Peptide capable of being analyzed and / or evaluated by mass spectrometry and use thereof

Abstract

The present inventors have devised a method for designing a unique sequence by modifying part of the sequence of an antigen-binding molecule, and analyzing and / or evaluating the sequence with a mass spectrometer. On the basis of said devised idea, the present application provides, for example: an antigen-binding molecule; a method for detecting and / or quantifying an antigen-binding molecule in a biological sample; a method for designing, selecting, or producing a modified antigen-binding molecule; a modified antigen-binding molecule designed, selected, or produced by the method; a peptide which is derived from an antigen-binding molecule and is capable of being analyzed and / or evaluated by mass spectrometry; and a set comprising two or more kinds of the peptide.

Description

Peptides that can be analyzed and / or evaluated by mass spectrometry and uses thereof

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

[0002] One method for detecting, quantifying, or examining the pharmacokinetics and distribution of antigen-binding molecules such as antibodies is to perform mass spectrometry on peptides with specific sequences contained in antigen-binding molecules (Patent Document 1). An example of measurement using a unique sequence contained in each antibody is described in Non-Patent Document 1. There is also a method for identifying antibodies and antigen-binding molecules by adding an amino acid sequence or the like that serves as a barcode to an antigen-binding molecule such as an antibody and measuring the barcode portion.

[0003] Cassette-dose testing (cocktail administration testing) is a method for simultaneously administering multiple drugs or samples to animals and evaluating their pharmacokinetics and distribution in the same animal. Because it allows for the evaluation of multiple samples in the same animal, cassette-dose testing has the advantages of facilitating comparison with a reference substance and reducing the number of animals used. Four types of cassette-dose testing have been performed on antibodies to date: (1) A method in which three different antibodies, each recognizing a different antigen, are mixed and assayed using the ECL method with the three different antigens (Non-Patent Document 1); (2) A method in which antibodies are labeled with different radioactive metals (Non-Patent Document 2); (3) A method in which a mass spectrometer is used to measure sequences in the variable region of an antibody, including sequences unique to that antibody (Non-Patent Document 3); and (4) A method in which a barcode amino acid sequence is added to the end of an antibody to measure the barcode site.

[0004] Pharmaceutical Research volume 38, 583-592 (2021).Methods Mol Biol. 2014; 1141: 147-157.Clin Vaccine Immunol. 2017 May; 24(5): e00545-16.

[0005] WO2012 / 155019WO2008 / 093762

[0006] The present application aims to provide a new method for analyzing and / or evaluating antigen-binding molecules using a mass spectrometer.

[0007] The present inventors have devised a method for designing unique sequences by modifying a portion of the sequence of an antigen-binding molecule and analyzing and / or evaluating the sequences using a mass spectrometer. Based on this invention, the present application provides antigen-binding molecules, methods for detecting and / or quantifying antigen-binding molecules in biological samples, methods for designing, selecting, or producing modified antigen-binding molecules, peptides derived from antigen-binding molecules that can be analyzed and / or evaluated by mass spectrometry, etc. For example, the present application provides the following.

[0008] [A1] An antigen-binding molecule comprising an antigen-binding domain, a CL domain, and a non-naturally occurring human IgG CH1 domain, wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-naturally occurring human IgG CH1 domain is different from the corresponding sequence in a natural human IgG of the same isotype as the non-naturally occurring human IgG. [A2] The antigen-binding molecule of [A1], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-naturally occurring human IgG CH1 domain has any one of the following characteristics: (1) an amino acid at one or more positions from 122 to 132 is different from the amino acid present at the corresponding position in a natural human IgG of the same isotype, and the amino acid at said position is an amino acid other than Lys, Arg, and Met (with the proviso that the amino acid at position 122 is not Pro); An amino acid other than Lys, Arg, or Met is inserted at one or more sites selected from 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 (however, the amino acid at the site between positions 121 and 122 is not Pro).[A3] The antigen-binding molecule of [A1] or [A2], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following features: (1) the amino acid at any one or more positions from 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one or more positions from 122 to 132 is Ile; (3) the amino acid at any one or more positions from 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one or more positions from 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one or more positions from 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 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 is inserted at one or more sites selected from the above.[A4] The antigen-binding molecule of [A1] or [A2], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following features: (1) the amino acid at any one of positions 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one of positions 122 to 132 is Ile; (3) the amino acid at any one of positions 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one of positions 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one of positions 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 [A5] The antigen-binding molecule of any one of [A1] to [A4], wherein the non-naturally occurring human IgG CH1 domain is a non-naturally occurring human IgG1 CH1 domain, a non-naturally occurring human IgG2 CH1 domain, a non-naturally occurring human IgG3 CH1 domain, or a non-naturally occurring human IgG4 CH1 domain, and wherein 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 is inserted into one site selected from the group consisting of: [A6] [A6] The antigen-binding molecule of any one of [A1] to [A5], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-native human IgG is any one of the amino acid sequences of SEQ ID NOs: 6 to 294 and 1565 to 1833.[A7] The antigen-binding molecule of any one of [A1] to [A6], wherein the antigen-binding domain comprises a VH domain and a VL domain. [A8] The antigen-binding molecule of [A7], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain. [A9] 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. [A10] 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. [A11] The antigen-binding molecule of any one of [A1] to [A10], further comprising an antibody Fc region. [A12] The antigen-binding molecule of any one of [A1] to [A11], wherein the antigen-binding molecule is a non-natural IgG antibody. [A13] The antigen-binding molecule of [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. [A14] The antigen-binding molecule of any one of [A1] to [A13], which is conjugated to a drug moiety.

[0009] [B1] A method for detecting a peptide, comprising the steps of: (a) preparing a sample containing a peptide consisting 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 differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG; and (b) performing mass spectrometry on the sample prepared in (a) to detect the peptide. [B2] A method for quantifying a peptide, comprising the following steps: (a) preparing a sample containing a peptide consisting 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 differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG; (b) subjecting the sample prepared in (a) to mass spectrometry to detect the peptide; and (c) quantifying the peptide based on the analysis results of (b). [B3] A method for detecting and / or quantifying antigen-binding molecules in a biological sample, comprising the following steps: (a) treating a biological sample containing antigen-binding molecules with a digestive enzyme to generate peptides from the antigen-binding molecules, wherein the antigen-binding molecules comprise an antigen-binding domain, a CL domain, and a CH1 domain of a non-natural human IgG, and the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-natural human IgG differs from the corresponding sequence in a 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 according to the EU index in the CH1 domain of the non-natural human IgG; (b) performing mass spectrometry on the biological sample treated in (a) to detect the peptides; and (c) detecting and / or quantifying the antigen-binding molecules based on the analysis results of (b).[B4] The method according to any one of [B1] to [B3], 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 has any one of the following characteristics: (1) an amino acid at one or more positions from 122 to 132 is different from the amino acid present at the corresponding position in a natural human IgG of the same isotype, and the amino acid at said position is an amino acid other than Lys, Arg, and Met (with the proviso that the amino acid at position 122 is not Pro); (2) An amino acid other than Lys, Arg, or Met is inserted at one or more sites selected from 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 (however, the amino acid at the site between positions 121 and 122 is not Pro).[B5] The method of any one of [B1] to [B3], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following characteristics: (1) the amino acid at any one or more positions from 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one or more positions from 122 to 132 is Ile; (3) the amino acid at any one or more positions from 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one or more positions from 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one or more positions from 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 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 is inserted at one or more sites selected from the above.[B6] The method according to any one of [B1] to [B3], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following characteristics: (1) the amino acid at any one of positions 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one of positions 122 to 132 is Ile; (3) the amino acid at any one of positions 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one of positions 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one of positions 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 [B7] The method of any one of [B1] to [B6], wherein the non-naturally occurring human IgG CH1 domain is a non-naturally occurring human IgG1 CH1 domain, a non-naturally occurring human IgG2 CH1 domain, a non-naturally occurring human IgG3 CH1 domain, or a non-naturally occurring human IgG4 CH1 domain, wherein 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 is inserted into one site selected from the group consisting of: [B8] [B8] The method according to any one of [B1] to [B6], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-native human IgG is any one of the amino acid sequences of SEQ ID NOs: 6 to 294 and 1565 to 1833.[B9] The method according to any one of [B3] to [B8], wherein the digestive enzyme is a protease. [B10] The method according to any one of [B3] to [B8], wherein the digestive enzyme is trypsin. [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, eluting the peptide-enriched biological sample, and subjecting the enriched biological sample to mass spectrometry. [B12] The method according to [B1] or [B2], wherein step (b) comprises contacting the sample prepared in step (a) with an affinity capture medium or a chromatographic adsorbent, eluting the peptide-enriched sample, and subjecting the enriched sample to mass spectrometry. [B13] The method of any one of [B3] to [B10], wherein the step (a) comprises contacting the biological sample with an affinity capture medium or a chromatographic adsorbent, and eluting the biological sample enriched in the antigen-binding molecule, and treating the enriched biological sample with a digestive enzyme. [B14] The method of any one of [B11] to [B13], wherein the affinity capture medium is Protein A or Protein G. [B15] The method of any one of [B11] to [B13], wherein the chromatographic adsorbent is a solid-phase extraction (SPE) adsorbent. [B16] The method of any one of [B1] to [B10], wherein the step (b) comprises subjecting the biological sample treated in step (a) or the sample prepared in step (a) to mass spectrometry in combination with liquid chromatography. [B17] The method according to any one of [B1] to [B10], wherein the step (b) comprises a step of separating a fraction containing the peptide from the biological sample treated in the step (a) or the sample prepared in the step (a) by liquid chromatography, and a step of subjecting the separated fraction to mass spectrometry.[B18] The method of any one of [B3] to [B10], wherein the step (a) comprises separating a fraction containing the antigen-binding molecule from the biological sample by liquid chromatography and treating the separated fraction with a digestive enzyme. [B19] The method of any one of [B1] to [B18], wherein mass analysis is performed by tandem mass spectrometry or multistage mass spectrometry. [B20] The method of any one of [B1] to [B18], wherein mass analysis is performed by tandem mass spectrometry or multistage mass spectrometry in selected reaction monitoring (SRM) measurement mode. [B21] The method of any one of [B3] to [B20], wherein the biological sample is serum, plasma, tissue, or cells from an animal treated with the antigen-binding molecule. [B22] The method of any one of [B3] to [B21], wherein the antigen-binding domain comprises a VH domain and a VL domain. [B23] The method according to [B22], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain. [B24] The method according to [B22] or [B23], 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. [B25] The method according to [B22] or [B23], 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. [B26] The method according to any one of [B3] to [B25], wherein the antigen-binding molecule further comprises an antibody Fc region. [B27] The method according to [B11] or [B13], wherein the antigen-binding molecule further comprises an antibody Fc region, and the affinity capture medium is an anti-Fc antibody. [B28] The method of any one of [B3] to [B27], wherein the antigen-binding molecule is a non-natural IgG antibody. [B29] The method of [B28], 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. [B30] The method of any one of [B3] to [B29], wherein the antigen-binding molecule is conjugated to a drug moiety.

