH1 domain of a natural human IgG cell between positions 121 and 133 according to the EU index; and one or more types of peptides consisting of an amino acid sequence in the CH1 domain of a non-natural human IgG cell between positions 121 and 133 according to the EU index, wherein

[0412] When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 6 to 294, and

[0413] When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 1565 to 1833.

[0414] [G11] The set according to any one of [G1] to [G5] is a set of two or more types of peptides selected from the group consisting of the following (1) to (26):

[0415] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0416] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0417] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0418] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0419] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 9;

[0420] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0421] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0422] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0423] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0424] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0425] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0426] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0427] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0428] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 38;

[0429] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 40;

[0430] (16) A peptide consisting of the amino acid sequence of SEQ ID NO: 42;

[0431] (17) A peptide consisting of the amino acid sequence of SEQ ID NO: 45;

[0432] (18) A peptide consisting of the amino acid sequence of SEQ ID NO: 46;

[0433] (19) A peptide consisting of the amino acid sequence of SEQ ID NO: 47;

[0434] (20) A peptide consisting of the amino acid sequence of SEQ ID NO: 73;

[0435] (21) A peptide consisting of the amino acid sequence of SEQ ID NO: 75;

[0436] (22) A peptide consisting of the amino acid sequence of SEQ ID NO: 76;

[0437] (23) A peptide consisting of the amino acid sequence of SEQ ID NO: 78;

[0438] (24) A peptide consisting of the amino acid sequence of SEQ ID NO: 79;

[0439] (25) A peptide consisting of the amino acid sequence of SEQ ID NO: 80; and

[0440] (26) A peptide consisting of the amino acid sequence of SEQ ID NO: 82.

[0441] [G12] The set according to any one of [G1] to [G5] is a set of two or more types of peptides selected from the group consisting of the following (1) to (13):

[0442] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0443] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0444] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0445] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0446] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 9;

[0447] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0448] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0449] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0450] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0451] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0452] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0453] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0454] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0455] [G13] The set according to any one of [G1] to [G5], wherein the set is a set of two types of peptides, and the two types of peptides are selected from the group consisting of the following (1) to (15):

[0456] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0457] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0458] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0459] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0460] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0461] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0462] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0463] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0464] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0465] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0466] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0467] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 7 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0468] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 10 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0469] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0470] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0471] [G14] The set according to any one of [G1] to [G5], wherein the set is a set of three types of peptides, and the three types of peptides are selected from the group consisting of the following (1) to (20):

[0472] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0473] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0474] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0475] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0476] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0477] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0478] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0479] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0480] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0481] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0482] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0483] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0484] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0485] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0486] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0487] (16) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0488] (17) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0489] (18) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0490] (19) A peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0491] (20) A peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0492] [G15] The set according to any one of [G1] to [G5], wherein the set comprises the activation of four types of peptides, and the four types of peptides are selected from the group consisting of the following (1) to (15):

[0493] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0494] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0495] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0496] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0497] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0498] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0499] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0500] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0501] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0502] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0503] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0504] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0505] (13) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0506] (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0507] (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0508] [G16] The set according to any one of [G1] to [G5], wherein the set is a set of five types of peptides, and the five types of peptides are selected from the group consisting of the following (1) to (6):

[0509] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0510] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0511] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0512] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0513] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0514] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 17 and a peptide consisting of the amino acid sequence of SEQ ID NO: 27.

[0515] [G17] The set according to any one of [G1] to [G5], which is a set of the following six types of peptides:

[0516] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0517] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0518] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0519] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0520] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 17; and

[0521] (6) A peptide consisting of SEQ ID NO: 27.

[0522] [G18] The set according to any one of [G1] to [G5], wherein the set comprises the activation of seven types of peptides, and the seven types of peptides are selected from the group consisting of the following (1) to (4):

[0523] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0524] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0525] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24; and

[0526] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0527] [G19] The set according to any one of [G1] to [G5], wherein the set is a set of eight types of peptides, and the eight types of peptides are selected from the group consisting of the following (1) to (6):

[0528] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0529] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0530] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0531] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0532] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; and

[0533] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0534] [G20] The set according to any one of [G1] to [G5], wherein the set is a set of nine types of peptides, and the nine types of peptides are selected from the group consisting of the following (1) to (4):

[0535] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0536] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36;

[0537] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; and

[0538] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0539] [G21] The set according to any one of [G1] to [G5], which is a set of the following ten types of peptides:

[0540] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0541] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0542] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0543] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0544] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0545] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 27;

[0546] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0547] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0548] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 24; and

[0549] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0550] [G22] The set according to any one of [G1] to [G5], wherein the set is a set of eleven types of peptides, and the eleven types of peptides are selected from the group consisting of the following (1) to (2):

[0551] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, a peptide consisting of the amino acid sequence of SEQ ID NO: 36, and a peptide consisting of the amino acid sequence of SEQ ID NO: 6; and

[0552] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 5, a peptide consisting of the amino acid sequence of SEQ ID NO: 7, a peptide consisting of the amino acid sequence of SEQ ID NO: 12, a peptide consisting of the amino acid sequence of SEQ ID NO: 10, a peptide consisting of the amino acid sequence of SEQ ID NO: 17, a peptide consisting of the amino acid sequence of SEQ ID NO: 27, a peptide consisting of the amino acid sequence of SEQ ID NO: 13, a peptide consisting of the amino acid sequence of SEQ ID NO: 14, a peptide consisting of the amino acid sequence of SEQ ID NO: 24, a peptide consisting of the amino acid sequence of SEQ ID NO: 36, and a peptide consisting of the amino acid sequence of SEQ ID NO: 8.

[0553] [G23] The set according to any one of [G1] to [G5], which is a set of the following twelve types of peptides:

[0554] (1) A peptide consisting of the amino acid sequence of SEQ ID NO: 5;

[0555] (2) A peptide consisting of the amino acid sequence of SEQ ID NO: 6;

[0556] (3) A peptide consisting of the amino acid sequence of SEQ ID NO: 7;

[0557] (4) A peptide consisting of the amino acid sequence of SEQ ID NO: 8;

[0558] (5) A peptide consisting of the amino acid sequence of SEQ ID NO: 10;

[0559] (6) A peptide consisting of the amino acid sequence of SEQ ID NO: 12;

[0560] (7) A peptide consisting of the amino acid sequence of SEQ ID NO: 13;

[0561] (8) A peptide consisting of the amino acid sequence of SEQ ID NO: 14;

[0562] (9) A peptide consisting of the amino acid sequence of SEQ ID NO: 17;

[0563] (10) A peptide consisting of the amino acid sequence of SEQ ID NO: 24;

[0564] (11) A peptide consisting of the amino acid sequence of SEQ ID NO: 27; and

[0565] (12) A peptide consisting of the amino acid sequence of SEQ ID NO: 36.

[0566] [G24] The set according to any one of [G1] to [G23], which is used in a method for detecting each of a variety of peptides derived from a variety of types of antigen-binding molecules in a biological sample by mass spectrometry, or in a method for detecting and / or quantifying each of a variety of types of antigen-binding molecules in a biological sample.

[0567] [G25] The set according to any one of [G1] to [G23], which is used in a method for detecting each of a variety of peptides derived from a variety of types of antigen-binding molecules in a biological sample by mass spectrometry, or for detecting and / or quantifying each of a variety of types of antigen-binding molecules in a biological sample.

[0568] [G26] A method for administering a mixture of multiple types of antigen-binding molecules to an individual or subject, each of the multiple types of antigen-binding molecules comprising a peptide according to any one of [G1] to [G23].

[0569] [G27] A method for selecting antigen-binding molecules, the method comprising:

[0570] (a) Detecting and / or quantifying each of the multiple types of antigen-binding molecules in a biological sample derived from an individual or subject who has been administered a mixture of multiple types of antigen-binding molecules, wherein each of the multiple types of antigen-binding molecules comprises a peptide according to any one of [G1] to [G23]; and

[0571] (b) Select antigen-binding molecules based on the results of the detection and / or quantification described in (a).

[0572] [G28] An antigen-binding molecule selected by means of the method described in accordance with [G27].

[0573] [G29] An antigen-binding molecule produced by altering the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of an antigen-binding molecule selected according to the method described in [G27] to the native sequence between positions 121 and 133 according to EU index in the CH1 domain of the same isotype of IgG antibody. Attached Figure Description

[0574] [Figure 1-1] Figure 1-1 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0575] [Figure 1-2] Figure 1-2 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0576] [Figure 1-3] Figure 1-3 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0577] [Figure 1-4] Figure 1-4 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0578] [Figure 1-5] Figure 1-5 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0579] [Figure 1-6] Figure 1-6 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0580] [Figure 1-7] Figure 1-7 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0581] [Figure 1-8] Figure 1-8 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0582] [Figure 1-9] Figure 1-9 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0583] [Figure 1-10] Figure 1-10 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0584] [Figure 1-11] Figure 1-11 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0585] [Figure 1-12] Figure 1-12 shows the internal calibration curves generated by mass spectrometry on antibody samples prepared to achieve mouse plasma concentrations ranging from 0.0200 to 5.12 μg / mL. The name of the target antibody is indicated in the upper left corner of the figure. The vertical axis represents the peak area ratio (peak area of ​​the target antibody sample / peak area of ​​the internal standard), and the horizontal axis represents the target antibody concentration (ng / mL).

[0586] [Figure 2-1] Figure 2-1 shows the SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the target peptide being measured is indicated in the upper left corner of each chromatogram. The numerical value in the upper right corner of each chromatogram indicates the peak intensity. The peak intensity indicated in the upper right corner of each chromatogram is set to 100% on the vertical axis of each chromatogram. The horizontal axis shows the elution time (min) in LC.

[0587] [Figure 2-2] Figure 2-2 shows the SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. The numerical value in the upper right corner of each chromatogram indicates the peak intensity. The peak intensity indicated in the upper right corner of each chromatogram is set to 100% on the vertical axis of each chromatogram. The horizontal axis shows the elution time (min) in LC.

[0588] [Figure 2-3] Figure 2-3 shows the SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the target peptide being measured is indicated in the upper left corner of each chromatogram. The numerical value in the upper right corner of each chromatogram indicates the peak intensity. The peak intensity indicated in the upper right corner of each chromatogram is set to 100% on the vertical axis of each chromatogram. The horizontal axis shows the elution time (min) in LC.

[0589] [Figure 2-4] Figure 2-4 shows the SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the target peptide being measured is indicated in the upper left corner of each chromatogram. IS stands for internal standard. The numerical value in the upper right corner of each chromatogram indicates the peak intensity. The peak intensity indicated in the upper right corner of each chromatogram is set to 100% on the vertical axis of each chromatogram. The horizontal axis shows the elution time (min) in LC.