[0010] [C1] A method for detecting each of multiple types of peptides in a sample, comprising the steps of: (a) preparing a sample containing multiple types of peptides, wherein the multiple types of peptides consist of amino acid sequences between positions 121 and 133 of the EU index in the CH1 domain of natural or non-natural human IgG, and the amino acid sequences differ among the multiple types of peptides; and (b) performing mass spectrometry on the sample prepared in (a) to detect each of the multiple types of peptides. [C2] A method for quantifying each of multiple types of peptides in a sample, comprising the steps of: (a) preparing a sample containing multiple types of peptides, wherein the multiple types of peptides consist of amino acid sequences between positions 121 and 133 of the EU index in the CH1 domain of natural or non-natural human IgG, and the amino acid sequences differ among the multiple types of peptides; (b) performing mass spectrometry on the sample prepared in (a) to detect each of the multiple types of peptides; and (c) quantifying each of the multiple types of peptides based on the analysis results of (b). [C3] A method for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample, the method comprising the following steps: (a) treating a biological sample containing multiple types of antigen-binding molecules with a digestive enzyme to generate multiple types of peptides from the multiple types of antigen-binding molecules, wherein the multiple types of antigen-binding molecules comprise antigen-binding domains, CL domains, and natural or non-natural human IgG CH1 domains, and the multiple types of antigen-binding molecules differ in the amino acid sequences between positions 121 and 133 according to the EU index in the human IgG CH1 domains of the multiple types of antigen-binding molecules, and the multiple types of peptides consist of amino acid sequences between positions 121 and 133 according to the EU index in the natural or non-natural human IgG CH1 domains, and the amino acid sequences differ among the multiple types of peptides; (b) performing mass spectrometry on the biological sample treated in (a) to detect each of the multiple types of peptides; and (c) detecting and / or quantifying each of the multiple types of antigen-binding molecules based on the analysis results of (b).[C4] The method according to any one of [C1] to [C3], wherein the multiple peptides include: (I) two or more peptides 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, which amino acid sequence differs from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG; or (II) one or more peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of natural human IgG, and a 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, which amino acid sequence differs from the corresponding sequence in natural human IgG of the same isotype as the non-natural human IgG. [C5] The method according to any one of [C1] to [C4], 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 has any one of the following characteristics: (1) an amino acid at one or more positions from 122 to 132 is different from an amino acid present at the corresponding position in a natural human IgG of the same isotype, and the amino acid at said position is an amino acid other than Lys, Arg, and Met (with the proviso that the amino acid at position 122 is not Pro); (2) [C6] The method according to any one of [C1] to [C4], wherein an amino acid other than Lys, Arg, or Met is inserted at one or more sites selected from 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 (with the proviso that the amino acid at the site between positions 121 and 122 is not Pro). [C7] The method according to any one of [C7] to [C74], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following characteristics:(1) the amino acid at any one or more of positions 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one or more of positions 122 to 132 is Ile; (3) the amino acid at any one or more of positions 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one or more of positions 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one or more of positions 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 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 is inserted at one or more sites selected from the above. [C7] The method of any one of [C1] to [C4], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following characteristics: (1) the amino acid at any one of positions 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one of positions 122 to 132 is Ile; (3) the amino acid at any one of positions 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one of positions 122 to 127 and 129 to 132 is Leu; (5)(6) The amino acid at any one of positions 122 to 125 and 127 to 132 is Phe; (7) The amino acid at any one of positions 121 to 122, 122 to 123, 123 to 124, 124 to 125, 125 to 126, 126 to 127, 127 to 128, 128 to 129, 129 to 130, 130 to 131, 131 to 132, or 132 to 133 [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, and wherein 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 is inserted into one site selected from the following: [C9] [C9] The method of any one of [C1] to [C7], wherein the plurality of peptides comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG are selected from the group consisting of SEQ ID NOs: 6 to 294 and 1565 to 1833. [C10] The method of any one of [C1] to [C7], wherein the plurality of peptides comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG are selected from the group consisting of SEQ ID NOs: 6 to 294. [C11] The method of any one of [C1] to [C7], wherein the plurality of peptides comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG are selected from the group consisting of SEQ ID NOs: 6 to 294.The method according to any one of [C1] to [C7], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain is selected from the group consisting of SEQ ID NOs: 1565 to 1833. [C12] The method according to any one of [C1] to [C7], wherein the plurality of peptides comprise one or more of the following: a peptide consisting of the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of native human IgG; and a peptide consisting of the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of non-naturally occurring human IgG; wherein, when the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of native human IgG is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 according to EU index in the non-naturally occurring human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 6 to 294; and when the amino acid sequence between positions 121 and 133 according to EU index in the CH1 domain of native human IgG is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 according to EU index in the non-naturally occurring human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 1565 to 1833. [C13] The method according to any one of [C1] to [C7], wherein the multiple types of peptides are two or more types of peptides selected from the group consisting of the following (1) to (26): (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 9; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; (13) a peptide consisting of the amino acid sequence of SEQ ID NO: 36; (14)(15) a peptide consisting of the amino acid sequence of SEQ ID NO: 38; (16) a peptide consisting of the amino acid sequence of SEQ ID NO: 42; (17) a peptide consisting of the amino acid sequence of SEQ ID NO: 45; (18) a peptide consisting of the amino acid sequence of SEQ ID NO: 46; (19) a peptide consisting of the amino acid sequence of SEQ ID NO: 47; (20) a peptide consisting of the amino acid sequence of SEQ ID NO: 73; (21) a peptide consisting of the amino acid sequence of SEQ ID NO: 75; (22) a peptide consisting of the amino acid sequence of SEQ ID NO: 76; (23) a peptide consisting of the amino acid sequence of SEQ ID NO: 78; (24) a peptide consisting of the amino acid sequence of SEQ ID NO: 79; (25) a peptide consisting of the amino acid sequence of SEQ ID NO: 80; and (26) a peptide consisting of the amino acid sequence of SEQ ID NO: 82. [C14] The method according to any one of [C1] to [C7], wherein the multiple types of peptides are two or more types of peptides selected from the group consisting of the following (1) to (13): (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 9; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and (13) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [C15] The method according to any one of [C1] to [C7], wherein the plurality of types of peptides are two types of peptides selected from the group consisting of the following (1) to (15): (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; (2)(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: 12; (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: 10; (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; (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; (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; (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; (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; (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; (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; (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; (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; (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 (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. [C16] The method according to any one of [C1] to [C7], wherein the multiple types of peptides are three types of peptides, and the three types of peptides are selected from the group consisting of the following (1) to (20): (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; (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; (3)(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, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (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; (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; (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; (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; (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; (10) (11) 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; (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; (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; (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; (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; (16)(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; (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; (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 (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. [C17] The method according to any one of [C1] to [C7], wherein the plurality of types of peptides are four types of peptides selected from the group consisting of the following (1) to (15): (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; (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; (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; (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; (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; (6)(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: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; (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; (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; (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; (11) (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; (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; (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 (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.[C18] The method according to any one of [C1] to [C7], wherein the plurality of types of peptides are five types of peptides, and the five types of peptides are selected from the group consisting of the following (1) to (6): (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; (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; (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; (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; (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 (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. [C19] The method according to any one of [C1] to [C7], wherein the plurality of types of peptides are the following six types of peptides: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (5)(6) a peptide consisting of the amino acid sequence of SEQ ID NO: 27. [C20] The method according to any one of [C1] to [C7], wherein the plurality of types of peptides are seven types of peptides, and the seven types of peptides are selected from the group consisting of the following (1) to (4): (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; (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; (3) (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: 24; and (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, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [C21] The method according to any one of [C1] to [C7], wherein the multiple types of peptides are eight types of peptides, and the eight types of peptides are selected from the group consisting of the following (1) to (6): (1)(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: 14; (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: 24; (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: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; (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: 24; 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 (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. [C22] The method according to any one of [C1] to [C7], wherein the plurality of peptides are nine peptides, and the nine peptides are selected from the group consisting of the following (1) to (4): (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; (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; (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 (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. [C23] A method according to any one of [C1] to [C7], wherein the multiple types of peptides are the following 10 types of peptides: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; and (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [C24] The method according to any one of [C1] to [C7], wherein the plurality of peptides are 11 peptides, and the 11 peptides are selected from the group consisting of the following (1) and (2): (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 (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. [C25] The method according to any one of [C1] to [C7], wherein the multiple types of peptides are the following 12 types of peptides: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [C26] The method according to any one of [C3] to [C25], wherein the digestive enzyme is a protease. [C27] The method according to any one of [C3] to [C25], wherein the digestive enzyme is trypsin. [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 to elute the biological sample enriched in peptides, and subjecting the enriched biological sample to mass spectrometry.[C29] The method of [C1] or [C2], wherein the step (b) comprises contacting the sample prepared in the step (a) with an affinity capture medium or a chromatographic adsorbent, eluting the sample enriched in the peptides, and subjecting the enriched sample to mass spectrometry. [C30] The method of any one of [C3] to [C27], wherein the step (a) comprises contacting the biological sample with an affinity capture medium or a chromatographic adsorbent, eluting the biological sample enriched in the antigen-binding molecules, and treating the enriched biological sample with a digestive enzyme. [C31] The method of any one of [C28] to [C30], wherein the affinity capture medium is Protein A or Protein G. [C32] The method of any one of [C28] to [C30], wherein the chromatographic adsorbent is a solid-phase extraction (SPE) adsorbent. [C33] The method of any one of [C1] to [C27], wherein the step (b) comprises subjecting the biological sample treated in step (a) or the sample prepared in step (a) to mass spectrometry in combination with liquid chromatography. [C34] The method of any one of [C1] to [C27], wherein the step (b) comprises separating a fraction containing the peptide from the biological sample treated in step (a) or the sample prepared in step (a) by liquid chromatography, and subjecting the separated fraction to mass spectrometry. [C35] The method of any one of [C3] to [C27], wherein the step (a) comprises separating a fraction containing the antigen-binding molecule from the biological sample by liquid chromatography, and treating the separated fraction with a digestive enzyme. [C36] The method of any one of [C1] to [C35], wherein mass spectrometry is performed by tandem mass spectrometry or multistage mass spectrometry. [C37] The method according to any one of [C1] to [C35], wherein the mass analysis is performed by tandem mass spectrometry or multistage mass spectrometry in selected reaction monitoring (SRM) measurement mode. [C38] The method according to any one of [C3] to [C37], wherein the biological sample is serum, plasma, tissue, or cells from an animal treated with the antigen-binding molecule.[C39] The method of any one of [C3] to [C38], wherein the antigen-binding domain comprises a VH domain and a VL domain. [C40] The method of [C39], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain. [C41] The method of [C39] or [C40], 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. [C42] The method of [C39] or [C40], 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. [C43] The method of any one of [C3] to [C42], wherein the antigen-binding molecule further comprises an antibody Fc region. [C44] 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. [C45] The method of any one of [C3] to [C44], wherein the antigen-binding molecule further comprises an antibody Fc region and the affinity capture medium is an anti-Fc antibody. [C46] The method of any one of [C45] to [C46], 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. [C47] The method of any one of [C3] to [C46], wherein the antigen-binding molecule is conjugated to a drug moiety.

[0011] [D1] A method for designing, selecting, or producing a modified antigen-binding molecule containing an artificially modified peptide moiety detectable by mass spectrometry, comprising the following steps: (I) identifying peptides obtainable after treating an antigen-binding molecule with a digestive enzyme, the peptides being detectable by mass spectrometry; (II) designing an artificially modified peptide by any of the following steps: (II-1) substituting one or more amino acid residues in the peptide identified in (I) with other amino acid residues or deleting them; (II-2) inserting one or more amino acid residues into the peptide identified in (I); (II-3) adding amino acid residues to the N-terminus or C-terminus of the peptide identified in (I); (II-4) a combination of (II-1) and (II-2); (II-5) a combination of (II-1) and (II-3); (II-6) a combination of (II-2) and (II-3); (II-7) a combination of (II-1), (II-2), and (II-3); (III) (IV-1) the Tm value of the modified antigen-binding molecule is 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 of (I); (IV-3) both (IV-1) and (IV-2); (V) the modified antigen-binding molecule selected in (IV) is selected when a portion corresponding to the peptide designed in (II) contained in the modified antigen-binding molecule is detectable by mass spectrometry after treating a biological sample containing the modified antigen-binding molecule with the digestive enzyme of (I).[D2] The method according to [D1], further comprising the steps of: (VI) treating a biological sample containing the antigen-binding molecule of (I) with the digestive enzyme of (I), followed by performing a pharmacokinetic test using mass spectrometry to obtain a pharmacokinetic profile or pharmacokinetic parameters; (VII) treating a biological sample containing the modified antigen-binding molecule selected in (V) with the digestive enzyme of (I), followed by performing a pharmacokinetic test using mass spectrometry to obtain a pharmacokinetic profile or pharmacokinetic parameters; and (VIII) selecting the modified antigen-binding molecule when the pharmacokinetic profile or pharmacokinetic parameter obtained in (VI) is equivalent to the pharmacokinetic profile or pharmacokinetic parameter obtained in (VII). [D3] The method according to [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 detectable by mass spectrometry is derived from the human IgG CH1 domain. [D4] The method according to [D3], wherein the peptide detectable by mass spectrometry consists of the amino acid sequence of positions 120 to 133 in the human IgG CH1 domain according to the EU index. [D5] The method according to [D3], wherein the peptide detectable by mass spectrometry consists of an amino acid sequence between positions 121 and 133 of the EU index in the CH1 domain of human IgG. [D6] The method according to any one of [D1] to [D5], wherein the other amino acid residue in (II-1) is Ala, Ile, Val, Leu, or Phe. [D7] The method according to any one of [D1] to [D6], wherein the inserted amino acid residue in (II-2) is 1, 2, or 3 residues. [D8] The method according to any one of [D1] to [D7], wherein in (II-2), the inserted amino acid residues are 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.[D9] The method of any one of [D1] to [D7], wherein in (II-2), amino acid residues are inserted at one, two, or three positions in the peptide identified in (I). [D10] The method of any one of [D1] to [D9], wherein in (II-3), the added amino acid residues are one, two, or three residues. [D11] The method of any one of [D1] to [D10], wherein the peptide designed in (II) does not have a site that is cleaved by the digestive enzyme of (I). [D12] The method of any one of [D1] to [D11], wherein the digestive enzyme is a protease. [D13] The method of any one of [D1] to [D11], wherein the digestive enzyme is trypsin. [D14] The method of any one of [D1] to [D13], wherein the mass spectrometry is mass spectrometry combined with liquid chromatography. [D15] The method of any one of [D1] to [D14], wherein the mass spectrometry is mass spectrometry by tandem mass spectrometry. [D16] The method of any one of [D1] to [D15], wherein the biological sample in steps (V) and (VII) is serum, plasma, tissue, or cells from an animal treated with the modified antigen-binding molecule, and the biological sample in step (VI) is serum, plasma, tissue, or cells from an animal treated with the antigen-binding molecule. [D17] The method of 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. [D18] The method of [D17], wherein the VH domain and the VL domain are humanized VH domain and humanized VL domain. [D19] The method of [D17] or [D18], 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. [D20] The method according to [D17] or [D18], 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.[D21] The method of any one of [D1] to [D20], wherein the antigen-binding molecule of step (I) further comprises an antibody Fc region. [D22] The method of any one of [D1] to [D21], wherein the antigen-binding molecule of step (I) is an IgG antibody. [D23] The method of [D22], wherein the IgG antibody is an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. [D24] The method of any one of [D1] to [D23], wherein the antigen-binding molecule of step (I) is conjugated to a drug moiety.

[0012] [E1] A modified antigen-binding molecule designed, selected, or produced by the method described in any one of [D1] to [D24]. [E2] A peptide designed in step (II) of the method described in any one of [D1] to [D24].

[0013] [F1] A peptide consisting 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 differs from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG. [F2] A peptide according to [F1], 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 has any one of the following characteristics: (1) an amino acid at one or more positions from 122 to 132 differs from the amino acid present at the corresponding position in a natural human IgG of the same isotype, and the amino acid at said position is an amino acid other than Lys, Arg, and Met (with the proviso that the amino acid at position 122 is not Pro); An amino acid other than Lys, Arg, or Met is inserted at one or more sites selected from 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 (however, the amino acid at the site between positions 121 and 122 is not Pro).[F3] The peptide according to [F1] or [F2], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following features: (1) the amino acid at any one or more positions from 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one or more positions from 122 to 132 is Ile; (3) the amino acid at any one or more positions from 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one or more positions from 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one or more positions from 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 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 is inserted at one or more sites selected from the above.[F4] The peptide according to [F1] or [F2], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following features: (1) an amino acid at any one of positions 122 to 128 and 130 to 132 is Ala; (2) an amino acid at any one of positions 122 to 132 is Ile; (3) an amino acid at any one of positions 122 to 124 and 126 to 132 is Val; (4) an amino acid at any one of positions 122 to 127 and 129 to 132 is Leu; (5) an amino acid at any one of positions 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 [F5] The peptide of any one of [F1] to [F4], wherein the non-naturally occurring human IgG CH1 domain is a non-naturally occurring human IgG1 CH1 domain, a non-naturally occurring human IgG2 CH1 domain, a non-naturally occurring human IgG3 CH1 domain, or a non-naturally occurring human IgG4 CH1 domain, and wherein 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 is inserted at one site selected from the following: [F6] The peptide of any one of [F6] to [F7], wherein the non-naturally occurring human IgG CH1 domain is a non-naturally occurring human IgG1 CH1 domain, a non-naturally occurring human IgG2 CH1 domain, a non-naturally occurring human IgG3 CH1 domain, or a non-naturally occurring human IgG4 CH1 domain. [F6] The peptide according to any one of [F1] to [F5], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of the non-native human IgG is any one of the amino acid sequences of SEQ ID NOs: 6 to 294 and 1565 to 1833.