[0590] [Figure 3A-1] Figure 3A-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3A-1 shows the measurement results of plasma samples with the addition of a single unmodified antibody. The peak intensity of the unmodified peptide (i.e., 4.24e4) is set to 100% on the vertical axis of each chromatogram in Figure 3A-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0591] [Figure 3A-2] Figure 3A-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3A-2 shows the measurement results of plasma samples with the addition of a single unmodified antibody. The peak intensity of the unmodified peptide (i.e., 4.24e4) is set to 100% on the vertical axis of each chromatogram in Figure 3A-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0592] [Figure 3A-3] Figure 3A-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3A-3 shows the measurement results of plasma samples with the addition of a single unmodified antibody. The peak intensity of the unmodified peptide (i.e., 4.24e4) is set to 100% on the vertical axis of each chromatogram in Figure 3A-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0593] [Figure 3B-1] Figure 3B-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3B-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0001 added individually. The peak intensity of MS0001 (i.e., 2.67e4) is set to 100% on the vertical axis of each chromatogram in Figure 3B-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0594] [Figure 3B-2] Figure 3B-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3B-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0001 added individually. The peak intensity of MS0001 (i.e., 2.67e4) is set to 100% on the vertical axis of each chromatogram in Figure 3B-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0595] [Figure 3B-3] Figure 3B-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3B-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0001 added individually. The peak intensity of MS0001 (i.e., 2.67e4) is set to 100% on the vertical axis of each chromatogram in Figure 3B-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0596] [Figure 3C-1] Figure 3C-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3C-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0002 added individually. The peak intensity of MS0002 (i.e., 1.23e5) is set to 100% on the vertical axis of each chromatogram in Figure 3C-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0597] [Figure 3C-2] Figure 3C-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3C-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0002 added individually. The peak intensity of MS0002 (i.e., 1.23e5) is set to 100% on the vertical axis of each chromatogram in Figure 3C-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0598] [Figure 3C-3] Figure 3C-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3C-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0002 added individually. The peak intensity of MS0002 (i.e., 1.23e5) is set to 100% on the vertical axis of each chromatogram in Figure 3C-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0599] [Figure 3D-1] Figure 3D-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3D-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0003 added individually. The peak intensity of MS0003 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3D-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0600] [Figure 3D-2] Figure 3D-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3D-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0003 added individually. The peak intensity of MS0003 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3D-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0601] [Figure 3D-3] Figure 3D-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3D-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0003 added individually. The peak intensity of MS0003 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3D-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0602] [Figure 3E-1] Figure 3E-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3E-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0005 added individually. The peak intensity of MS0005 (i.e., 4.55e5) is set to 100% on the vertical axis of each chromatogram in Figure 3E-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0603] [Figure 3E-2] Figure 3E-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3E-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0005 added individually. The peak intensity of MS0005 (i.e., 4.55e5) is set to 100% on the vertical axis of each chromatogram in Figure 3E-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0604] [Figure 3E-3] Figure 3E-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3E-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0005 added individually. The peak intensity of MS0005 (i.e., 4.55e5) is set to 100% on the vertical axis of each chromatogram in Figure 3E-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0605] [Figure 3F-1] Figure 3F-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3F-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0007 added individually. The peak intensity of MS0007 (i.e., 1.68e5) is set to 100% on the vertical axis of each chromatogram in Figure 3F-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0606] [Figure 3F-2] Figure 3F-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3F-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0007 added individually. The peak intensity of MS0007 (i.e., 1.68e5) is set to 100% on the vertical axis of each chromatogram in Figure 3F-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0607] [Figure 3F-3] Figure 3F-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3F-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0007 added individually. The peak intensity of MS0007 (i.e., 1.68e5) is set to 100% on the vertical axis of each chromatogram in Figure 3F-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0608] [Figure 3G-1] Figure 3G-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3G-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0008 added individually. The peak intensity of MS0008 (i.e., 4.17e4) is set to 100% on the vertical axis of each chromatogram in Figure 3G-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0609] [Figure 3G-2] Figure 3G-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3G-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0008 added individually. The peak intensity of MS0008 (i.e., 4.17e4) is set to 100% on the vertical axis of each chromatogram in Figure 3G-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0610] [Figure 3G-3] Figure 3G-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3G-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0008 added individually. The peak intensity of MS0008 (i.e., 4.17e4) is set to 100% on the vertical axis of each chromatogram in Figure 3G-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0611] [Figure 3H-1] Figure 3H-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3H-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0009 added individually. The peak intensity of MS0009 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3H-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0612] [Figure 3H-2] Figure 3H-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3H-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0009 added individually. The peak intensity of MS0009 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3H-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0613] [Figure 3H-3] Figure 3H-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3H-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0009 added individually. The peak intensity of MS0009 (i.e., 4.48e4) is set to 100% on the vertical axis of each chromatogram in Figure 3H-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0614] [Figure 3I-1] Figure 3I-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3I-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0012 added individually. The peak intensity of MS0012 (i.e., 1.88e5) is set to 100% on the vertical axis of each chromatogram in Figure 3I-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0615] [Figure 3I-2] Figure 3I-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3I-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0012 added individually. The peak intensity of MS0012 (i.e., 1.88e5) is set to 100% on the vertical axis of each chromatogram in Figure 3I-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0616] [Figure 3I-3] Figure 3I-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3I-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0012 added individually. The peak intensity of MS0012 (i.e., 1.88e5) is set to 100% on the vertical axis of each chromatogram in Figure 3I-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0617] [Figure 3J-1] Figure 3J-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3J-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0019 added individually. The peak intensity of MS0019 (i.e., 4.30e4) is set to 100% on the vertical axis of each chromatogram in Figure 3J-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0618] [Figure 3J-2] Figure 3J-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3J-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0019 added individually. The peak intensity of MS0019 (i.e., 4.30e4) is set to 100% on the vertical axis of each chromatogram in Figure 3J-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0619] [Figure 3J-3] Figure 3J-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3J-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0019 added individually. The peak intensity of MS0019 (i.e., 4.30e4) is set to 100% on the vertical axis of each chromatogram in Figure 3J-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0620] [Figure 3K-1] Figure 3K-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3K-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0022 added individually. The peak intensity of MS0022 (i.e., 2.60e5) is set to 100% on the vertical axis of each chromatogram in Figure 3K-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0621] [Figure 3K-2] Figure 3K-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3K-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0022 added individually. The peak intensity of MS0022 (i.e., 2.60e5) is set to 100% on the vertical axis of each chromatogram in Figure 3K-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0622] [Figure 3K-3] Figure 3K-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3K-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0022 added individually. The peak intensity of MS0022 (i.e., 2.60e5) is set to 100% on the vertical axis of each chromatogram in Figure 3K-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0623] [Figure 3L-1] Figure 3L-1 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3L-1 shows the measurement results of plasma samples with a modified antibody corresponding to MS0031 added individually. The peak intensity of MS0031 (i.e., 9.49e4) is set to 100% on the vertical axis of each chromatogram in Figure 3L-1. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0624] [Figure 3L-2] Figure 3L-2 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3L-2 shows the measurement results of plasma samples with a modified antibody corresponding to MS0031 added individually. The peak intensity of MS0031 (i.e., 9.49e4) is set to 100% on the vertical axis of each chromatogram in Figure 3L-2. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0625] [Figure 3L-3] Figure 3L-3 shows the SRM chromatograms of plasma samples with a single antibody standard solution (1.28 μg / mL mouse plasma concentration) added to confirm specificity. The horizontal axis of each chromatogram shows the elution time (min) in LC. Figure 3L-3 shows the measurement results of plasma samples with a modified antibody corresponding to MS0031 added individually. The peak intensity of MS0031 (i.e., 9.49e4) is set to 100% on the vertical axis of each chromatogram in Figure 3L-3. The name of the target peptide measured is indicated in the upper left corner of each chromatogram. Each black arrow indicates the expected position of the peak of the target peptide measured.

[0626] [Figure 4-1] Figure 4-1 shows the plasma antibody concentration-time curves of H54-SG181 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0627] [Figure 4-2] Figure 4-2 shows the plasma antibody concentration-time curves of H54-SG181.MS2 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0628] [Figure 4-3] Figure 4-3 shows the plasma antibody concentration-time curves of H54-SG181.MS5 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0629] [Figure 4-4] Figure 4-4 shows the plasma antibody concentration-time curves of H54-SG181.MS7 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0630] [Figure 4-5] Figure 4-5 shows the plasma antibody concentration-time curves of H54-SG181.MS12 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0631] [Figure 4-6] Figure 4-6 shows the plasma antibody concentration-time curves of H54-SG181.MS22 after mixed administration and single administration to mice. The title in the figure is the name of the target antibody measured, the horizontal axis is the time (days) elapsed after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL).

[0632] [Figure 5] Figure 5 shows the plasma antibody concentration-time curves for antibodies containing unmodified or alanine-modified sequences after co-administration to mice. The horizontal axis represents the time (days) elapsed since drug administration, and the vertical axis represents the plasma concentration of the target antibody (ng / mL). Detailed Implementation

[0633] I. Definition

[0634] The term “and / or” in this document is used to refer to any one of the subjects presented before and after “and / or”, or any combination thereof. For example, “A, B and / or C” includes single objects “A”, “B”, and “C”, as well as combinations of “A and B”, “A and C”, “B and C”, and “A and B and C”. The term “and / or” may be used interchangeably with “both or either” or “all or either”.

[0635] In this document, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant (epitope). In one embodiment, the antigen-binding molecule is an antibody, an antibody fragment, or an antibody derivative. In one embodiment, the antigen-binding molecule is a non-antibody protein, a fragment thereof, or a derivative thereof. In one embodiment, the antigen-binding molecule is an antibody-like molecule, a fragment thereof, or a derivative thereof. In one embodiment, the antigen-binding molecule is a multispecific antigen-binding molecule (e.g., a bispecific antigen-binding molecule), a fragment thereof, or a derivative thereof. In one embodiment, the antigen-binding molecule is an antibody fragment comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). In one embodiment, the antigen-binding molecule is an antibody fragment comprising Fab. In one embodiment, the antigen-binding molecule is an IgG antibody.

[0636] The antigen-binding molecule in this application is not limited in its source and can be human antibody, mouse antibody, rat antibody, etc. It can also be a genetically modified antibody, such as a chimeric antibody and a humanized antibody.

[0637] In this document, the term "multispecific antigen-binding molecule" refers to an antigen-binding molecule comprising two or more distinct antigen-binding domains that are specific to different antigens or epitopes. In one embodiment, the multispecific antigen-binding molecule of this application is a multispecific antibody. Multispecific antibodies are not particularly limited, but monoclonal antibodies are preferred.

[0638] In this document, an "antigen-binding domain" refers to a region that specifically binds to and complements an intact antigen or a portion thereof. In this document, antigen-binding molecules contain antigen-binding domains. When the molecular weight of the antigen is large, the antigen-binding domain can only bind to a specific portion of the antigen. Such a specific portion is called an "epitope." In one embodiment, the antigen-binding domain contains an antibody fragment that binds to a specific antigen. The antigen-binding domain may be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "single-chain Fv (scFv)", "single-chain antibody", "Fv", and "single-chain Fv2 (scFv2)".

[0639] In this article, "specific binding" means that one of the specifically binding molecules binds to one or more binding partner molecules, without showing any significant binding to molecules other than binding partner molecules. It is also used when the antigen-binding domain is specific to a particular epitope among multiple epitopes contained in an antigen. When the epitope bound to the antigen-binding domain is contained in multiple different antigens, an antigen-binding molecule having that antigen-binding domain can bind to various antigens having that epitope.

[0640] The term "antibody" is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and modified antibodies, as long as they exhibit the desired antigen-binding activity.

[0641] The term "binding activity" refers to the strength of the sum of non-covalent interactions between one or more binding sites of a molecule (e.g., an antibody) and its partner (e.g., an antigen). Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). For example, when members of a binding pair exhibit a monovalent 1:1 interaction, binding activity is referred to as intrinsic binding affinity ("affinity"). When members of a binding pair can bind both monovalently and multivalently, binding activity is the sum of these binding forces. The binding activity of molecule X with its partner Y is typically expressed as a dissociation constant (KD) or "the amount of analyte bound per unit amount of ligand". Binding activity can be measured using conventional methods known in the art, including those described herein.

[0642] An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0643] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0644] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0645] In one embodiment of this application, the constant region is preferably an antibody constant region, more preferably a constant region of IgG1, IgG2, IgG3, and IgG4 type antibodies, and even more preferably a constant region of human IgG1, IgG2, IgG3, and IgG4 type antibodies. In another embodiment of this application, the constant region is preferably a heavy chain constant region, more preferably a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4 type antibodies, and even more preferably a heavy chain constant region of human IgG1, IgG2, IgG3, and IgG4 type antibodies. The amino acid sequences of the human IgG1 constant region, human IgG2 constant region, human IgG3 constant region, and human IgG4 constant region are known. For the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotropic sequences based on gene polymorphism are described in the protein sequences of immunological interest, NIH Publication No. 91-3242. Any of these sequences may be used in this application. The amino acid-modified constant region of this application may contain other amino acid mutations or modifications, as long as they include the amino acid mutations of this application.

[0646] The term "hinge region" refers to the polypeptide portion of the antibody heavy chain in the wild-type antibody heavy chain that connects the CH1 and CH2 domains, for example, from approximately position 216 to approximately position 230 according to the EU numbering system, or from approximately position 226 to approximately position 243 according to the Kabat numbering system. It is known that in native IgG antibodies, a cysteine ​​residue at position 220 according to the EU number in the hinge region forms a disulfide bond with a cysteine ​​residue at position 214 in the antibody light chain. It is also known that disulfide bonds are formed between two antibody heavy chains by cysteine ​​residues at position 226 according to the EU number in the hinge region and by cysteine ​​residues at position 229. In this document, the hinge region includes the wild-type hinge region and its variants resulting from the substitution, addition, or deletion of amino acid residues in the wild-type hinge region.

[0647] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At、 131 I, 125 I, 90 Y、 186 Re、 188 Re、 153 Sm、 212 Bi、 32 P, 212 Radioactive isotopes of Pb and Lu; chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as lysozymes; antibiotics; toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various antitumor or anticancer agents disclosed below.

[0648] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0649] The term “Fc region” used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. This 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 C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0650] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0651] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0652] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny cells may not have completely identical nucleic acid contents to the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0653] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a human antibody library or other human antibody-encoding sequence. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0654] The "human common framework" is a framework that represents the most frequently occurring amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the sequence subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda, MD (1991), Volumes 1-3. In one embodiment, for VL, this subgroup is subgroup κ I as described in Kabat et al. (ibid.). In one embodiment, for VH, this subgroup is subgroup III as described in Kabat et al. (ibid.).

[0655] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody will substantially contain at least one of all, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0656] As used herein, the term "hypervariant region" or "HVR" refers to each of the following: a region of antibody variable domain that is hypervariable in sequence ("complementarity-determining region" or "CDR") and / or forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs in this document include:

[0657] (a) Hyperchromatic rings present 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));

[0658] (b) CDRs located 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 edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)).

[0659] (c) Antigen contact sites located 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

[0660] (d) Combinations of (a), (b) and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0661] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are referenced in this paper according to Kabat et al., ibid.

[0662] "Immune conjugates" are antibodies conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0663] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses, and pigs), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats, and guinea pigs). In some embodiments, the individual or subject is a non-human animal. In a particular embodiment, the individual or subject is a human.

[0664] "Isolated" antibodies are antibodies that have been separated from components in their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99% by means of, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0665] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0666] "Isolated nucleic acid encoding antibody" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0667] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of a monoclonal antibody formulation, such variants are typically present in small quantities), the individual antibodies constituting this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody intended for use according to this application can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0668] "Naked antibody" refers to an antibody that is not conjugated to a heterologous part (e.g., a cytotoxic part) or a radiolabel. Naked antibodies can be found in pharmaceutical preparations.

[0669] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be classified into one of two types based on the amino acid sequence of their constant domains, called kappa (κ) and lamuda (λ).

[0670] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENTYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0671] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0672] When using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

[0673] 100 multiplied by the score X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B will not be equal to the amino acid sequence identity % of B to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0674] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding that antigen to screen libraries of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0675] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures, as well as vectors that have been introduced into the genome of a host cell. Some vectors can direct the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors." Vectors can be introduced into host cells using methods such as viral methods and electroporation, but the introduction of vectors is not limited to in vitro introduction and can also be introduced directly into the body.

[0676] In a further aspect, the antibody according to any of the above embodiments may incorporate, alone or in combination, any of the features described in Sections 1-7 below:

[0677] 1. Antibody affinity

[0678] In some embodiments, the antibodies provided herein have a concentration of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 nM). -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M).

[0679] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using the Fab form of the target antibody and its antigen. For example, by using the minimum concentration in the presence of a series of unlabeled antigen titrations. 125 I) The labeled antigen was equilibrated with Fab, and then the bound antigen was captured using a plate coated with anti-Fab antibody to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish the assay conditions, MICROTITER (trademark) multiwell plates (ThermoScientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6) and then blocked at room temperature (approximately 23°C) for two to five hours with 2% (w / v) bovine serum albumin in PBS. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] The antigen was mixed with a serially diluted solution of the target Fab (e.g., following the evaluation of anti-VEGF antibody (Fab-12) in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab was then incubated overnight; however, incubation may be prolonged (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate had dried, 150 μl / well of scintillation agent (MICROSCINT-20™; Packard) was added, and the plate was counted for several tens of minutes on a TOPCOUNT™ γ counter (Packard). The concentration of each Fab that yielded a maximum binding of less than or equal to 20% was selected for use in the competitive binding assay.

[0680] According to another embodiment, Kd was measured using the BIACORE (registered trademark) surface plasmon resonance assay. For example, the assay using BIACORE-2000 or BIACORE-3000 (BIAcore, Inc., Piscatave, NJ) was performed at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 μl / min to obtain a conjugated protein of approximately 10 response units (RU). Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25°C, Fab was injected in serially diluted 2-fold solutions (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μl / min. The association rate (kJ / kJ) was calculated using a simple one-to-one Langmuir binding model (BIACORE Evaluation Software version 3.2) by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (k offThe equilibrium dissociation constant (Kd) is calculated as the ratio k. off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate obtained by the above surface plasmon resonance determination exceeds 10... 6 M -1 s -1 The association rate can be determined by using fluorescence quenching techniques, i.e., by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of gradually increasing antigen concentration using a spectrometer such as an Aviv Instruments spectrophotometer equipped with a flow stop or a ThermoSpectronic 8000 series SLM-AMINCO™ spectrophotometer with a stirring cuvette.

[0681] 2. Antibody fragments

[0682] In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); also see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing rescue receptor-binding epitope residues and having an extended in vivo half-life, see U.S. Patent No. 5,869,046.

[0683] A single-domain antibody is an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Massachusetts; see, for example, U.S. Patent No. 6,248,516 B1).

[0684] Antibody fragments can be prepared using a variety of techniques, including but not limited to the proteolytic digestion of intact antibodies and the production of recombinant host cells (such as E. coli or bacteriophages), as described herein.

[0685] 3. Chimeric and humanized antibodies

[0686] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0687] In some embodiments, the chimeric antibody is a humanized antibody. Typically, nonhuman antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent nonhuman antibody. Generally, humanized antibodies comprise one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from the nonhuman antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0688] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface rework”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR reorganization”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes the “guided selection” method for FR reorganization).

[0689] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a “best fit” method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequences of human antibodies from specific subgroups of light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok ... Chem. 271:22611-22618(1996)).

[0690] 4. Human antibodies

[0691] In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol. 5: 368-74 (2001) and Lonberg, Curr Opin Immunol. 20:450-459 (2008).

[0692] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or complete antibodies with human variable regions in response to antigen stimulation. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HuMab (registered trademark) technology; U.S. Patent No. 7,041,870 describing KM MOUSE (registered trademark) technology; and U.S. Patent Application Publication No. US 2007 / 0061900 describing VelociMouse (registered trademark) technology. The human variable region derived from intact antibodies produced by such animals can be further modified, for example, by combining it with different human constant regions.

[0693] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, XiandaiMianyixue, 26(4):265-268 (2006) (which describes human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0694] Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human phage display libraries. These variable domain sequences can then be bound to the desired human constant domain. The technique for selecting human antibodies from antibody libraries is described below.

[0695] 5. Antibodies derived from a library

[0696] The antibodies of this application can be isolated by screening a combinatorial library for antibodies possessing one or more of the desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., editors, Human Press, Totoa, NJ, 2001) and further described in the following literature: for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, editors, Human Press, Totoa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34); 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0697] In some phage display methods, all components of the VH and VL genes are independently cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, from which antigen-binding phages can then be screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, the initial library can also be synthesized by cloning the unrearranged V gene segment from stem cells; and by using PCR primers containing random sequences to encode the highly variable CDR3 region and perform in vitro rearrangement, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0698] In this paper, antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

[0699] 6. Multispecific antibodies

[0700] In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, one of the binding specificities is against any antigen, and the other is against any other antigen. In some embodiments, a bispecific antibody may bind to two different epitopes of an antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0701] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)) and mortar-and-mortar engineering (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared using the following techniques: engineered electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using “dimeric antibody” techniques to prepare bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol. 152:5368). (1994)); and prepare trispecific antibodies according to, for example, the description in Tutt et al. J. Immunol. 147: 60 (1991).

[0702] This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0703] The antibodies or fragments mentioned in this article also include “dual-action Fab” or “DAF” (see, for example, US 2008 / 0069820).

[0704] 7. Antibody variants

[0705] In some embodiments, amino acid sequence variants of the antibodies provided herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0706] a) Substitution, insertion, and deletion variants

[0707] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVR and FR. Conservative substitutions are shown under the heading “Preferred Substitutions” in Table 1. Further substantial changes are provided under the heading “Exemplary Substitutions” in Table 1 and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities (e.g., preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0708] (Table 1)

[0709]

[0710] Amino acids can be grouped based on common side-chain characteristics:

[0711] (1) Hydrophobic: Leucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile);

[0712] (2) Neutral hydrophilic: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln);

[0713] (3) Acidic: Aspartic acid (Asp), glutamic acid (Glu);

[0714] (4) Alkaline: Histidine (His), Lysine (Lys), Arginine (Arg);

[0715] (5) Residues that affect chain orientation: glycine (Gly) and proline (Pro);

[0716] (6) Aromatics: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0717] Non-conservative substitution would require swapping members of one of these categories for members of another category.

[0718] One type of substitution variant involves replacing one or more highly variable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more resulting variants selected for further research will alter (e.g., improve) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody, relative to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0719] For example, HVR can be altered (e.g., substituted) to improve antibody affinity. Such alterations can be made in HVR “hotspots,” namely residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues in contact with the antigen, where binding affinity is tested for the resulting variant VH or VL. Affinity maturation achieved by constructing and reselecting from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totoa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods, such as error-prone PCR, strand shuffling, or site-directed oligonucleotide mutagenesis. A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4 to 6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutations or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0720] In some embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such changes do not substantially reduce the antigen-binding ability of the antibody. For example, conserved changes (e.g., conserved substitutions as provided herein) that do not substantially reduce binding affinity may be made in the HVRs. Such changes may occur outside the antigen-contact residues of the HVRs. In some embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0721] A method used to identify antibody residues or regions that can be targeted for mutation is called "alanine scan mutation," as described in Cunningham and Wells, (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

[0722] Amino acid sequence insertions include the fusion of amino and / or carboxyl termini of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include the fusion of an enzyme (e.g., for ADEPT) or a peptide that increases the plasma half-life of the antibody with the N- or C-terminus of the antibody.

[0723] b) Glycosylated variants

[0724] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0725] When an antibody contains an Fc region, the carbohydrates attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of this application can be modified to produce antibody variants with certain improved properties.

[0726] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose linked (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose intrinsically present at Asn297 within the glycan chain relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU number of the Fc region residue) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Antibody variants that are "defucosylated" or "fucose-deficient" include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13CHO cells with protein fucosylation defects (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines such as CHO cells with α-1,6-fucosylation gene (FUT8) knockout (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0727] Further antibody variants with dimeric oligosaccharides are provided, for example, wherein the diangular oligosaccharide linked to the Fc region of the antibody is dimeric with GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0728] c) Fc region variant

[0729] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0730] In some embodiments, this application considers antibody variants having some, but not all, effector functions, making them desirable candidates for application where the half-life of the antibody in vivo is important while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l. Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l. Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, the ACT1™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA); and the CytoTox 96 (registered trademark) non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in animal models (such as those disclosed in Clynes et al., Proc. Nat'l. Acad. Sci. USA 95:652-656 (1998)). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402.To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0731] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acids at positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0732] Certain antibody variants with increased or decreased binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0733] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbers of residues) in the Fc region.

[0734] In some embodiments, such as those described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), alterations are made in the Fc region resulting in altered (i.e., increased or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC).

[0735] Antibodies with extended half-lives and increased binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934A1 (Hinton et al.), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain an Fc region with one or more substitutions that increase the binding of the Fc region to the FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0736] For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0737] d) Cysteine-engineered antibody variants

[0738] In some embodiments, it is desirable to generate cysteine-engineered antibodies, such as “thioMAbs”, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. As further described herein, by replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to conjugate the antibody to other parts (such as a pharmaceutical part or a linker-pharmaceutical part) to produce an immunoconjugate. In some embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat number) of the light chain; A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0739] e) Antibody derivatives

[0740] In some embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for a limited therapy.

[0741] In another embodiment, a conjugate of an antibody and a non-protein portion that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature at which cells proximal to the antibody-non-protein portion are killed.

[0742] Antibodies can be generated using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising a VL of the antibody and / or an amino acid sequence comprising a VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., having been transformed with): (1) a vector comprising nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody; or (2) a first vector and a second vector, the first vector comprising nucleic acid encoding an amino acid sequence comprising a VL of the antibody and the second vector comprising nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp2 / O cells). In one embodiment, a method for preparing an antibody is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0743] For recombinant antibody production, nucleic acids encoding antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of antibodies).

[0744] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, HumanaPress, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*.) Antibodies can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified.

[0745] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. These eukaryotic microorganisms include fungal and yeast strains whose glycosylation pathways have been "humanized," thereby enabling the production of antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0746] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0747] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES™ technology for generating antibodies in transgenic plants).

[0748] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed from SV40; human embryonic kidney lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (as described, for example, in Mather et al., Annals NYAcad. Sci.). (As described in 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, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0749] In some embodiments, this application provides an antibody that can be obtained by the methods described above.

[0750] The physical / chemical properties and / or biological activity of the antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

[0751] In one respect, the antibodies of this application are tested for antigen-binding activity by known methods such as ELISA and Western blotting.

[0752] This application also provides an immunoconjugate comprising the antibody described herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal-derived protein toxins, enzyme-active toxins, or fragments thereof), or radioisotopes.