[0014] [G1] A set comprising two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of natural or non-natural human IgG, wherein the amino acid sequences differ between the two or more peptides. [G2] The set according to [G1], comprising: (I) two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences differ from corresponding sequences in natural human IgG of the same isotype as the non-natural human IgG, or (II) one or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of natural human IgG and peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences differ from corresponding sequences in natural human IgG of the same isotype as the non-natural human IgG. [G3] The set according to [G1] or [G2], 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 has any one of the following characteristics: (1) an amino acid at any one or more positions from 122 to 132 is different from the amino acid present at the corresponding position in a natural human IgG of the same isotype, and the amino acid at said position is an amino acid other than Lys, Arg, and Met (with the proviso that the amino acid at position 122 is not Pro); (2) An amino acid other than Lys, Arg, or Met is inserted at one or more sites selected from the group consisting of a site between positions 121 and 122, a site between positions 122 and 123, a site between positions 123 and 124, a site between positions 124 and 125, a site between positions 125 and 126, a site between positions 126 and 127, a site between positions 127 and 128, a site between positions 128 and 129, a site between positions 129 and 130, a site between positions 130 and 131, a site between positions 131 and 132, and a site between positions 132 and 133 (with the proviso that the amino acid at the site between positions 121 and 122 is not Pro). [G4] EU in the non-native human IgG CH1 domainThe set described in [G1] or [G2], wherein the amino acid sequence between positions 121 and 133 according to the index has any one of the following characteristics: (1) the amino acid at any one or more positions from 122 to 128 and 130 to 132 is Ala; (2) the amino acid at any one or more positions from 122 to 132 is Ile; (3) the amino acid at any one or more positions from 122 to 124 and 126 to 132 is Val; (4) the amino acid at any one or more positions from 122 to 127 and 129 to 132 is Leu; (5) the amino acid at any one or more positions from 122 to 125 and 127 to 132 is Phe; (6) Sites between positions 121 and 122, sites between positions 122 and 123, sites between positions 123 and 124, sites between positions 124 and 125, sites between positions 125 and 126, sites between positions 126 and 127, sites between positions 127 and 128, sites between positions 128 and 129, sites between positions 129 and 130, sites between positions 130 and 131, sites between positions 131 and 132, sites between positions 132 and 133 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 is inserted at one or more sites selected from the above. [G5] The set according to [G1] or [G2], wherein the amino acid sequence between positions 121 and 133 according to the EU index in the non-native human IgG CH1 domain has any one of the following features: (1) an amino acid at any one of positions 122 to 128 and 130 to 132 is Ala; (2) an amino acid at any one of positions 122 to 132 is Ile; (3) an amino acid at any one of positions 122 to 124 and 126 to 132 is Val; (4)(5) the amino acid at any one of positions 122 to 127 and 129 to 132 is Leu; (6) the amino acid at any one of positions 122 to 125 and 127 to 132 is Phe; (7) the amino acid at a site between positions 121 and 122, between positions 122 and 123, between positions 123 and 124, between positions 124 and 125, between positions 125 and 126, between positions 126 and 127, between positions 127 and 128, between positions 128 and 129, between positions 129 and 130, between positions 130 and 131, between positions 131 and 132, or between positions 132 and 133 [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, and wherein 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 is inserted at one site selected from the group consisting of: [G7] [G7] The set according to any one of [G1] to [G5], wherein the set comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG are selected from the group consisting of SEQ ID NOs: 6 to 294 and 1565 to 1833. [G8] The set according to any one of [G1] to [G5], wherein the set comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG, wherein the amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG are selected from the group consisting of SEQ ID NOs: 6 to 294. [G9] The set comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in the CH1 domain of non-natural human IgG.The set of any one of [G1] to [G5], comprising two or more peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in the 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 selected from the group consisting of SEQ ID NOs: 1565 to 1833. [G10] The set according to any one of [G1] to [G5], comprising one or more peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of native human IgG, and an amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of non-naturally occurring human IgG, wherein when the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of native human IgG is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 according to the EU index in the non-naturally occurring human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 6 to 294, and when the amino acid sequence between positions 121 and 133 according to the EU index in the CH1 domain of native human IgG is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 according to the EU index in the non-naturally occurring human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 1565 to 1833. [G11] A set according to any one of [G1] to [G5], which is a set of two or more peptides selected from the group consisting of (1) to (26) below: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 9; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 27;(13) A peptide consisting of the amino acid sequence of SEQ ID NO: 36; (14) A peptide consisting of the amino acid sequence of SEQ ID NO: 38; (15) A peptide consisting of the amino acid sequence of SEQ ID NO: 40; (16) A peptide consisting of the amino acid sequence of SEQ ID NO: 42; (17) A peptide consisting of the amino acid sequence of SEQ ID NO: 45; (18) A peptide consisting of the amino acid sequence of SEQ ID NO: 46; (19) A peptide consisting of the amino acid sequence of SEQ ID NO: 47; (20) A peptide consisting of the amino acid sequence of SEQ ID NO: 73; (21) A peptide consisting of the amino acid sequence of SEQ ID NO: 75; (22) A peptide consisting of the amino acid sequence of SEQ ID NO: 76; (23) A peptide consisting of the amino acid sequence of SEQ ID NO: 78; (24) A peptide consisting of the amino acid sequence of SEQ ID NO: 79; (25) A peptide consisting of the amino acid sequence of SEQ ID NO: 80; and (26) A peptide consisting of the amino acid sequence of SEQ ID NO: 82. [G12] A set of two or more peptides selected from the group consisting of (1) to (13) below, which is a set according to any one of [G1] to [G5]: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 9; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and (13) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [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): (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; (2)(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: 12; (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: 10; (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; (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; (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; (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; (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; (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; (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; (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; (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; (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 (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. [G14] A 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): (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; (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; (3)(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, and a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (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; (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; (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; (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; (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; (10) (11) 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; (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; (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; (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; (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; (16)(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; (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; (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 (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. [G15] A set according to any one of [G1] to [G5], wherein the set is a set of four types of peptides, and the four types of peptides are selected from the group consisting of the following (1) to (15): (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; (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; (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; (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; (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; (6)(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: 10, and a peptide consisting of the amino acid sequence of SEQ ID NO: 27; (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; (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; (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; (11) (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; (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; (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 (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.[G16] A set of five types of peptides, the five types of peptides being selected from the group consisting of the following (1) to (6): (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; (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; (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; (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; (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 (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. [G17] A set of the following six peptides, which is a set of any one of [G1] to [G5]: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (5)(6) a peptide consisting of the amino acid sequence of SEQ ID NO: 27. [G18] A set according to any one of [G1] to [G5], wherein the set is a set of seven types of peptides, and the seven types of peptides are selected from the group consisting of the following (1) to (4): (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; (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; (3) (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: 24; and (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, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [G19] A 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 (1) to (6) below: (1)(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: 14; (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: 24; (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: 13, and a peptide consisting of the amino acid sequence of SEQ ID NO: 36; (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: 24; 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 (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. [G20] A set of nine types of peptides, the nine types of peptides being selected from the group consisting of (1) to (4) below, the set being described in any one of [G1] to [G5]: (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; (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; (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 (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. [G21] A set of the following 10 types of peptides, which is a set described in any one of [G1] to [G5]: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; and (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [G22] A set according to any one of [G1] to [G5], wherein the set is a set of 11 types of peptides, and the 11 types of peptides are selected from the group consisting of the following (1) and (2): (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 (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. [G23] A set of the following 12 types of peptides, which is a set described in any one of [G1] to [G5]: (1) a peptide consisting of the amino acid sequence of SEQ ID NO: 5; (2) a peptide consisting of the amino acid sequence of SEQ ID NO: 6; (3) a peptide consisting of the amino acid sequence of SEQ ID NO: 7; (4) a peptide consisting of the amino acid sequence of SEQ ID NO: 8; (5) a peptide consisting of the amino acid sequence of SEQ ID NO: 10; (6) a peptide consisting of the amino acid sequence of SEQ ID NO: 12; (7) a peptide consisting of the amino acid sequence of SEQ ID NO: 13; (8) a peptide consisting of the amino acid sequence of SEQ ID NO: 14; (9) a peptide consisting of the amino acid sequence of SEQ ID NO: 17; (10) a peptide consisting of the amino acid sequence of SEQ ID NO: 24; (11) a peptide consisting of the amino acid sequence of SEQ ID NO: 27; and (12) a peptide consisting of the amino acid sequence of SEQ ID NO: 36. [G24] The set according to any one of [G1] to [G23] for use in a method for detecting each of multiple types of peptides derived from multiple types of antigen-binding molecules in a biological sample by mass spectrometry, or a method for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample. [G25] Use of the set according to any one of [G1] to [G23] for detecting each of multiple types of peptides derived from multiple types of antigen-binding molecules in a biological sample by mass spectrometry, or for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample.[G26] A method of administering to an individual or a subject a mixture of multiple types of antigen-binding molecules, each comprising a peptide in the set described in any one of [G1] to [G23]. [G27] A method of selecting an antigen-binding molecule, comprising the following steps: (a) detecting and / or quantifying each of the multiple types of antigen-binding molecules in a biological sample derived from an individual or a subject to whom a mixture of multiple types of antigen-binding molecules, each comprising a peptide in the set described in any one of [G1] to [G23], has been administered; and (b) selecting an antigen-binding molecule based on the detection and / or quantification results of (a). [G28] An antigen-binding molecule selected by the method described in [G27]. [G29] An antigen-binding molecule, in which the amino acid sequence between positions 121 and 133 according to the EU index in the IgG CH1 domain of an antigen-binding molecule selected by the method described in [G27] has been changed to a native sequence between positions 121 and 133 according to the EU index in the CH1 domain of an IgG antibody of the same isotype.