[0753] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more drugs, including but not limited to maytansines (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); aurestatin, such as the drug portions DE and DF (MMAE and MMAF) of monomethylaurestatin (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolasstatin; cazithromycin or derivatives thereof (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines, such as daunorubicin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and US Patent No. 6,630,579); methotrexate; vinca; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and oxadecane; trichothecotoxins and CC1065.

[0754] In another embodiment, the immunoconjugate comprises an antibody conjugated herein to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, absinthecin A chain, senna root toxin A chain, α-arbusculin, tung oil protein, caryophyllein protein, pokeweed antiviral protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcumin, crotonin, soapwort inhibitor, gelatin, mitochondriin, localized aspergillin, phenolmycin, enoxacin, and trichosporine.

[0755] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated with a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce the radioconjugate. Examples include... 211 At、 131 I, 125 I, 90 Y、 186 Re、 188 Re、 153 Sm、 212 Bi、 32 P, 212 Radioactive isotopes of Pb and Lu. When radioconjugates are used for detection, they may contain radioactive atoms used in scintillation studies, such as Tc-99m or... 123 I, or spin markers used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0756] A variety of bifunctional protein conjugates, such as N-succinimino-3-(2-pyridyldithio)propionate (SPDP), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), iminothiacyclopentane (IT), bifunctional derivatives of imino esters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene), can be used to prepare conjugates of antibodies and cytotoxic agents. For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The adapter can be a “cleavable adapter” that promotes the release of cytotoxic drugs from cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Res. 52:127-131 (1992); US Patent No. 5,208,020).

[0757] The immunoconjugates or ADCs discussed in this article are explicitly considered, but not limited to, such conjugates prepared with cross-linking agents, including but not limited to commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, sulfon-SMPB, and SVSB (succinimino-(4-vinyl sulfone)benzoate).

[0758] The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinantly designing the nucleic acid encoding the antibody. Therefore, a composition containing an antibody having an Fc region according to this application may comprise an antibody having G446 to K447, an antibody having G446 but not K447, an antibody with all G446 to K447 removed, or a mixture of all three types of antibodies described above.

[0759] A “functional Fc region” possesses the “effective function” of the native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the Fc region in combination with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed in the definitions herein.

[0760] The “natural sequence Fc region” contains the same amino acid sequence as the Fc region found in nature. The natural sequence human Fc region includes the natural sequence human IgG1 Fc region (non-A and A allotypes); the natural sequence human IgG2 Fc region; the natural sequence human IgG3 Fc region; and the natural sequence human IgG4 Fc region and its naturally occurring variants.

[0761] The antigen-binding molecules of this application include, for example, the antigen-binding molecules described in WO2020 / 027330. For instance, the antigen-binding molecules of this application include antigen-binding molecules comprising a first antigen-binding domain and a second antigen-binding domain, wherein the antigen-binding domains are linked to each other via one or more bonds.

[0762] In one embodiment, at least one of the bonds connecting the two antigen-binding domains is a covalent bond. In some embodiments, the covalent bond is formed by directly crosslinking amino acid residues in the first antigen-binding domain and amino acid residues in the second antigen-binding domain. The type of amino acid residue to be crosslinked is, for example, cysteine, and the covalent bond formed is, for example, a disulfide bond.

[0763] In another specific embodiment, amino acid residues in the first antigen-binding domain and amino acid residues in the second antigen-binding domain are cross-linked via a cross-linking agent to form covalent bonds. The cross-linking agent is, for example, an amine-reactive cross-linking agent, and the type of amino acid residue to be cross-linked is, for example, lysine.

[0764] In one embodiment, at least one of the bonds connecting one or more antigen-binding domains is a non-covalent bond. In some embodiments, the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond. For example, an ionic bond is formed between an acidic amino acid and a basic amino acid. The acidic amino acid is, for example, aspartic acid (Asp) or glutamic acid (Glu). The basic amino acid is, for example, histidine (His), lysine (Lys), or arginine (Arg).

[0765] The amino acid residues from which the bonds between antigen-binding domains (bonds connecting two antigen-binding domains) originate are located in the first and second antigen-binding domains, respectively, and the bonds between the antigen-binding domains are formed by linking these amino acid residues. In one embodiment, at least one amino acid residue generating the bond between the antigen-binding domains is an artificially introduced mutated amino acid residue, and for example, an artificially introduced cysteine ​​residue. Such a mutated amino acid residue can be introduced into the wild-type antigen-binding domain by, for example, amino acid substitution. This specification discloses sites of amino acid residues that can generate the bond between the antigen-binding domains when the antigen-binding domain contains, for example, an antibody fragment and, for example, a cysteine ​​residue can be introduced into such sites, for each of the CH1, CL, and hinge regions as constant regions and the VH, VL, and VHH regions as variable regions.

[0766] In one embodiment, at least one of the first antigen-binding domain and the second antigen-binding domain is itself active in binding to an antigen (i.e., a single antigen-binding domain independently has antigen-binding activity). In some embodiments, each of the first antigen-binding domain and the second antigen-binding domain is itself active in binding to an antigen.

[0767] In one embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain can be formed by linking amino acid residues at the same position in the first antigen-binding domain and the second antigen-binding domain to each other, or by linking amino acid residues at different positions to each other.

[0768] In one embodiment, at least one of the amino acid residues that form the bond between the antigen-binding domains is present in the constant region. In some embodiments, the amino acid residues in the CH1 region are present, for example, at any of the positions 119 to 123, 131 to 140, 148 to 150, 155 to 167, 174 to 178, 188 to 197, 201 to 214, and 218 to 219 in the CH1 region according to EU numbering. In some embodiments, the amino acid residues are located in the CH1 region at positions selected from the group consisting of EU numbers 119, 122, 123, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 148, 150, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 167, 174, 176, 177, 178, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 201, 203, 205, 206, 207, 208, 211, 212, 213, 214, 218, and 219. In some embodiments, the amino acid residues are located at positions 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 in the CH1 region according to EU numbering.

[0769] In one embodiment, the constant region is derived from human. In some embodiments, the subclass of the heavy chain constant region is any one of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the subclass of the CH1 region is any one of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[0770] In one embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by connecting amino acid residues in the CH1 region of the first antigen-binding domain and amino acid residues in the CH1 region of the second antigen-binding domain. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 119, 120, 121, 122, and 123 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 148, 149, and 150 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 174, 175, 176, 177, and 178 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214 according to EU numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 218 and 219 according to EU numbers.

[0771] In one embodiment, the difference in the positions of the bond-forming amino acid residues in each of the first and second antigen-binding domains is three amino acids or less. In one embodiment, this means that when comparing the positions of the bond-forming amino acid residues in the CH1 region of the first antigen-binding domain and the CH1 region of the second antigen-binding domain according to EU numbers, the difference is three amino acids or less. In some embodiments, at least one bond connecting the first and second antigen-binding domains is formed by connecting an amino acid residue at position 135 according to EU numbers in the CH1 region of the first antigen-binding domain and an amino acid residue at any of positions 132 to 138 according to EU numbers in the CH1 region of the second antigen-binding domain. In some embodiments, at least one bond connecting the first and second antigen-binding domains is formed by connecting an amino acid residue at position 136 according to EU numbers in the CH1 region of the first antigen-binding domain and an amino acid residue at any of positions 133 to 139 according to EU numbers in the CH1 region of the second antigen-binding domain. In some embodiments, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by connecting an amino acid residue at position 191 (EU number) in the CH1 region of the first antigen-binding domain and an amino acid residue at any of positions 188 to 194 (EU number) in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue at position 135 (EU number) in the CH1 region of the two antigen-binding domains to each other. In an exemplary embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue at position 136 (EU number) in the CH1 region of the two antigen-binding domains to each other. In an exemplary embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by linking an amino acid residue at position 191 (EU number) in the CH1 region of the two antigen-binding domains to each other.

[0772] In one embodiment, at least one of the amino acid residues that form bonds between the antigen-binding domains is present in the CL region, for example, at any of the following positions in the CL region according to Kabat numbering: 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213. In some embodiments, the amino acid residues are present at positions selected from the group consisting of: positions in the CL region according to Kabat numbering: 108, 109, 112, 121, 123, 126, 128, 151, 152, 153, 156, 184, 186, 188, 189, 190, 195, 196, 200, 201, 202, 203, 208, 210, 211, 212, and 213. In some embodiments, the amino acid residue is located at position 126 in the CL region according to the Kabat number.

[0773] In one embodiment, the constant region is derived from humans. In some embodiments, the subclass of the CL region is κ or λ.

[0774] In one embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by connecting amino acid residues in the CL region of the first antigen-binding domain and amino acid residues in the CL region of the second antigen-binding domain. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 108, 109, 110, 111, and 112 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 121, 122, 123, 124, 125, 126, 127, and 128 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 151, 152, 153, 154, 155, and 156 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 184, 185, 186, 187, 188, 189, and 190 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 195 and 196 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 200, 201, 202, and 203 according to Kabat numbers. In some embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of: positions 208, 209, 210, 211, 212, and 213 according to Kabat numbers.

[0775] In one embodiment, the difference in the positions of the bond-forming amino acid residues in each of the first and second antigen-binding domains is three amino acids or less. In one embodiment, this means that when comparing the positions of the bond-forming amino acid residues in the CL region of the first antigen-binding domain and the CL region of the second antigen-binding domain according to EU numbers, the difference is three amino acids or less. In an exemplary embodiment, at least one bond connecting the first and second antigen-binding domains is formed by linking amino acid residues at position 126 according to Kabat numbers in the CL regions of the two antigen-binding domains to each other.

[0776] In one embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by connecting amino acid residues in the CH1 region of the first antigen-binding domain and amino acid residues in the CL region of the second antigen-binding domain. In some embodiments, the amino acid residues in the CH1 region of the first antigen-binding domain are selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 according to EU numbers, and the amino acid residues in the CL region of the second antigen-binding domain are selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 according to Kabat numbers. In an exemplary embodiment, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is formed by connecting amino acid residues at position 191 according to EU numbers in the CH1 region of the first antigen-binding domain and amino acid residues at position 126 according to Kabat numbers in the CL region of the second antigen-binding domain.

[0777] In one embodiment, at least one of the amino acid residues that form the bond between the antigen-binding domains is present in the variable region. In some embodiments, the amino acid residues are present in the VH region at any position selected from the group consisting of: positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b according to Kabat numbers in the VH region. In some embodiments, the aforementioned amino acid residues are present in the VL region at any position selected from the group consisting of: positions 21, 27, 58, 77, 100, 105, and 107 according to Kabat numbers in the VL region (κ subclass) and positions 6, 19, 33, and 34 according to Kabat numbers in the VL region (λ subclass). In some embodiments, the amino acid residues are present in the VHH region at any position selected from the group consisting of: positions 4, 6, 7, 8, 9, 10, 11, 12, 14, 15, 17, 20, 24, 27, 29, 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 67, 69, 71, 78, 80, 82, 82c, 85, 88, 91, 93, 94, and 107 according to Kabat numbering in the VHH region.

[0778] In one embodiment, the first and / or second antigen-binding domain comprises a hinge region. In some embodiments, at least one cysteine ​​residue present in the wild-type hinge region has been replaced by another amino acid residue. Such cysteine ​​residues are, for example, present at positions 226 and / or 229 according to EU numbers in the wild-type hinge region. In some embodiments, at least one amino acid residue that forms the bond between the antigen-binding domains is present in the hinge region, for example, at any position in the hinge region selected from the group consisting of positions 216, 218, and 219 according to EU numbers.

[0779] In one embodiment, the first antigen-binding domain and the second antigen-binding domain are connected to each other via two or more bonds.

[0780] In some embodiments, at least one amino acid residue that forms the bond between the antigen-binding domains is an amino acid residue present in the wild-type sequence, such as a cysteine ​​residue in the wild-type hinge region. In some embodiments, at least one of the bonds connecting the first and second antigen-binding domains is a disulfide bond formed by crosslinking between cysteine ​​residues present in the wild-type hinge region. Such cysteine ​​residues are, for example, present at positions 226 and / or 229 according to EU numbering in the wild-type hinge region.

[0781] In some embodiments, at least one of the amino acid residues that form the bond between the antigen-binding domains is present within the antibody fragment, and at least one is present within the hinge region. In an exemplary embodiment, the antigen-binding molecule of this disclosure is F(ab')2, wherein both the first and second antigen-binding domains contain Fab and a hinge region.

[0782] The antigen-binding molecules in this application include, for example, those described in WO2018 / 097307.