[0015] Figure 1-1 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that their mouse plasma concentrations ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-2 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that their mouse plasma concentrations ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-3 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that their mouse plasma concentrations ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-4 shows an internal calibration curve prepared by mass spectrometry of antibody samples prepared so that the antibody concentrations in mouse plasma range from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-5 shows an internal calibration curve prepared by mass spectrometry of antibody samples prepared so that the antibody concentrations in mouse plasma range from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-6 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that the antibody concentrations in mouse plasma ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure, and the vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), while the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-7 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that the antibody concentrations in mouse plasma ranged from 0.0200 to 5.12 μg / mL.The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-8 shows an internal calibration curve prepared by mass spectrometry of antibody samples prepared so that the antibody concentrations in mouse plasma range from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-9 shows an internal calibration curve prepared by mass spectrometry of antibody samples prepared so that the antibody concentrations in mouse plasma range from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure. The vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), and the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-10 shows the internal calibration curves prepared by mass spectrometry analysis of antibody samples prepared so that the antibody concentrations in mouse plasma ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure, and the vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), while the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-11 shows the internal calibration curves prepared by mass spectrometry analysis of antibody samples prepared so that the antibody concentrations in mouse plasma ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure, and the vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), while the horizontal axis shows the concentration of the target antibody (ng / mL). Figure 1-12 shows the internal calibration curves generated by mass spectrometry analysis of antibody samples prepared so that the antibody concentrations in mouse plasma ranged from 0.0200 to 5.12 μg / mL. The name of the target antibody is shown in the upper left of the figure, and the vertical axis shows the peak area ratio (peak area of ​​the target antibody sample ÷ peak area of ​​the internal standard), while the horizontal axis shows the target antibody concentration (ng / mL). Figure 2-1 shows SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the peptide being measured is shown in the upper left of each chromatogram. The numbers in the upper right of each figure indicate peak intensity. The vertical axis of each chromatogram represents the peak intensity in the upper right of each figure as 100%, and the horizontal axis represents the LC elution time (minutes).Figure 2-2 shows SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the peptide being measured is indicated in the upper left of each chromatogram. The numbers in the upper right of each figure indicate peak intensity. The vertical axis of each chromatogram represents the peak intensity in the upper right of each figure as 100%, and the horizontal axis represents the LC elution time (minutes). Figure 2-3 shows SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the peptide being measured is indicated in the upper left of each chromatogram. The numbers in the upper right of each figure indicate peak intensity. The vertical axis of each chromatogram represents the peak intensity in the upper right of each figure as 100%, and the horizontal axis represents the LC elution time (minutes). Figure 2-4 shows SRM chromatograms of the mixed administration group 5 minutes after administration. The name of the peptide being measured is indicated in the upper left of each chromatogram. IS stands for internal standard. The numbers in the upper right of each figure indicate peak intensity. The vertical axis of each chromatogram represents the peak intensity in the upper right corner of each figure as 100%, and the horizontal axis represents the LC elution time (minutes). Figure 3A-1 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3A-1 shows the measurement results of a plasma sample spiked with unmodified antibody alone. The vertical axis of each chromatogram in Figure 3A-1 represents the peak intensity of the unmodified peptide, 4.24e4, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3A-2 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3A-2 shows the results of measuring a plasma sample spiked with unmodified antibody alone. The vertical axis of each chromatogram in Figure 3A-2 represents the peak intensity of the unmodified peptide, 4.24e4, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide's peak. Figure 3A-3 shows an SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes).Figure 3A-3 shows the results of measurements of a plasma sample spiked with the unmodified antibody alone. The vertical axis of each chromatogram in Figure 3A-3 represents the peak intensity of the unmodified peptide, 4.24e4, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be located. Figure 3B-1 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3B-1 shows the results of measurements of a plasma sample spiked with the corresponding modified antibody of MS0001 alone. The vertical axis of each chromatogram in Figure 3B-1 represents the peak intensity of MS0001, 2.67e4, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be located. Figure 3B-2 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3B-2 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0001 alone. The vertical axis of each chromatogram in Figure 3B-2 represents the MS0001 peak intensity of 2.67e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3B-3 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3B-3 shows the results of measuring a plasma sample spiked with the corresponding engineered antibody against MS0001 alone. The vertical axis of each chromatogram in Figure 3B-3 represents the MS0001 peak intensity of 2.67e4, which is set to 100%. The name of the peptide being measured is indicated in the upper left corner of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3C-1 shows the SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity.The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3C-1 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0002 alone. The vertical axis of each chromatogram in Figure 3C-1 represents the MS0002 peak intensity of 1.23e5, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide's peak. Figure 3C-2 shows an SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3C-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0002 alone. The vertical axis of each chromatogram in Figure 3C-2 represents the MS0002 peak intensity of 1.23e5, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3C-3 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3C-3 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0002 alone. The vertical axis of each chromatogram in Figure 3C-3 represents the MS0002 peak intensity of 1.23e5, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3D-1 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3D-1 shows the measurement results for a plasma sample spiked with the corresponding modified antibody of MS0003 alone. The vertical axis of each chromatogram in Figure 3D-1 represents the MS0003 peak intensity of 4.48e4, set at 100%. The name of the peptide being measured is shown in the upper left of each chromatogram. The black arrow indicates the position where the peak of the peptide being measured should be.Figure 3D-2 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3D-2 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0003 alone. The vertical axis of each chromatogram in Figure 3D-2 represents the MS0003 peak intensity of 4.48e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3D-3 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3D-3 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0003 alone. The vertical axis of each chromatogram in Figure 3D-3 represents the peak intensity of MS0003, 4.48e4, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3E-1 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3E-1 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0005 alone. The vertical axis of each chromatogram in Figure 3E-1 represents the peak intensity of MS0005, 4.55e5, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3E-2 shows the SRM chromatogram of a plasma sample spiked with a standard antibody solution (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3E-2 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0005 alone. The vertical axis of each chromatogram in Figure 3E-2 represents the MS0005 peak intensity of 4.55e5, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram.The black arrow indicates the position where the peak of the target peptide should be located. Figure 3E-3 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3E-3 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0005 alone. The vertical axis of each chromatogram in Figure 3E-3 represents the MS0005 peak intensity of 4.55e5, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3F-1 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3F-1 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0007 alone. The vertical axis of each chromatogram in Figure 3F-1 represents the peak intensity of MS0007, 1.68e5, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3F-2 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3F-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0007 alone. The vertical axis of each chromatogram in Figure 3F-2 represents the peak intensity of MS0007, 1.68e5, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3F-3 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3F-3 shows the results of measurements of a plasma sample spiked with the corresponding modified antibody of MS0007 alone. The vertical axis of each chromatogram in Figure 3F-3 represents the MS0007 peak intensity of 1.68e5, which is set to 100%.The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3G-1 shows an SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3G-1 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0008 alone. The vertical axis of each chromatogram in Figure 3G-1 represents the MS0008 peak intensity of 4.17e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3G-2 shows an SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3G-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0008 alone. The vertical axis of each chromatogram in Figure 3G-2 represents the MS0008 peak intensity of 4.17e4, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3G-3 shows an SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3G-3 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0008 alone. The vertical axis of each chromatogram in Figure 3G-3 represents the MS0008 peak intensity of 4.17e4, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3H-1 shows an SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes).Figure 3H-1 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0009 alone. The vertical axis of each chromatogram in Figure 3H-1 represents the MS0009 peak intensity of 4.48e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3H-2 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3H-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0009 alone. The vertical axis of each chromatogram in Figure 3H-2 represents the MS0009 peak intensity of 4.48e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected peak position of the target peptide. Figure 3H-3 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3H-3 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0009 alone. The vertical axis of each chromatogram in Figure 3H-3 represents the MS0009 peak intensity of 4.48e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected peak position of the target peptide. Figure 3I-1 shows SRM chromatograms of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3I-1 shows the results of measuring a plasma sample spiked with the corresponding modified antibody against MS0012 alone. The vertical axis of each chromatogram in Figure 3I-1 represents the MS0012 peak intensity of 1.88e5, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram. The black arrow indicates the expected position of the target peptide's peak. Figure 3I-2 shows the SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity.The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3I-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0012 alone. The vertical axis of each chromatogram in Figure 3I-2 represents the MS0012 peak intensity of 1.88e5, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram. The black arrow indicates the expected peak location of the target peptide. Figure 3I-3 shows an SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3I-3 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0012 alone. The vertical axis of each chromatogram in Figure 3I-3 represents the MS0012 peak intensity of 1.88e5, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3J-1 shows an SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3J-1 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS00019 alone. The vertical axis of each chromatogram in Figure 3J-1 represents the MS0019 peak intensity of 4.30e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the target peptide's peak should be. Figure 3J-2 shows an SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with an antibody standard solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3J-2 shows the measurement results for a plasma sample spiked with the corresponding modified antibody for MS00019 alone. The vertical axis of each chromatogram in Figure 3J-2 represents the MS0019 peak intensity of 4.30e4, set at 100%. The name of the peptide being measured is shown in the upper left of each chromatogram. The black arrow indicates the position where the peak of the peptide being measured should be.Figure 3J-3 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3J-3 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS00019 alone. The vertical axis of each chromatogram in Figure 3J-3 represents the MS0019 peak intensity of 4.30e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3K-1 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3K-1 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0022 alone. The vertical axis of each chromatogram in Figure 3K-1 represents the peak intensity of MS0022, 2.60e5, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3K-2 shows an SRM chromatogram of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3K-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0022 alone. The vertical axis of each chromatogram in Figure 3K-2 represents the peak intensity of MS0022, 2.60e5, as 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be located. Figure 3K-3 shows the SRM chromatogram of a plasma sample spiked with a standard antibody solution (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3K-3 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0022 alone. The vertical axis of each chromatogram in Figure 3K-3 represents the MS0022 peak intensity of 2.60e5, which is set to 100%. The name of the target peptide is indicated in the upper left corner of each chromatogram.The black arrow indicates the expected peak position of the target peptide. Figure 3L-1 shows the SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3L-1 shows the measurement results of a plasma sample spiked with the corresponding modified antibody of MS0031 alone. The vertical axis of each chromatogram in Figure 3L-1 represents the MS0031 peak intensity of 9.49e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected peak position of the target peptide. Figure 3L-2 shows the SRM chromatogram of a plasma sample (1.28 μg / mL in mouse plasma) spiked with a standard antibody solution alone to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3L-2 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0031 alone. The vertical axis of each chromatogram in Figure 3L-2 represents the MS0031 peak intensity of 9.49e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the expected position of the target peptide peak. Figure 3L-3 shows SRM chromatograms of a plasma sample spiked with a standard antibody solution alone (1.28 μg / mL in mouse plasma) to confirm specificity. The horizontal axis of each chromatogram represents the LC elution time (minutes). Figure 3L-3 shows the results of measurements of a plasma sample spiked with the corresponding engineered antibody to MS0031 alone. The vertical axis of each chromatogram in Figure 3L-3 represents the MS0031 peak intensity of 9.49e4, which is set to 100%. The name of the target peptide is indicated in the upper left of each chromatogram. The black arrow indicates the position where the peak of the target peptide should be. Figure 4-1 shows the plasma antibody concentration-time curve of H54-SG181 after mixed administration and single administration to mice. The title of the figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figure 4-2 shows the plasma antibody concentration-time curve of H54-SG181.MS2 after mixed administration and single administration to mice.The title of the figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figure 4-3 shows the plasma antibody concentration-time curves of H54-SG181.MS5 after mixed administration and single administration to mice. The title of the figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figure 4-4 shows the plasma antibody concentration-time curves of H54-SG181.MS7 after mixed administration and single administration to mice. The title of the figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figure 4-5 shows the plasma antibody concentration-time curves of H54-SG181.MS12 after mixed administration and single administration to mice. The title of each figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figures 4-6 show the plasma antibody concentration-time curves of H54-SG181.MS22 after co-administration and single administration to mice. The title of each figure is the name of the antibody being measured, the horizontal axis is the time (days) after drug administration, and the vertical axis is the plasma concentration of the target antibody (ng / mL). Figure 5 shows the plasma antibody concentration-time curves of antibodies containing the unmodified sequence and the alanine-modified sequence after co-administration to mice. The horizontal axis of each figure is the time (days) after drug administration, and the vertical axis is the plasma concentration of the antibody (ng / mL).