[0783] For example, the antigen-binding molecule of this application relates to an antigen-binding molecule as a polypeptide, wherein the polypeptide comprises an antigen-binding domain and a carrier portion, wherein the carrier portion comprises an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and wherein the half-life of the antigen-binding domain in the blood is shorter than that of the carrier portion.

[0784] The carrier portion has an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain. In this document, the term "inhibitory domain" is limited only by the inhibition of the antigen-binding activity of the antigen-binding domain. The inhibitory domain can be any domain with any structure, as long as the domain used can inhibit the antigen-binding activity of the antigen-binding domain. Examples of such inhibitory domains include, but are not limited to, antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), pre-B cell receptors, and single-domain antibodies. The inhibitory domain may constitute all or part of the carrier portion.

[0785] In some embodiments, the antigen-binding domain released from the peptide exhibits higher antigen-binding activity than before release. In other words, when the antigen-binding domain is not released from the peptide, its antigen-binding activity is inhibited by the inhibition domain. Whether the antigen-binding activity of the antigen-binding domain is inhibited by the inhibition domain is confirmed using methods such as FACS (Fluorescence Activated Cell Sorting), ELISA (Enzyme-Linked Immunosorbent Assay), ECL (Electrochemiluminescence), SPR (Surface Plasmon Resonance) (Biacore), and BLI (Biolayer Interference) (Octet). In some embodiments, the antigen-binding activity of the antigen-binding domain released from the peptide is equal to or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, or 3,000 times that of the antigen-binding domain not released from the peptide. In some more specific embodiments, when the antigen-binding activity of the antigen-binding domain is measured using one of the methods selected above, no binding of the antigen-binding domain prior to release to the antigen is observed.

[0786] In some respects, the cleavage site is cleaved, allowing the antigen-binding domain to be released from the peptide. Therefore, in such respects, antigen-binding activity can be compared before and after peptide cleavage. Specifically, the antigen-binding activity measured using the cleaved peptide is a value equal to or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, or 3,000 times that measured using the uncleaved peptide. In some more specific embodiments, when antigen-binding activity was measured using one of the methods selected above, no binding of the antigen-binding domain of the uncleaved peptide to the antigen was observed.

[0787] In some respects, the cleavage site is cleaved by a protease. Therefore, in such respects, the antigen-binding activity of the peptide before and after protease treatment can be compared. Specifically, the antigen-binding activity measured using the peptide after protease treatment is equal to or greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, or 3,000 times that measured using the peptide without protease treatment. In some more specific embodiments, when antigen-binding activity was measured using one of the methods selected above, no binding of the antigen-binding domain of the untreated peptide to the antigen was observed.

[0788] In this application, peptides comprising an antigen-binding domain and a carrier moiety have a longer blood half-life compared to peptides containing only an antigen-binding domain. In some embodiments, the carrier moiety is designed to have a longer blood half-life for the peptide's longer half-life. Examples of methods for extending the blood half-life of the carrier moiety in such embodiments include, but are not limited to, a high molecular weight of the carrier moiety, FcRn binding activity of the carrier moiety, albumin binding activity of the carrier moiety, and PEGylation of the carrier moiety. In some embodiments, the carrier moiety has a longer blood half-life compared to the half-life of the antigen-binding domain (in other words, the antigen-binding domain has a shorter blood half-life compared to the half-life of the carrier moiety).

[0789] In this application, it is preferable to compare the half-life of the individual antigen-binding domain and peptide, or the half-life of the antigen-binding domain and the carrier portion in the blood, with respect to their half-life in human blood. If it is difficult to measure the half-life in human blood, the half-life in human blood can be predicted based on its half-life in the blood of mice (e.g., normal mice, transgenic mice expressing human antigens, and transgenic mice expressing human FcRn) or monkeys (e.g., cynomolgus monkeys).

[0790] In one embodiment, a method for prolonging the half-life of the carrier portion in the blood includes using a large molecular weight of the carrier portion. In one embodiment, a method for making the half-life of the carrier portion in the blood longer than the half-life of the antigen-binding domain includes using a molecular weight of the carrier portion that is greater than the molecular weight of the antigen-binding domain.

[0791] In one embodiment, a method for prolonging the half-life of the carrier portion in blood includes the carrier portion possessing FcRn binding activity. The carrier portion typically possesses FcRn binding activity by establishing an FcRn binding region within the carrier portion. An FcRn binding region refers to a region possessing FcRn binding activity and can have any structure, as long as the region used has FcRn binding activity.

[0792] Carrier regions containing FcRn-binding domains can be absorbed into cells and then returned to the plasma via the FcRn salvage pathway. For example, IgG molecules have a relatively long plasma circulation time (slow disappearance) because FcRn is known as the salvage receptor for IgG molecule function. IgG molecules taken into endosomes via pineal action bind to FcRn expressed in endosomes under acidic conditions. IgG molecules that fail to bind to FcRn are moved to lysosomes and degraded therein, while IgG molecules that bind to FcRn are transferred to the cell surface and then dissociate from FcRn in the plasma under neutral conditions, thus being returned to the plasma.

[0793] The FcRn binding region is preferably a region that binds directly to FcRn. Preferred examples of FcRn binding regions may include the antibody Fc region. However, regions capable of binding to peptides with FcRn-binding ability, such as albumin or IgG, can bind indirectly to FcRn via albumin, IgG, etc. Therefore, the FcRn binding region can be a region that binds to such peptides with FcRn-binding ability.

[0794] The binding activity of FcRn binding regions to FcRn, particularly human FcRn, can be measured using methods known to those skilled in the art, as described in the section on binding activity above. The conditions can be appropriately determined by those skilled in the art. Binding activity with human FcRn can be assessed as KD (dissociation constant), apparent KD (apparent dissociation constant), kd (dissociation rate), or apparent kd (apparent dissociation rate), etc. These values ​​can be measured using methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plots, flow cytometry, etc., can be used.

[0795] The conditions for measuring the binding activity of the FcRn binding region to FcRn are not particularly limited and can be appropriately selected by those skilled in the art. Binding activity can be measured under conditions involving, for example, MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the binding activity of the FcRn binding region to FcRn can be measured using methods known to those skilled in the art, and can be measured using, for example, Biacore (GE Healthcare). When measuring the binding activity of the FcRn binding region to FcRn, FcRn and the FcRn binding region, or a carrier portion containing the FcRn binding region, can be injected as analytes onto a chip on which the FcRn binding region or a carrier portion containing the FcRn binding region and FcRn are respectively immobilized, and then evaluated.

[0796] Regarding the pH used for measurement under certain conditions, the binding affinity of the FcRn binding region to FcRn can be evaluated at any pH from 4.0 to 6.5. Preferably, a pH from 5.8 to 6.0 (which is close to the pH in early endosomal tissue) is used to determine the binding affinity of the FcRn binding region to human FcRn. Regarding the temperature used for measurement under certain conditions, the binding affinity of the FcRn binding region to FcRn can be evaluated at any temperature from 10°C to 50°C. Preferably, a temperature from 15°C to 40°C is used to determine the binding affinity of the FcRn binding region to human FcRn. More preferably, any temperature from 20°C to 35°C (e.g., any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, and 35°C) can be used to determine the binding affinity of the FcRn binding region to FcRn. The temperature of 25°C is a non-limiting example of the embodiments.

[0797] The presence of FcRn binding activity in the carrier portion does not imply the absence of FcRn binding activity in the antigen-binding domain. In embodiments where the half-life of the carrier portion in blood is longer than the half-life of the antigen-binding domain, the antigen-binding domain may, of course, lack FcRn binding activity, or it may possess FcRn binding activity, provided that the FcRn binding activity is weaker than the half-life of the carrier portion.

[0798] In one embodiment, a method for prolonging the blood half-life of the carrier portion involves binding the carrier portion to albumin. Since albumin is not excreted by the kidneys and has FcRn-binding activity, its half-life in the blood is as long as 17 to 19 days (J Clin Invest. August 1953; 32(8): 746-768). Therefore, it has been reported that proteins bound to albumin become bulky and capable of indirect binding to FcRn, and thus have an increased half-life in the blood (Antibodies 2015, 4(3), 141-156).

[0799] In one embodiment, an alternative method for extending the blood half-life of the carrier portion involves PEGylation of the carrier portion. PEGylation of proteins is thought to make the proteins bulky and also to inhibit their degradation by proteases in the blood, thereby extending the blood half-life of the proteins (J Pharm Sci. Oct 2008; 97(10): 4167-83).

[0800] In some embodiments, the carrying portion contains an antibody Fc region. In a specific embodiment, the carrying portion contains the CH2 and CH3 domains of a human IgG antibody. In a specific embodiment, the carrying portion contains a portion spanning from Cys226 or Pro230 of the human IgG1 antibody heavy chain to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

[0801] In some embodiments, the carrying portion contains an antibody constant region. In a more preferred embodiment, the carrying portion contains an IgG antibody constant region. In another preferred embodiment, the carrying portion contains a human IgG antibody constant region.

[0802] In some further embodiments, the carrying portion includes: a region structurally similar to the constant region of the antibody heavy chain; and a region structurally similar to the antibody light chain, which is connected to the region via covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0803] In this article, "a polypeptide containing an antigen-binding domain and a carrier portion" is generally a series of polypeptides linked by amide bonds, or a protein containing multiple polypeptides linked by amide bonds.

[0804] In some embodiments, the antigen-binding domain is capable of being released from the peptide, and the released antigen-binding domain exhibits higher antigen-binding activity. Herein, the term "release" refers to the separation of the two parts of the peptide. The release of the antigen-binding domain from the peptide can be attributed to the cancellation of the interaction between the antigen-binding domain and the carrier moiety. The antigen-binding activity of the antigen-binding domain incorporated into the peptide is inhibited. Therefore, the release of the antigen-binding domain from the peptide can be confirmed by measuring the antigen-binding activity of a subject and comparing it with the antigen-binding activity of the antigen-binding domain incorporated into the peptide.

[0805] In some embodiments, the polypeptide includes a cleavage site, and the cleavage site is cleaved to release the antigen-binding domain from the polypeptide. The cleavage site can be cleaved by, for example, an enzyme, reduced by a reducing agent, or photodegraded. The cleavage site can be located anywhere in the polypeptide, as long as the antigen-binding domain can be released and does not lose its antigen-binding activity after release. The polypeptide may further contain additional cleavage sites besides the cleavage site for releasing the antigen-binding domain. In one embodiment, the cleavage site comprises a protease-cleaving sequence and can be cleaved by a protease.

[0806] In this paper, the term "cleaved" refers to the state in which the antigen-binding domain and the carrier moiety are separated from each other after alteration of the cleavage site of the protease, reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site, and / or photoactivation. The term "uncleaved" refers to the state in which the antigen-binding domain and the carrier moiety are connected in the absence of protease cleavage at the cleavage site, reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site, and / or the absence of light.

[0807] Cleavage can be detected by subjecting a solution containing a polypeptide with a cleavage site to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragment or detecting the change in molecular weight before and after cleavage.

[0808] The cleavage site can be determined by reagents (i.e., proteases, reducing agents, or light) at a range of approximately 0.001 to 1500 x 10⁻⁶. 4 M -1 S -1Or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S -1 Rate-specific modifications (cleavage, reduction, or photodegradation).

[0809] Specific cleavage by a protease occurs through contact between the protease and a cleavage site or a molecule containing a cleavage site. The cleavage site can be cleaved in the presence of sufficient enzymatic activity. Sufficient enzymatic activity refers to the enzyme's ability to produce cleavage upon contact with the cleavage site.

[0810] In this document, the term "protease" refers to an enzyme that hydrolyzes peptide bonds, such as an endopeptidase or exopeptidase, typically an endopeptidase. The protease used in this application is limited only to those capable of cleaving protease cleavage sequences, and is not particularly limited by its type. In some embodiments, target tissue-specific proteases are used. Target tissue-specific proteases may refer to, for example, any of the following:

[0811] (1) The protease was expressed at a higher level in the target tissue compared to normal tissue.

[0812] (2) Proteases exhibiting higher activity in target tissues compared to normal tissues.

[0813] (3) Proteases expressed at higher levels in target cells compared to normal cells, and

[0814] (4) Proteases with higher activity in target cells compared to normal cells.

[0815] In a more specific embodiment, a cancer-specific protease or an inflammatory tissue-specific protease is used.

[0816] In this document, the term "target tissue" means tissue containing at least one target cell. In some embodiments, the target tissue is cancerous tissue. In some embodiments, the target tissue is inflamed tissue.

[0817] The term "cancer tissue" refers to tissue containing at least one type of cancer cell. Therefore, considering, for example, that cancer tissue contains both cancer cells and blood vessels, the term refers to every cell type that contributes to the formation of a tumor mass, including cancer cells and endothelial cells. In this document, a tumor mass refers to a lesion of tumor tissue. The term "tumor" is generally used to refer to either benign or malignant growths.

[0818] In this article, examples of "inflammatory tissue" include the following:

[0819] - Joint tissues in rheumatoid arthritis or osteoarthritis,

[0820] - Lung (alveolar) tissue in patients with bronchial asthma or COPD.

[0821] - Digestive organ tissues in inflammatory bowel disease, Crohn's disease, or ulcerative colitis.

[0822] - Fibrotic tissue in the liver, kidneys, or lungs.

[0823] - Tissues in organ transplant rejection

[0824] - Blood vessels or cardiac (myocardial) tissue in arteriosclerosis or heart failure.

[0825] - Visceral adipose tissue in metabolic syndrome

[0826] - Skin tissue in atopic dermatitis and other dermatitis, and

[0827] - Spinal nerve tissue in cases of herniated discs or chronic low back pain.

[0828] For certain types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are considered to be associated with the disease status of the target tissue (target tissue-specific proteases), are known. For example, international publications WO2013 / 128194, WO2010 / 081173 and WO2009 / 025846 disclose proteases that are specifically expressed in cancerous tissues. In addition, J Inflamm (Lond). 2010; 7: 45, Nat Rev Immunol. July 2006; 6 (7): 541-50; Nat Rev Drug Discov. December 2014; 13 (12): 904-27; Respir Res. March 4, 2016; 17: 23, Dis Model Mech. February 2014; 7 (2): 193-203 and Biochim Biophys Acta. January 2012; 1824 (1): 133-45 disclose proteases that are considered to be associated with inflammation.

[0829] In addition to proteases specifically expressed in target tissues, there are also proteases specifically activated in target tissues. For example, proteases can be expressed in an inactive form and then converted to an active form. Many tissues contain substances that inhibit active proteases, and activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 July; 3(7): 489-501). In target tissues, active proteases can be specifically activated by evading inhibition.

[0830] Active proteases can be measured by using an antibody that recognizes the active protease (PNAS 2013 Jan 2; 110 (1): 93-98) or by fluorescently labeling a peptide that can be recognized by the protease, such that the fluorescence is quenched before cleavage but emitted after cleavage (Nat Rev Drug Discov. 2010 Sep; 9 (9): 690-701. doi: 10.1038 / nrd3053).

[0831] From one perspective, the term "target tissue-specific protease" can refer to any of the following:

[0832] (i) Proteases expressed at higher levels in target tissues compared to normal tissues.

[0833] (ii) Proteases exhibiting higher activity in target tissues compared to normal tissues.

[0834] (iii) Proteases expressed at higher levels in target cells compared to normal cells, and

[0835] (iv) Proteases with higher activity in target cells compared to normal cells.

[0836] Specific examples of proteases include, but are not limited to, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartic proteases (cathepsin D, E, K, O, etc.), serine proteases (including matrix proteases (including MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, protease 3, thrombin, kallikrein, trypsin, and chymotrypsin), metalloproteinases (metalloproteinases (MMP1-28), including membrane-bound forms (MMP14-17 and MMP24-25) and secreted forms (MMP1-13, MMP18-23 and MMP26-28), deintegrin metalloproteinases (ADAM), deintegrin metalloproteinases containing platelet-reactive protein motifs (ADAMTS), transmembrane peptidases (transmembrane peptidases α and β), CD10, etc. (CALLA), Prostate-Specific Antigen (PSA), Bean Pod Protein, TMPRSS3, TMPRSS4, Human Neutrophil Elastase (HNE), β-Secretase (BACE), Fibroblast Activator Protein α (FAP), Granase B, Guanidinylbenzoic Acidase (GB), Serine Protease, Enkephalin, NS3 / 4A, HCV-NS3 / 4, Caloplasmin, ADAMDEC1, Renin, Cathepsin C, Cathepsin V / L2, Cathepsin X / Z / P, Cruzipain, Otubain 2, Kallikrein-related Peptidases (KLK (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14)), Bone Morphogenetic Protein 1 (BMP-1), Activator Protein C, Coagulation-related Proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa) And factor XIIa), HtrA1, lactoferrin, malathione, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, pepsin, calpain 2, glutamate carboxypeptidase 2, AMSH-like protease, AMSH, γ-secretase, anti-fibrinolytic cleavage enzyme (APCE), decysin 1, N-acetylated α-linked acidic dipeptidase-like 1 (NAALADL1) and furin.

[0837] From another perspective, target tissue-specific proteases can refer to proteases specific to cancerous tissues or proteases specific to inflamed tissues.

[0838] Examples of cancer tissue-specific proteases include those specifically expressed in cancer tissues as disclosed in international publications WO2013 / 128194, WO2010 / 081173 and WO2009 / 025846.

[0839] Regarding the type of cancer tissue-specific protease, proteases with higher expression specificity in the cancer tissue to be treated are more effective in reducing adverse reactions. Preferably, the concentration of the cancer tissue-specific protease in cancer tissue is at least 5 times, more preferably at least 10 times, further preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times, its concentration in normal tissue. Furthermore, the activity of the preferred cancer tissue-specific protease in cancer tissue is at least 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, further preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times, its activity in normal tissue.

[0840] Cancer-specific proteases can be in the form of being bound to the cancer cell membrane or can be secreted extracellularly without being bound to the cell membrane. When cancer-specific proteases are not bound to the cancer cell membrane, it is preferable that the cancer-specific proteases be present within or near the cancer cell membrane for cancer cell-specific immune cell-mediated cytotoxicity. In this document, "near the cancer cell membrane" means falling within the range where the cancer-specific protease cleavage sequence is cleaved, allowing the antigen-binding domain to exert its antigen-binding activity. However, within this range, it is preferable to minimize damage to normal cells.

[0841] From another perspective, cancer tissue-specific proteases are any of the following:

[0842] (i) Proteases expressed at higher levels in cancerous tissues compared to normal tissues.

[0843] (ii) Proteases exhibiting higher activity in cancerous tissue compared to normal tissue.

[0844] (iii) Proteases expressed at higher levels in cancer cells compared to normal cells, and

[0845] (iv) Proteases with higher activity in cancer cells compared to normal cells.

[0846] A single type of cancer tissue-specific protease can be used, or two or more types of cancer tissue-specific proteases can be combined. The number of cancer tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the type of cancer to be treated.

[0847] From these perspectives, among the proteases listed above, cancer tissue-specific proteases are preferably serine proteases or metalloproteinases, more preferably matrix enzymes (including MT-SP1), urokinase (uPA), or metalloproteinases, and even more preferably MT-SP1, uPA, MMP2, or MMP9.

[0848] Regarding the type of inflammatory tissue-specific protease, proteases with higher expression specificity in the inflamed tissue to be treated are more effective in reducing adverse reactions. Preferably, the concentration of the inflammatory tissue-specific protease in the inflamed tissue is at least 5 times, more preferably at least 10 times, further preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times, its concentration in normal tissue. Furthermore, the activity of the preferred inflammatory tissue-specific protease in the inflamed tissue is at least 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, further preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times, its activity in normal tissue.

[0849] Inflammatory tissue-specific proteases can be in a form that binds to the inflammatory cell membrane or can be secreted extracellularly without binding to the cell membrane. When inflammatory tissue-specific proteases are not bound to the inflammatory cell membrane, it is preferable that the proteases be present within or near the inflammatory tissue for cytotoxicity mediated by immune cells specific to inflammatory cells. In this document, "near the inflammatory tissue" means falling within the range where the protease cleavage sequence specific to the inflammatory tissue is cleaved, allowing the antigen-binding domain to exert its antigen-binding activity. However, within this range, it is preferable to minimize damage to normal cells.

[0850] From another perspective, the inflammatory tissue-specific protease is any one of the following:

[0851] (i) Proteases expressed at higher levels in inflamed tissues compared to normal tissues.

[0852] (ii) Proteases exhibiting higher activity in inflamed tissues compared to normal tissues.

[0853] (iii) Proteases expressed at higher levels in inflammatory cells compared to normal cells, and

[0854] (iv) Proteases with higher activity in inflammatory cells compared to normal cells.

[0855] One type of inflammatory tissue-specific protease can be used alone, or two or more types of inflammatory tissue-specific proteases can be combined. The number and types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the pathological condition to be treated.

[0856] From these perspectives, the tissue-specific proteases for inflammation are preferably metalloproteinases among the proteases listed above. More preferably, the metalloproteinases are ADAMTS5, MMP2, MMP7, MMP9, or MMP13.

[0857] The protease cleavage sequence is a specific amino acid sequence that is specifically recognized by the target tissue-specific protease when the polypeptide is hydrolyzed in aqueous solution by the target tissue-specific protease.

[0858] From the perspective of reducing adverse reactions, the preferred protease cleavage sequence is an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue or cells to be treated.

[0859] Specific examples of protease cleavage sequences include target sequences for the specific hydrolysis of the aforementioned proteases specifically expressed in cancerous tissues, as disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, and proteases specific to inflamed tissues. Sequences that are artificially altered, for example, by introducing appropriate amino acid mutations into target sequences known for their specific hydrolysis by proteases can also be used. Alternatively, protease cleavage sequences identified by methods known to those skilled in the art can be used, as described in Nature Biotechnology 19, 661-667 (2001).

[0860] In addition, naturally occurring protease cleavage sequences can be used. For example, TGFβ is converted to its potential form via protease cleavage. Similarly, protease cleavage sequences that alter the molecular form of a protein through protease cleavage can also be used.

[0861] Examples of usable protease cleavage sequences include, but are not limited to, International Publication Nos. WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, US Patent Publication Nos. US2016 / 0289324, US Patent Publication No. US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228, and Beilstein J. The sequence is disclosed in Nanotechnol. (2016) 7: 364-373.

[0862] The protease cleavage sequence is more preferably an amino acid sequence specifically hydrolyzed by a suitable target tissue-specific protease as described above.

[0863] In one embodiment, the flexible adapter is further attached to one or both ends of the protease cleavage sequence. The flexible adapter at one end of the protease cleavage sequence may be referred to as a first flexible adapter, and the flexible adapter at the other end may be referred to as a second flexible adapter. In a particular embodiment, the protease cleavage sequence and the flexible adapter have any of the following formulas:

[0864] (Protein cleavage sequence)

[0865] (First flexible linker) - (Protein cleavage sequence)

[0866] (Protein cleavage sequence) - (Second flexible adaptor) and

[0867] (First flexible linker) - (Protein cleavage sequence) - (Second flexible linker).

[0868] The flexible linker according to this embodiment is preferably a peptide linker. The first and second flexible linkers are each independent and arbitrary, and may be the same or different flexible linkers, each containing at least one flexible amino acid (Gly, etc.). The flexible linker contains, for example, a sufficient number of residues (arbitrarily selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., especially Gly, Ser, Asp, Asn, and Ala, particularly Gly and Ser, especially Gly, etc.) to enable the protease cleavage sequence to achieve the desired protease accessibility.

[0869] Flexible linkers suitable for use at both ends of protease cleavage sequences are typically flexible linkers that improve protease access to the cleavage sequence and enhance cleavage efficiency. Suitable flexible linkers can be readily selected, and these linkers are preferably selected from different lengths, such as 1 amino acid (Gly, etc.) to 20 amino acids, 2 amino acids to 15 amino acids, or 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. In some embodiments, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0870] Examples of flexible joints include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS: SEQ ID NO: 1834)n and (GGGS: SEQ ID NO: 1835)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible joints well known in conventional techniques.

[0871] Among them, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and can easily act as neutral chain links between components.

[0872] In this article, "association" can refer to, for example, the state in which two or more polypeptide regions interact with each other. Typically, hydrophobic bonds, hydrogen bonds, ionic bonds, etc., are formed between the intended polypeptide regions to form an association. As a common example of association, antibodies, represented by natural antibodies, are known to retain the paired structure of the heavy chain variable region (VH) and the light chain variable region (VL) through non-covalent bonds, etc.

[0873] In some embodiments, the inhibitory domain of the carrier portion associates with the antigen-binding domain. The inhibitory domain may constitute part of or all of the carrier portion. Alternatively, the inhibitory domain may also be defined as the portion of the carrier portion that associates with the antigen-binding domain.

[0874] In a more specific embodiment, the antigen-binding domain of the single-domain antibody and the inhibitory domain as VL, VH, or VHH form an association as found between antibody VH and antibody VL. In another specific embodiment, the antigen-binding domain of the single-domain antibody and the inhibitory domain as VL, VH, or VHH are found to form an association between antibody VH and antibody VL, and in the thus formed association state, the inhibitory domain conformation inhibits the binding of the antigen-binding domain to the antigen or conformationally alters the antigen-binding site of the antigen-binding domain, such that the antigen-binding activity of the single-domain antibody is inhibited by VL, VH, or VHH. In embodiments using VHH as a single-domain antibody, when CDR3 (the major antigen-binding site of VHH) or its adjacent site is present at the interface with the inhibitory domain, the binding of VHH to the antigen is considered to be inhibited by the conformation of the inhibitory domain.