[0016] I. Definitions The term "and / or" is used herein to refer to each of the objects listed before and after "and / or" or any combination thereof. For example, "A, B and / or C" includes each of the objects "A," "B," and "C," as well as the combinations "A and B," "A and C," "B and C," and "A and B and C." The term "and / or" is used interchangeably with "both or either" or "all or any one."

[0017] As used herein, the term "antigen-binding molecule" refers, in its broadest sense, 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.

[0018] The antigen-binding molecule of the present application is not limited to its origin, and may be a human antibody, mouse antibody, rat antibody, etc. It may also be a genetically modified antibody such as a chimeric antibody or a humanized antibody.

[0019] As used herein, the term "multispecific antigen-binding molecule" refers to an antigen-binding molecule comprising two or more antigen-binding domains specific for different antigens or epitopes. In one embodiment, the multispecific antigen-binding molecule of the present application is a multispecific antibody. The multispecific antibody is not particularly limited, but is preferably a monoclonal antibody.

[0020] As used herein, the term "antigen-binding domain" refers to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, the antigen-binding domain can bind only to a specific part of the antigen. This specific part is called an epitope. In one embodiment, the antigen-binding domain comprises an antibody fragment that binds to a specific antigen. The antigen-binding domain can be provided by one or more antibody variable domains. In a non-limiting embodiment, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," and "scFv2 (single chain Fv 2)."

[0021] As used herein, the term "specifically binds" refers to a state in which one of the specifically binding molecules binds without showing any significant binding to any molecules other than the one or more molecules to which it binds. The term also applies to cases in which an antigen-binding domain is specific for a particular epitope among multiple epitopes contained in an antigen. Furthermore, when the epitopes to which the antigen-binding domain binds are contained in multiple different antigens, an antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

[0022] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and modified antibodies, so long as they exhibit the desired antigen-binding activity.

[0023] "Binding activity" refers to the strength of the total noncovalent interactions between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Here, binding activity is not strictly limited to 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of these binding forces. The binding activity of a molecule X to its partner Y can generally be expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Binding activity can be measured by conventional methods known in the art, including those described herein.

[0024] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0025] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0026] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0027] In one embodiment of the present application, the constant region is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 antibody constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 antibody constant region. In another embodiment of the present application, the constant region is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and even more preferably a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. 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 allotype sequences due to genetic polymorphisms are described in "Sequences of proteins of immunological interest," NIH Publication No. 91-3242, and any of these may be used in the present application. Note that the amino acid-altered constant regions of the present application may also contain other amino acid mutations or modifications, as long as they contain the amino acid mutations of the present application.

[0028] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that links the CH1 domain and the CH2 domain in a wild-type antibody heavy chain, 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. In naturally occurring IgG antibodies, the cysteine ​​residue at EU numbering position 220 in the hinge region is known to form a disulfide bond with the cysteine ​​residue at EU numbering position 214 in the antibody light chain. Furthermore, it is known that the cysteine ​​residues at EU numbering positions 226 and 229 in the hinge region of two antibody heavy chains form disulfide bonds. As used herein, the term "hinge region" encompasses not only the wild-type but also variants in which amino acid residues have been substituted, added, or deleted from the wild-type.

[0029] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb and Lu radioactive isotopes); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitory agents; enzymes such as nucleases and fragments thereof; antibiotics; toxins such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents, as disclosed below.

[0030] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the 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; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0031] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0032] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0033] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0034] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0035] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0036] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0037] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0038] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR"), and / or forms structurally defined loops (the "hypervariable loops"), and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) 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).Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0039] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0040] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses, pigs), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats, guinea pigs). In one embodiment, the individual or subject is a non-human animal. In a particular embodiment, the individual or subject is a human.

[0041] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0042] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0043] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0044] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are typically present in small amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present application may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0045] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0046] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy 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 contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ).

[0047] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0048] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0049] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, each containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0050] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of mediating the expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by viral or electroporation methods, but vector introduction is not limited to ex vivo introduction; vectors can also be introduced directly into a living body.

[0051] In further aspects, antibodies according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1 to 7 below.

[0052] 1. Antibody Affinity In certain embodiments, the antibodies provided herein have an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10-8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 It has a dissociation constant (Kd) of 1 M.

[0053] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured using the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0054] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve protein binding of approximately 10 response units (RU). After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (Kd) is given by k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds 100 kJ / s, the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0055] 2. Antibody Fragments In certain 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 specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); in addition, see WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0056] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0057] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0058] 3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Specific 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 example, a chimeric antibody contains 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 a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0059] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0060] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and also 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); U.S. Pat. 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) grafting); Padlan, Mol. Immunol. 28:489-498 (1991). (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guide selection" approach for FR shuffling).

[0061] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0062] 4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0063] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are 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, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0064] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., 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 generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). 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).

[0065] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0066] 5. Library-Derived Antibodies The antibodies of the present application may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, for example, in 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, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, 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).

[0067] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0068] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0069] 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one binding specificity is for a given antigen and the other is for a different antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of a given antigen. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing the antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0070] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1994); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0071] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0072] Antibodies or fragments herein also include "dual-acting Fabs" or "DAFs" (see, e.g., US Patent Application Publication No. 2008 / 0069820).

[0073] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. 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 may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0074] a) Substitutional, Insertional, and Deletional Variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into an antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0075] (Table 1)

[0076] Amino acids can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) acidic: aspartic acid (Asp), glutamic acid (Glu); (4) basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Nonconservative substitutions involve exchanging a member of one of these classes for another.

[0077] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further study will have modified (e.g., improved) specific biological properties compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0078] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0079] In certain embodiments, substitutions, insertions, or deletions can be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs. Such modifications can be, for example, outside the antigen contact residues of HVRs. In certain embodiments of the above-mentioned mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0080] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants can be screened to determine whether they contain desired properties.

[0081] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0082] b) Glycosylation Variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0083] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present application may be performed to create antibody variants with specific improved properties.

[0084] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose 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 in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. 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 Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0085] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides added to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide added to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0086] c) Fc Region Variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating 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.

[0087] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated herein, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., 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); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (see, e.g., 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)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0088] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0089] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0090] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0091] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0092] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those 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 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0093] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0094] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. Substituting these residues with cysteine ​​places reactive thiol groups at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0095] e) Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatizing antibodies 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0096] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0097] Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells)). In one aspect, a method of making an antibody is provided, comprising culturing a host cell comprising nucleic acid encoding the antibody, as described above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0098] For recombinant production of an antibody, nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0099] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For bacterial expression of antibody fragments and polypeptides, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0100] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0101] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly transformation of Spodoptera frugiperda cells.

[0102] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0103] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68). (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0104] In certain aspects, the present application provides antibodies obtainable by the above methods.

[0105] The antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0106] In one aspect, the antibodies of the present application are tested for their antigen binding activity by known methods, such as ELISA, Western blot, and the like.

[0107] The present application also provides immunoconjugates comprising the antibodies herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0108] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. 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 daunomycin or doxorubicin (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 U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0109] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0110] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes include Pb and Lu. When radioactive conjugates are used for detection, they are used in combination with radioactive atoms for scintigraphic examinations (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0111] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0112] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0113] 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 the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present application can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0114] A "functional Fc region" comprises an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors (BCRs)); and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, for example, as disclosed within the definitions herein.

[0115] A "native-sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes), native-sequence human IgG2 Fc regions, native-sequence human IgG3 Fc regions, and native-sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0116] Antigen-binding molecules of the present application include, for example, antigen-binding molecules described in WO2020 / 027330. For example, antigen-binding molecules of the present 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.

[0117] In one embodiment, at least one of the one or more bonds linking the two antigen-binding domains of the antigen-binding molecule of the present application is a covalent bond. In a specific embodiment, the covalent bond is formed by directly cross-linking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain. The type of cross-linked amino acid residue is, for example, cysteine, and the covalent bond formed is, for example, a disulfide bond. In another specific embodiment, the covalent bond is formed by cross-linking an amino acid residue in the first antigen-binding domain with an amino acid residue in the second antigen-binding domain via a cross-linking agent. The cross-linking agent is, for example, an amine-reactive cross-linking agent, and the type of cross-linked amino acid residue is, for example, lysine.

[0118] In one embodiment, at least one of the one or more bonds connecting the antigen-binding domains is a non-covalent bond. In a specific embodiment, the non-covalent bond is an ionic bond, a hydrogen bond, or a hydrophobic bond. The ionic bond is formed, for example, between an acidic amino acid and a basic amino acid. The acidic amino acid is, for example, aspartic acid (Asp) or glutamic acid (Glu), and the basic amino acid is, for example, histidine (His), lysine (Lys), or arginine (Arg).

[0119] The bond between the antigen-binding domains (the bond connecting the two antigen-binding domains) is formed by linking amino acid residues that serve as the origin of the bond in each of the first and second antigen-binding domains. In one embodiment, at least one of the amino acid residues that serve as the origin of the bond between the antigen-binding domains is an artificially introduced mutated amino acid residue, for example, an artificially introduced cysteine ​​residue. Such a mutated amino acid residue can be introduced into a wild-type antigen-binding domain by techniques such as amino acid substitution. When the antigen-binding domain comprises, for example, an antibody fragment, amino acid residues that can serve as the origin of the bond between the antigen-binding domains are disclosed herein in the CH1 region, CL region, and hinge region as constant regions, and the VH region, VL region, and VHH region as variable regions. For example, cysteine ​​residues can be introduced into these sites.

[0120] In one embodiment, at least one of the first and second antigen-binding domains has antigen-binding activity alone (i.e., one antigen-binding domain alone has antigen-binding activity). In a particular embodiment, both the first and second antigen-binding domains have antigen-binding activity alone.

[0121] In one embodiment, the at least one bond linking the first antigen-binding domain and the second antigen-binding domain may be formed by linking amino acid residues located at the same position on the first antigen-binding domain and the second antigen-binding domain, respectively, or may be formed by linking amino acid residues located at different positions on the first antigen-binding domain and the second antigen-binding domain, respectively.