[0875] The association between antigen-binding and inhibitory domains can be canceled, for example, by cleavage of the cleavage site. The cancellation of association can be used interchangeably with, for example, the cancellation of interactions between two or more polypeptide regions. The interactions between two or more polypeptide regions can be completely canceled, or they can be partially canceled.

[0876] In this document, "interface" generally refers to the face of two regions that associate or interact with each other. The amino acid residues forming the interface are typically one or more amino acid residues contained in each polypeptide region undergoing association, and more preferably, amino acid residues that are close to each other and participate in the interaction during association. Specifically, the interaction includes non-covalent bonds, such as hydrogen bonds, electrostatic interactions, or the formation of salt bridges between amino acid residues that are close to each other during association.

[0877] In this document, "amino acid residues forming the interface" specifically refers to amino acid residues contained in the polypeptide regions constituting the interface. As an example, the polypeptide region constituting the interface refers to a polypeptide region in antibodies, ligands, receptors, substrates, etc., responsible for selective intramolecular or intermolecular binding. Specific examples of such polypeptide regions in antibodies may include heavy chain variable regions and light chain variable regions. In some embodiments, examples of such polypeptide regions may include antigen-binding domains and inhibitory domains.

[0878] Examples of amino acid residues that form an interface include, but are not limited to, amino acid residues that are close to each other during association. For example, amino acid residues that are close to each other during association can be identified by analyzing the conformation of a polypeptide and examining the amino acid sequence of the polypeptide region that forms the interface during polypeptide association.

[0879] In some embodiments, amino acid residues involved in association within the antigen-binding domain or in association within the repressor domain may be modified to promote association between the antigen-binding and repressor domains. In another specific embodiment, amino acid residues forming the interface between the antigen-binding domain and the repressor domain, or vice versa, may be modified. In a preferred embodiment, the amino acid residues forming the interface may be modified by introducing mutations into the interface amino acid residues, such that the two or more amino acid residues forming the interface have different charges. Modifying amino acid residues to produce different charges includes changing positively charged amino acid residues to negatively charged or uncharged amino acid residues, changing negatively charged amino acid residues to positively charged or uncharged amino acid residues, and changing uncharged amino acid residues to positively or negatively charged amino acid residues. Such amino acid modifications are made for the purpose of promoting association and are not limited to the position or type of amino acid modified, as long as the purpose of promoting association is achieved. Examples of modifications include, but are not limited to, substitution.

[0880] In some embodiments, the VHH, serving as the antigen-binding domain, associates with the VL, serving as the inhibitory domain. The amino acid residues in the VHH involved in the association with the VL can refer to, for example, amino acid residues that form an interface between the VHH and the VL. Examples of amino acid residues in the VHH involved in the association with the VL include, but are not limited to, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). The activity of the VHH is inhibited by promoting association between the VHH and the VL. Similarly, the amino acid residues in the VL involved in the association with the VHH can refer to, for example, amino acid residues that form an interface between the VHH and the VL.

[0881] The amino acid residues in VHH involved in association with VL can be altered to promote association between VHH and VL. Examples of such amino acid substitutions include, but are not limited to, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, and / or S47W. A VHH originally having amino acid residues 37V, 44G, 45L, and / or 47W can be used instead of altering every residue in the VHH.

[0882] The amino acid residues involved in association with VL in VHH can be changed instead of the VHH amino acids, and amino acid changes can also be introduced into both VHH and VL, as long as the purpose of promoting association between VHH and VL can be achieved.

[0883] In some alternative embodiments, the antigen-binding domain and the repressive domain can associate with each other by using VHH as the antigen-binding domain and using VH or VHH as the repressive domain. The amino acid residues in VHH that participate in association with VH or VHH that serve as the repressive domain can be identified and modified to promote association between the antigen-binding domain VHH and the repressive domain VH or VHH. Furthermore, the amino acid residues in VH or VHH that participate in association with VHH that serves as the antigen-binding domain can be identified and modified.

[0884] When using single-domain antibodies other than VHH as antigen-binding domains, it is also possible to identify and modify amino acid residues in association-participating, antigen-binding, or inhibitory domains similar to those described above.

[0885] In some embodiments, the carrier portion and the antigen-binding domain are fused via a linker. In a more specific embodiment, the carrier portion and the antigen-binding domain are fused via a linker containing a cleavage site. In an alternative specific embodiment, the carrier portion and the antigen-binding domain are fused via a linker, and the resulting fusion protein contains a cleavage site.

[0886] In another embodiment, the carrier portion and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino bond is formed between the N-terminal amino acid of the carrier portion and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The formed fusion protein contains a cleavage site. In a particular embodiment, one or more N-terminal amino acids of the carrier portion and / or one or more C-terminal amino acids of the antigen-binding domain are altered, and the N-terminus of the carrier portion and the C-terminus of the antigen-binding domain are fused to form a cleavage site near the fusion site. More specifically, the cleavage site can be formed, for example, by converting four C-terminal amino acids of the antigen-binding domain to the LSGR (SEQ ID NO: 1836) sequence and four N-terminal amino acids of the carrier portion to the SDNH (SEQ ID NO: 1837) sequence.

[0887] In some embodiments, the cleavage site of the polypeptide comprising a portion and an antigen-binding domain includes a protease cleavage sequence. The protease cleavage sequence can be placed at any position on the polypeptide, as long as the antigen-binding domain is released by protease cleavage and does not lose its antigen-binding activity after release.

[0888] In some embodiments, the carrying portion includes an antibody constant region, and the N-terminus of the antibody constant region and the C-terminus of the antigen-binding domain are fused via a linker or without a linker.

[0889] In a particular embodiment, the protease cleavage sequence is located within the antibody constant region contained in the carrier portion. In this case, the protease cleavage sequence may be located within the antibody constant region, such that the antigen-binding domain is released by protease cleavage. In a particular embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region contained in the carrier portion, and more specifically, on the side of the antigen-binding domain relative to amino acid position 140 (EU number) in the antibody heavy chain constant region, preferably on the side of the antigen-binding domain relative to amino acid position 122 (EU number) in the antibody heavy chain constant region. In an alternative embodiment, the protease cleavage sequence is located within the antibody light chain constant region contained in the carrier portion, and more specifically, on the side of the antigen-binding domain relative to amino acid position 130 (EU number) (Kabat number position 130) in the antibody light chain constant region, preferably on the side of the antigen-binding domain relative to amino acid position 113 (EU number) (Kabat number position 113) in the antibody light chain constant region.

[0890] In some embodiments, the antigen-binding domain is a single-domain antibody, and the C-terminus and the N-terminus of the carrying portion of the single-domain antibody are fused via a linker or not fused with a linker.

[0891] In a particular embodiment, the protease cleavage sequence is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody prepared from VH or VHH, and the protease cleavage sequence is located on the carrier side of the single-domain antibody relative to amino acid position 35b (Kabat number), preferably on the carrier side of the single-domain antibody relative to amino acid position 95 (Kabat number), and more preferably on the carrier side of the single-domain antibody relative to amino acid position 109 (Kabat number). In an alternative embodiment, the single-domain antibody is a single-domain antibody prepared from VL, and the protease cleavage sequence is located on the carrier side of the single-domain antibody relative to amino acid position 32 (Kabat number), preferably on the carrier side of the single-domain antibody relative to amino acid position 91 (Kabat number), and more preferably on the carrier side of the single-domain antibody relative to amino acid position 104 (Kabat number).

[0892] In some embodiments, the carrying portion includes an antibody constant region, the antigen-binding domain is a single-domain antibody, and the antibody constant region and the single-domain antibody are fused via a linker or without a linker. In a more specific embodiment, the N-terminus of the antibody constant region and the C-terminus of the single-domain antibody are fused via a linker or without a linker. In an alternative embodiment, the C-terminus of the antibody constant region and the N-terminus of the single-domain antibody are fused via a linker or without a linker.

[0893] In a particular embodiment, the protease cleavage sequence is located within the antibody constant region included in the carrying portion. In a more specific embodiment, the protease cleavage sequence is located on the antibody heavy chain constant region on the single-domain antibody side relative to amino acid position 140 (EU number), preferably on the antibody heavy chain constant region on the single-domain antibody side relative to amino acid position 122 (EU number). In an alternative embodiment, the protease cleavage sequence is located on the antigen-binding domain side relative to amino acid position 130 (EU number) (Kabat number position 130) in the antibody light chain constant region, preferably on the antigen-binding domain side relative to amino acid position 113 (EU number) (Kabat number position 113) in the antibody light chain constant region.

[0894] In a particular embodiment, the protease cleavage sequence is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody prepared from VH or VHH, and the protease cleavage sequence is located on the antibody constant region side of the single-domain antibody relative to amino acid position 35b (Kabat number), preferably on the antibody constant region side of the single-domain antibody relative to amino acid position 95 (Kabat number), and more preferably on the antibody constant region side of the single-domain antibody relative to amino acid position 109 (Kabat number). In an alternative embodiment, the single-domain antibody is a single-domain antibody prepared from VL, and the protease cleavage sequence is located on the antibody constant region side of the single-domain antibody relative to amino acid position 32 (Kabat number), preferably on the antibody constant region side of the single-domain antibody relative to amino acid position 91 (Kabat number), and more preferably on the antibody constant region side of the single-domain antibody relative to amino acid position 104 (Kabat number).

[0895] In a particular embodiment, the protease cleavage sequence is located near the boundary between the antigen-binding domain and the carrier moiety. The phrase "near the boundary between the antigen-binding domain and the carrier moiety" refers to a portion located upstream or downstream of the junction site between the antigen-binding domain and the carrier moiety, and which largely does not affect the secondary structure of the antigen-binding domain.

[0896] In a more specific embodiment, the antigen-binding domain is linked to an antibody constant region contained in the carrying portion, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. The phrase "near the boundary between the antigen-binding domain and the antibody constant region" can refer to the boundary between the antigen-binding domain and the antibody heavy chain constant region, or the boundary between the antigen-binding domain and the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody prepared from VH or VHH and linked to the antibody heavy chain constant region, the phrase "near the boundary between the antigen-binding domain and the antibody constant region" can refer to the amino acid position 101 (Kabat number) of the single-domain antibody and amino acid position 140 (EU number) of the antibody heavy chain constant region, and preferably can refer to the amino acid position 109 (Kabat number) of the single-domain antibody and amino acid position 122 (EU number) of the antibody heavy chain constant region. When the antigen-binding domain is a single-domain antibody prepared from VH or VHH and is linked to the constant region of the antibody light chain, the phrase "near the boundary between the antigen-binding domain and the constant region of the antibody light chain" can refer to the area between amino acid position 101 (Kabat number) of the single-domain antibody and amino acid position 130 (EU number) of the constant region of the antibody light chain (Kabat number position 130), and preferably between amino acid position 109 (Kabat number) of the single-domain antibody and amino acid position 113 (EU number) of the constant region of the antibody light chain (Kabat number position 113). When the antigen-binding domain is a single-domain antibody prepared from VL, the phrase "near the boundary between the antigen-binding domain and the constant region of the antibody" refers to the area between amino acid position 96 (Kabat number) of the single-domain antibody and a specified position of the constant region of the antibody, preferably between amino acid position 104 (Kabat number) of the single-domain antibody and a specified position of the constant region of the antibody.

[0897] In some embodiments, the polypeptide is an IgG antibody-like molecule. Examples of such embodiments include, but are not limited to: embodiments in which the carrying portion comprises an IgG antibody constant region, a single-domain antibody serving as an antigen-binding domain replaces the VH of the IgG antibody, and antigen-binding activity is inhibited by VL; embodiments in which the carrying portion comprises an IgG antibody constant region, a single-domain antibody serving as an antigen-binding domain replaces the VL of the IgG antibody, and antigen-binding activity is inhibited by VH; and embodiments in which the carrying portion comprises an IgG antibody constant region, a single-domain antibody serving as an antigen-binding domain replaces one of the VH and VL of the IgG antibody, and a further single-domain antibody inhibits the antigen-binding activity of the antigen-binding domain, replacing other domains of the IgG antibody.

[0898] The term "IgG antibody-like molecule" as used herein is used to define a molecule having portions of a structure substantially similar to constant domains or constant regions in an IgG antibody, and portions of a structure substantially similar to variable domains or variable regions in an IgG antibody, and having a conformation substantially similar to that of an IgG antibody. However, in this context, "IgG antibody-like molecules" may or may not exhibit antigen-binding activity while retaining a structure similar to that of an IgG antibody.

[0899] A peptide may contain one or more antigen-binding domains. One or more inhibitory domains may inhibit the antigen-binding activity of multiple antigen-binding domains. Multiple antigen-binding domains may each associate with an inhibitory domain. Multiple antigen-binding domains may each fuse with a carrier moiety. Multiple antigen-binding domains may each be capable of being released from the peptide. One or more cleavage sites for releasing multiple antigen-binding domains may be multiple cleavage sites corresponding to the number of antigen-binding domains.

[0900] In some embodiments, the antigen-binding domain is further linked to a second antigen-binding domain. Examples of the second antigen-binding domain include, but are not limited to, single-domain antibodies, antibody fragments, modules containing an A domain of approximately 35 amino acids in the in vivo cell membrane protein avimer (International Publications WO2004 / 044011 and WO2005 / 040229); adnectin containing a 10Fn3 domain that acts as a protein-binding domain, derived from the glycoprotein fibronectin expressed on the cell membrane (International Publication WO2002 / 032925); affinity molecules containing an IgG-binding domain scaffold that forms a triple-helix bundle of 58 amino acids of protein A (International Publication WO1995 / 001937); and DARPins (designed ankyrin repeat proteins), which are molecular surface exposed regions of ankyrin repeat sequences (AR), each region having 33 subunits folded into turns, two antiparallel helices, and loops. The structure consists of 10 amino acid residues (International Publication No. WO2002 / 020565); an anticarrier protein having four loop regions connected to eight antiparallel chains bent toward the central axis at one end of a barrel-shaped structure, which is highly conserved in lipid carrier protein molecules such as neutrophil gelatinase-associated carrier protein (NGAL) (International Publication No. WO2003 / 029462); and a recessed region in the horseshoe-shaped folded inner parallel sheet-like structure of the variable lymphocyte receptor (VLR) with a non-immunoglobulin structure, consisting of repeating leucine-rich repeat (LRR) modules (International Publication No. WO2008 / 016854). In a preferred embodiment, the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. In a preferred embodiment, the molecular weight of the connected antigen-binding domain and the second antigen-binding domain is 60 kDa or less.

[0901] In some more specific embodiments, the antigen-binding domain and the second antigen-binding domain are single-domain antibodies with different antigen-binding specificities. The linked antigen-binding domain and the second antigen-binding domain are releaseable from the peptide, and upon release, they form a bispecific antigen-binding molecule. Examples of such bispecific antigen-binding molecules include, but are not limited to, bispecific antigen-binding molecules having an antigen-binding domain that specifically binds to antigens on the surface of target cells and a second antigen-binding domain that specifically binds to antigens on the surface of immune cells; bispecific antigen-binding molecules having an antigen-binding domain and a second antigen-binding domain that bind to different subunits of the same antigen; and bispecific antigen-binding molecules having an antigen-binding domain and a second antigen-binding domain that bind to different epitopes of the same antigen. Such bispecific antigen-binding molecules can recruit immune cells to the vicinity of target cells and are therefore considered for use in treating diseases caused by target cells.

[0902] The antigen-binding activity of the second antigen-binding domain may or may not be inhibited by the carrier portion. The second antigen-binding domain may or may not associate with a portion of the carrier portion. Specifically, when the antigen-binding domain and the second antigen-binding domain differ in antigen-binding specificity, the unreleased antigen-binding domain cannot exert antigen-binding activity, even if the antigen-binding activity of the second antigen-binding domain is not inhibited, and even if the second antigen-binding domain does not associate with a portion of the carrier portion. Such a bispecific antigen-binding molecule containing an antigen-binding domain linked to a second antigen-binding domain cannot function as a bispecific binding agent for both types of antigens.

[0903] The antigen-binding molecules in this application include, for example, those described in WO2018 / 097308.

[0904] In one aspect, the antigen-binding molecule of this application relates to an antigen-binding molecule as a ligand-binding molecule capable of binding to a ligand, wherein the molecule is a polypeptide having at least one cleavage site, and wherein the ligand binding of the molecule is weakened when the molecule is in a cleaved state at at least one cleavage site.

[0905] Ligand-binding molecules are molecules capable of binding to ligands, particularly those that are unclessed. In this context, "binding" typically refers to binding primarily through interactions based on non-covalent bonds (such as electrostatic forces, van der Waals forces, or hydrogen bonds). Preferred examples of ligand-binding forms of ligand-binding molecules include, but are not limited to, antigen-antibody reactions, in which antigen-binding regions, antigen-binding molecules, antibodies, antibody fragments, etc., bind to antigens.

[0906] The phrase "capable of binding to ligands" means that the ligand-binding molecule can bind to the ligand even if the ligand-binding molecule and the ligand are different molecules; it does not imply that the ligand-binding molecule and the ligand are connected by a covalent bond. For example, the phrase "capable of binding to ligands" does not mean that the ligand and the ligand-binding molecule are connected by a covalent bond via a linker. Furthermore, the phrase "weakened ligand binding" means that the binding abilit...

Claims

1. An antigen-binding molecule comprising an antigen-binding domain, a CL domain, and a non-natural human IgG CH1 domain, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-natural human IgG CH1 domain is different from the corresponding sequence in a natural human IgG having the same isotype as the non-natural human IgG.

2. The antigen-binding molecule according to claim 1, wherein the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index has any of the following characteristics: (1) The amino acid at any one or more sites at positions 122 to 128 and 130 to 132 is Ala; (2) The amino acid at any one or more sites from position 122 to 132 is Ile; (3) The amino acid at any one or more sites at positions 122 to 124 and positions 126 to 132 is Val; (4) The amino acid at any one or more sites at positions 122 to 127 and positions 129 to 132 is Leu; (5) The amino acid at any one or more sites at positions 122 to 125 and positions 127 to 132 is Phe; (6) Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala has been inserted at one or more of the following sites: the site between positions 121 and 122, the site between positions 122 and 123, the site between positions 123 and 124, the site between positions 124 and 125, the site between positions 125 and 126, the site between positions 126 and 127, the site between positions 127 and 128, the site between positions 128 and 129, the site between positions 129 and 130. The loci between positions 130 and 131, between positions 131 and 132, and between positions 132 and 133.

3. A method for detecting and / or quantifying antigen-binding molecules in a biological sample, the method comprising: (a) Treating a biological sample containing an antigen-binding molecule with a digestive enzyme to produce a peptide from the antigen-binding molecule, wherein The antigen-binding molecule comprises an antigen-binding domain, a CL domain, and a non-natural human IgG CH1 domain. The amino acid sequence between positions 121 and 133 of the CH1 domain of the non-natural human IgG, according to the EU index, differs from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG, and The peptide consists of the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index; (b) Analyze the biological sample treated in (a) by mass spectrometry to detect the peptide; as well as (c) Detect and / or quantify the antigen-binding molecules based on the analytical results of (b).

4. A method for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample, the method comprising: (a) Treating a biological sample containing multiple types of antigen-binding molecules with a digestive enzyme to produce multiple types of peptides from said multiple types of antigen-binding molecules, wherein The various types of antigen-binding molecules include antigen-binding molecules containing an antigen-binding domain, a CL domain, and a natural or non-natural human IgG CH1 domain. The amino acid sequence between positions 121 and 133 of the human IgG CH1 domain, according to the EU index, differs among the various types of antigen-binding molecules. The various types of peptides consist of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural or non-natural human IgG according to the EU index, and the amino acid sequence is different among the various types of peptides; (b) Analyze the biological sample treated in (a) by mass spectrometry to detect each of the multiple types of peptides; as well as (c) Based on the analytical results of (b), detect and / or quantify each of the multiple types of antigen-binding molecules.

5. The method of claim 4, wherein the plurality of peptides comprises: (I) Two or more types of peptides comprising an amino acid sequence in the CH1 domain of a non-natural human IgG, between positions 121 and 133 according to the EU index, wherein the amino acid sequence differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG, or (II) A peptide consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural human IgG according to the EU index; and one or more types of peptides consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of non-natural human IgG according to the EU index, wherein the amino acid sequence is different from the corresponding sequence in natural human IgG having the same isotype as the non-natural human IgG.

6. A method for designing, selecting, or generating a modified antigen-binding molecule, said modified antigen-binding molecule comprising an artificially modified peptide moiety detectable by mass spectrometry, wherein the method comprises: (I) Identification of peptides obtained by treating an antigen-binding molecule with a digestive enzyme and detectable by mass spectrometry; (II) Design artificially modified peptides using any of the following methods: (II-1) Delete one or more amino acid residues in the peptide identified in (I) or replace one or more amino acid residues in the peptide identified in (I) with other amino acid residues; (II-2) Insert one or more amino acid residues into the peptide identified in (I); (II-3) Add an amino acid residue to the N-terminus or C-terminus of the peptide identified in (I); (II-4) Combinations of (II-1) and (II-2); (II-5) Combinations of (II-1) and (II-3); Combinations of (II-6), (II-2), and (II-3); Combinations of (II-7), (II-1), (II-2), and (II-3); (III) Modify the peptide portion identified in (I) of the antigen-binding molecule of (I) in (II) to produce a modified antigen-binding molecule; (IV) The modified antigen-binding molecule produced in (III) is selected if it satisfies any of the following: (IV-1) The modified antigen-binding molecule has a Tm value of 50°C or higher; (IV-2) The antigen-binding activity of the modified antigen-binding molecule is 10% or higher of the antigen-binding activity of the antigen-binding molecule in (I); and (IV-3) Both (IV-1) and (IV-2) mentioned above; and (V) The modified antigen-binding molecule is selected if the portion of the modified antigen-binding molecule selected in (IV) that corresponds to the peptide designed in (II) can be detected by mass spectrometry after the biological sample containing the modified antigen-binding molecule is treated with the digestive enzyme of (I).

7. The method according to claim 6, wherein in (II-2), the inserted amino acid residue is Ala, Ile, Val, Leu, Phe, Ala-Ala, Ile-Ile, Val-Val, Leu-Leu, Phe-Phe, Ala-Ile, Ala-Val, Ala-Leu, Ala-Phe, Ile-Val, Ile-Leu, Ile-Phe, Val-Leu, Val-Phe, Leu-Phe, or Ala-Ala-Ala.

8. A modified antigen-binding molecule designed, selected, or generated by the method according to claim 6 or 7.

9. A peptide comprising an amino acid sequence in the CH1 domain of a non-natural human IgG between positions 121 and 133 according to the EU index, wherein the amino acid sequence is different from the corresponding sequence in a natural human IgG having the same isotype as the non-natural human IgG.

10. A collection comprising two or more types of peptides, said two or more types of peptides consisting of an amino acid sequence between positions 121 and 133 of the CH1 domain of natural or non-natural human IgG according to the EU index, wherein said amino acid sequence is distinct between said two or more types of peptides.

11. The collection of claim 10, wherein the collection comprises two or more types of peptides, the two or more types of peptides being composed of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of a non-natural human IgG, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG is selected from the group consisting of SEQ ID NO: 6 to 294 and 1565 to 1833.

12. The set according to claim 10, wherein the set comprises: a peptide consisting of an amino acid sequence in the CH1 domain of a natural human IgG cell between positions 121 and 133 according to the EU index; and one or more types of peptides consisting of an amino acid sequence in the CH1 domain of a non-natural human IgG cell between positions 121 and 133 according to the EU index, wherein When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 6 to 294, and When the amino acid sequence between positions 121 and 133 of the natural human IgG CH1 domain according to the EU index is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 of the non-natural human IgG CH1 domain according to the EU index is selected from the group consisting of SEQ ID NO: 1565 to 1833.

13. The set according to claim 10, for use in a method for detecting each of a plurality of peptides derived from a plurality of types of antigen-binding molecules in a biological sample by mass spectrometry, or in a method for detecting and / or quantifying each of a plurality of types of antigen-binding molecules in a biological sample.

14. A method for administering a mixture of multiple types of antigen-binding molecules to an individual or subject, each of the multiple types of antigen-binding molecules comprising a peptide from any one of claims 10 to 13.

15. A method for selecting antigen-binding molecules, the method comprising: (a) Detecting and / or quantifying each of the plurality of antigen-binding molecules in a biological sample from an individual or subject to which a mixture of the plurality of antigen-binding molecules has been administered, wherein each of the plurality of antigen-binding molecules comprises a peptide from the set according to any one of claims 10 to 13; and (b) Select antigen-binding molecules based on the results of the detection and / or quantification described in (a).

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