[0122] In one embodiment, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is present in the constant region. In a specific embodiment, the amino acid residue is present in the CH1 region, for example, at any of 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 (EU numbering) in the CH1 region. In certain embodiments, the amino acid residues are located at positions 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, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 33 The amino acid residue is located at any one of positions selected from the group consisting of: 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 a specific embodiment, the amino acid residue is located at position 134, 135, 136, 137, 191, 192, 193, 194, 195, or 196 (EU numbering) in the CH1 region. In a specific embodiment, the amino acid residue is present at position 135, 136, or 191 (EU numbering) in the CH1 region. In one embodiment, the constant region is of human origin. In a specific embodiment, the subclass of the heavy chain constant region is any of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In a specific embodiment, the subclass of the CH1 region is any of γ1, γ2, γ3, γ4, α1, α2, μ, δ, and ε.

[0123] In one embodiment, at least one bond connecting the first and second antigen-binding domains is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CH1 region of the second antigen-binding domain. In a specific embodiment, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of EU numbering positions 119, 120, 121, 122, and 123. In a specific embodiment, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of EU numbering positions 131, 132, 133, 134, 135, 136, 137, 138, 139, and 140. In certain embodiments, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 148, 149, and 150. In certain embodiments, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. In certain embodiments, amino acid residues in the first antigen-binding domain and the second antigen-binding domain are each independently selected from the group consisting of EU numbering positions 174, 175, 176, 177, and 178. In certain 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 EU numbering positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197. In certain 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 EU numbering positions 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, and 214.In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of EU numbering positions 218 and 219.

[0124] In one embodiment, the difference in the position of the amino acid residue that serves as the binding origin in the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. In one embodiment, this means that when comparing the position of the amino acid residue that serves as the binding origin in the CH1 region of the first antigen-binding domain and the position of the amino acid residue that serves as the binding origin in the CH1 region of the second antigen-binding domain, the difference is within 3 amino acids, respectively, according to EU numbering. In a specific embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 135 (EU numbering) in the CH1 region of the first antigen-binding domain with any amino acid residue at positions 132 to 138 (EU numbering) in the CH1 region of the second antigen-binding domain. In a specific embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at EU numbering position 136 in the CH1 region of the first antigen-binding domain to any amino acid residue from EU numbering positions 133 to 139 in the CH1 region of the second antigen-binding domain. In a specific embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at EU numbering position 191 in the CH1 region of the first antigen-binding domain to any amino acid residue from EU numbering positions 188 to 194 in the CH1 region of the second antigen-binding domain. In an exemplary embodiment, at least one bond linking the first and second antigen-binding domains is formed by linking the amino acid residue at EU numbering position 135 in the CH1 regions of the two antigen-binding domains. In an exemplary embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 136 (EU numbering) in the CH1 regions of the two antigen-binding domains.In an exemplary embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 191 (EU numbering) in the CH1 regions of the two antigen-binding domains.

[0125] In one aspect, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is located in the CL region, for example, at any one of positions 108 to 112, 121 to 128, 151 to 156, 184 to 190, 195 to 196, 200 to 203, and 208 to 213 according to Kabat numbering in the CL region. In a specific embodiment, the amino acid residue is located at any one selected from the group consisting of positions 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 the Kabat numbering of the CL region. In a specific embodiment, the amino acid residue is located at position 126 in the Kabat numbering of the CL region. In one embodiment, the constant region is derived from a human. In a specific embodiment, the subclass of the CL region is kappa or lambda.

[0126] In one embodiment, at least one bond connecting the first and second antigen-binding domains is formed by linking an amino acid residue in the CL region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a specific embodiment, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of positions 108, 109, 110, 111, and 112 (Kabat numbering). In a specific embodiment, the amino acid residues in the first and second antigen-binding domains are independently selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 (Kabat numbering). In certain embodiments, 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 (Kabat numbering). In certain embodiments, 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 (Kabat numbering). In certain embodiments, 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 (Kabat numbering). In certain embodiments, 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 (Kabat numbering). In certain embodiments, the amino acid residues in the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of positions 208, 209, 210, 211, 212, and 213 according to the Kabat numbering system.

[0127] In one embodiment, the difference in the position of the amino acid residue that serves as the binding origin in the first antigen-binding domain and the second antigen-binding domain is within 3 amino acids. In one embodiment, this means that when comparing the position of the amino acid residue that serves as the binding origin in the CL region of the first antigen-binding domain and the position of the amino acid residue that serves as the binding origin in the CL region of the second antigen-binding domain, the difference is within 3 amino acids, respectively, according to EU numbering. In an exemplary embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residues at position 126 (Kabat numbering) in the CL regions of the two antigen-binding domains.

[0128] In one embodiment, at least one bond connecting the first and second antigen-binding domains is formed by linking an amino acid residue in the CH1 region of the first antigen-binding domain with an amino acid residue in the CL region of the second antigen-binding domain. In a specific embodiment, the amino acid residue in the CH1 region of the first antigen-binding domain is selected from the group consisting of positions 188, 189, 190, 191, 192, 193, 194, 195, 196, and 197 (EU numbering), and the amino acid residue in the CL region of the second antigen-binding domain is selected from the group consisting of positions 121, 122, 123, 124, 125, 126, 127, and 128 (Kabat numbering). In an exemplary embodiment, at least one bond linking the first antigen-binding domain and the second antigen-binding domain is formed by linking the amino acid residue at position 191 (EU numbering) in the CH1 region of the first antigen-binding domain with the amino acid residue at position 126 (Kabat numbering) in the CL region of the second antigen-binding domain.

[0129] In one embodiment, at least one of the amino acid residues that serve as the origin of binding between the antigen-binding domains is located in a variable region. In a specific embodiment, the amino acid residue is located in the VH region, e.g., at positions 6, 8, 16, 20, 25, 26, 28, 74, and 82b according to the Kabat numbering system of the VH region. In a specific embodiment, the amino acid residue is located in the VL region, e.g., at positions 21, 27, 58, 77, 100, 105, and 107 according to the Kabat numbering system of the VL region (subclass κ) and 6, 19, 33, and 34 according to the Kabat numbering system of the VL region (subclass λ). In certain embodiments, the amino acid residue is present in the VHH region, for example, 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.

[0130] In one embodiment, the first and / or second antigen-binding domains comprise a hinge region. In a specific embodiment, at least one of the cysteine ​​residues present in the wild-type hinge region is substituted with another amino acid residue. Such a cysteine ​​residue is, for example, present at EU numbering positions 226 and / or 229 in the wild-type hinge region. In a specific embodiment, at least one of the amino acid residues that serves as the origin of binding between the antigen-binding domains is present in the hinge region, for example, at any amino acid residue selected from the group consisting of EU numbering positions 216, 218, and 219 in the hinge region.

[0131] In one embodiment, the first antigen-binding domain and the second antigen-binding domain are linked to each other via two or more bonds.

[0132] In certain embodiments, at least one of the amino acid residues that form the bond between the antigen-binding domains is an amino acid residue present in the wild-type sequence, for example, a cysteine ​​residue in the wild-type hinge region. In certain embodiments, at least one bond connecting the first antigen-binding domain and the second antigen-binding domain is a disulfide bond formed by cross-linking cysteine ​​residues present in the wild-type hinge region. Such cysteine ​​residues are present, for example, at positions 226 and / or 229 (EU numbering) in the wild-type hinge region.

[0133] In a specific embodiment, at least one of the amino acid residues that form the bond between the antigen-binding domains is present in the antibody fragment, and at least one is present in the hinge region. In an exemplary embodiment, the antigen-binding molecule of the present disclosure is an F(ab')2 fragment, in which both the first and second antigen-binding domains comprise an Fab and hinge region.

[0134] The antigen-binding molecules of the present application include, for example, the antigen-binding molecules described in WO2018 / 097307. For example, the antigen-binding molecules of the present application relate to polypeptides, which comprise an antigen-binding domain and a delivery moiety, the delivery moiety having an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain having a shorter half-life in blood than the delivery moiety.

[0135] The delivery moiety has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain. As used herein, the term "inhibition domain" is limited only to its ability to inhibit the antigen-binding activity of the antigen-binding domain. The inhibition domain may have any structure as long as it can inhibit the antigen-binding activity of the antigen-binding domain. Examples of such inhibition 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 inhibition domain may consist of the entire delivery moiety or a portion of the delivery moiety.

[0136] In some embodiments, the antigen-binding activity of the antigen-binding domain increases when released from the polypeptide. In other words, when the antigen-binding domain is not released from the polypeptide, its antigen-binding activity is inhibited by the inhibitory domain. Methods for confirming that the antigen-binding activity of the antigen-binding domain is inhibited by the inhibitory domain include fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), electrogenerated chemiluminescence (ECL), surface plasmon resonance (SPR) (Biacore), and bio-layer interferometry (BLI) (Octet). In some embodiments, the antigen-binding activity of the antigen-binding domain released from the polypeptide is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more compared to the binding activity of the antigen-binding domain when not released from the polypeptide. In more specific embodiments, when the antigen-binding activity of the antigen-binding domain before release is measured by one of the above methods, no binding between the antigen-binding domain and the antigen is observed. In some embodiments, cleavage of the cleavage site allows the antigen-binding domain to be released from the polypeptide, and therefore, in such embodiments, the antigen-binding activity can be compared by comparing the antigen-binding activity before and after cleavage of the polypeptide.That is, compared to the antigen-binding activity measured using the uncleaved polypeptide, the antigen-binding activity measured using the cleaved polypeptide is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more. In more specific embodiments, when the antigen-binding activity of the uncleaved polypeptide is measured using one of the above methods, no binding between the antigen-binding domain and the antigen is observed. In some embodiments, the cleavage site is cleaved by a protease, and therefore, in such embodiments, the antigen-binding activity can be compared by comparing the antigen-binding activity of the polypeptide before and after protease treatment. That is, compared to the antigen-binding activity measured using a polypeptide that has not been treated with a protease, the antigen-binding activity measured using a protease-treated polypeptide is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more. In more specific embodiments, when the antigen-binding activity of a polypeptide that has not been treated with a protease is measured by a method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed.

[0137] In the present application, a polypeptide comprising an antigen-binding domain and a delivery moiety has a longer serum half-life than an antigen-binding domain alone. To prolong the half-life of the polypeptide, in some embodiments, the delivery moiety is designed to have a longer serum half-life. Examples of embodiments that extend the serum half-life of the delivery moiety include, but are not limited to, a delivery moiety with a larger molecular weight, a delivery moiety that binds to FcRn, a delivery moiety that binds to albumin, or a delivery moiety that is PEGylated. In some embodiments, the delivery moiety has a longer serum half-life than the antigen-binding domain (i.e., the antigen-binding domain has a shorter serum half-life than the delivery moiety).

[0138] In the present application, the half-life of the antigen-binding domain alone and that of the polypeptide, or that of the antigen-binding domain and the carrier moiety, is preferably compared based on the blood half-life in humans. If it is difficult to measure the blood half-life in humans, the blood half-life in humans can be predicted based on the blood half-life in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0139] In one embodiment, the serum half-life of the delivery moiety is increased by increasing the molecular weight of the delivery moiety. In one embodiment, the serum half-life of the delivery moiety is increased relative to the serum half-life of the antigen-binding domain by increasing the molecular weight of the delivery moiety relative to the molecular weight of the antigen-binding domain.

[0140] One embodiment for extending the blood half-life of a delivery moiety is to confer FcRn binding ability to the delivery moiety. Conferring FcRn binding ability to a delivery moiety is typically achieved by incorporating an FcRn-binding domain into the delivery moiety. An FcRn-binding domain is a region that has binding ability to FcRn, and any structure can be used as long as it has FcRn binding ability. Delivery moieties containing an FcRn-binding domain can return to plasma after being taken up into cells via the FcRn salvage pathway. For example, the relatively long plasma retention (slow elimination) of IgG molecules is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions within the endosome. IgG molecules that fail to bind to FcRn proceed to lysosomes where they are degraded, whereas IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral plasma conditions, returning to the plasma. The FcRn-binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn-binding region is the Fc region of an antibody. However, since regions capable of binding to polypeptides capable of binding to FcRn, such as albumin and IgG, can bind to FcRn indirectly via albumin, IgG, or the like, the FcRn-binding region may also be a region that binds to such polypeptides capable of binding to FcRn.

[0141] The binding activity of an FcRn-binding region to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described above in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (dissociation constant), apparent KD (apparent dissociation constant), dissociation rate kd (dissociation rate), apparent kd (apparent dissociation rate), or the like. These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plots, flow cytometers, etc. can be used.

[0142] The conditions for measuring the FcRn-binding activity of an FcRn-binding region can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009 / 125825, measurements can be performed in MES buffer at 37°C. Furthermore, the FcRn-binding activity of an FcRn-binding region can be measured by methods known to those skilled in the art, such as using a Biacore (GE Healthcare). The binding activity of an FcRn-binding region to FcRn can be assessed by passing FcRn, the FcRn-binding region, or the FcRn-binding region as an analyte through a chip on which the FcRn-binding region or a carrier moiety containing the FcRn-binding region is immobilized, or FcRn is immobilized.

[0143] The pH used as a measurement condition for evaluating the binding affinity between the FcRn-binding region and FcRn may be any pH between pH 4.0 and pH 6.5. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn-binding region and human FcRn. The temperature used as a measurement condition for evaluating the binding affinity between the FcRn-binding region and FcRn may be any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn-binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn-binding region and FcRn. A temperature of 25°C is a non-limiting example embodiment.

[0144] Having FcRn-binding ability in the delivery moiety does not mean that the antigen-binding domain does not have FcRn-binding ability. In an embodiment in which the blood half-life of the delivery moiety is made longer than that of the antigen-binding domain, the antigen-binding domain may not have FcRn-binding ability, or even if the antigen-binding domain has FcRn-binding ability, the delivery moiety may have weaker FcRn-binding ability.

[0145] One embodiment for extending the blood half-life of a delivery moiety is to conjugate it to albumin. Albumin is not excreted renally and has FcRn-binding activity, resulting in a long blood half-life of 17 to 19 days (J Clin Invest. 1953 Aug; 32(8): 746-768.). It has been reported that proteins bound to albumin become bulky and are able to indirectly bind to FcRn, thereby increasing their blood half-life (Antibodies 2015, 4(3), 141-156).

[0146] Furthermore, one embodiment for extending the blood half-life of a delivery moiety is to PEGylate the delivery moiety. PEGylation of a protein increases the protein's bulkiness and simultaneously inhibits its degradation by proteases in the blood, which is thought to extend the blood half-life of the protein (J Pharm Sci. 2008 Oct;97(10):4167-83).

[0147] In some embodiments, the transport moiety comprises an antibody Fc region. In one specific embodiment, the transport moiety comprises the CH2 and CH3 domains of a human IgG antibody. In one specific embodiment, the transport moiety comprises a portion of a human IgG1 antibody heavy chain extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, with the proviso that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

[0148] In some embodiments, the delivery moiety comprises an antibody constant region. In more preferred embodiments, the delivery moiety comprises an IgG antibody constant region. In a more preferred embodiment, the delivery moiety comprises a human IgG antibody constant region.

[0149] In some further embodiments, the delivery moiety comprises a region having a structure substantially similar to that of an antibody heavy chain constant region, and a region having a structure substantially similar to that of an antibody light chain, bound to said region by covalent bonds, such as disulfide bonds, or non-covalent bonds, such as hydrogen bonds or hydrophobic interactions.

[0150] As used herein, a "polypeptide comprising an antigen-binding domain and a transport moiety" is typically a sequence of polypeptides linked by amide bonds, or a protein comprising multiple sequences of polypeptides linked by amide bonds.

[0151] In some embodiments, the antigen-binding domain is releasable from the polypeptide, and release of the antigen-binding domain from the polypeptide increases the antigen-binding activity. As used herein, the term "release" refers to the separation of two portions of a polypeptide from each other. Release of the antigen-binding domain from the polypeptide can be caused by the dissolution of the interaction between the antigen-binding domain and the delivery moiety. Since the antigen-binding activity of an antigen-binding domain incorporated into a polypeptide is suppressed, release of the antigen-binding domain from the polypeptide can be confirmed by measuring the antigen-binding activity of the target substance and comparing it with the antigen-binding activity of the antigen-binding domain incorporated into the polypeptide.

[0152] In some embodiments, the polypeptide contains a cleavage site, and cleavage of the cleavage site releases the antigen-binding domain from the polypeptide. The cleavage site can be cleaved, for example, by an enzyme, by reduction with a reducing agent, or by photolysis. The cleavage site can be located anywhere in the polypeptide, as long as it allows the antigen-binding domain to be released and does not abolish the antigen-binding activity of the released antigen-binding domain. Furthermore, the polypeptide may contain another cleavage site in addition to the cleavage site for releasing the antigen-binding domain. In one embodiment, the cleavage site contains a protease cleavage sequence and can be cleaved by a protease.

[0153] As used herein, the term "cleaved" refers to a state in which the antigen-binding domain and the transport moiety are separated after modification of the cleavage site by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site and / or photoactivation. As used herein, the term "uncleaved" refers to a state in which the antigen-binding domain and the transport moiety are linked in the absence of cleavage of the cleavage site by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site and / or in the absence of light.

[0154] Cleavage at the cleavage site can be detected by subjecting a solution containing the cleavage site-containing polypeptide to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragments, or by detecting the change in molecular weight before and after cleavage.

[0155] The cleavage site varies from approximately 0.001 to 1500×10 depending on the agent (i.e., protease, reducing agent, light). ４ M －１ S －１ or 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 can be specifically modified (cleaved, reduced or photolyzed) at rates of .

[0156] Specific cleavage by a protease requires contact between the protease and the cleavage site or a molecule containing the cleavage site. In the presence of sufficient enzymatic activity, the cleavage site can be cleaved. Sufficient enzymatic activity can refer to the ability of the enzyme to contact the cleavage site and effect cleavage.

[0157] As used herein, the term "protease" refers to an enzyme, such as an endopeptidase or exopeptidase, that hydrolyzes peptide bonds, typically an endopeptidase. The protease used herein is limited only by its ability to cleave the protease cleavage sequence, and its type is not particularly limited. In some embodiments, a target tissue-specific protease is used. A target tissue-specific protease can refer to, for example, any of the following: (1) a protease expressed at a higher level in a target tissue than in normal tissue; (2) a protease having higher activity in a target tissue than in normal tissue; (3) a protease expressed at a higher level in a target cell than in normal cells; or (4) a protease having higher activity in a target cell than in normal cells. In more specific embodiments, a cancer tissue-specific protease or an inflamed tissue-specific protease is used.

[0158] As used herein, the term "target tissue" refers to a tissue that contains at least one target cell. In some embodiments, the target tissue is cancerous tissue. In some embodiments, the target tissue is inflamed tissue.

[0159] The term "cancer tissue" refers to tissue containing at least one cancer cell. Thus, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing both cancer cells and blood vessels. As used herein, a tumor mass refers to a foci of tumor tissue. The term "tumor" is generally used to refer to benign or malignant neoplasms.

[0160] As used herein, "inflammatory tissue" refers to, for example, the following: - joints in rheumatoid arthritis and osteoarthritis - lungs (alveoli) in bronchial asthma and COPD - digestive organs in inflammatory bowel disease, Crohn's disease and ulcerative colitis - fibrotic tissues in fibrosis of the liver, kidneys and lungs - tissues undergoing rejection in organ transplants - blood vessels and heart (myocardium) in arteriosclerosis and heart failure - visceral fat in metabolic syndrome - skin tissue in atopic dermatitis and other dermatitis - spinal nerves in herniated discs and chronic lower back pain

[0161] Proteases that are specifically expressed or specifically activated in several types of target tissues or that are thought to be associated with the disease state of the target tissues (target tissue-specific proteases) are known. For example, International Publication Nos. WO 2013 / 128194, WO 2010 / 081173, and WO 2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul; 6(7): 541-50., Nat Rev Drug Discov. 2014 Dec; 13(12): 904-27., Respir Res. 2016 Mar 4; 17: 23., Dis Model Mech. 2014 Feb; 7(2): 193-203., and Biochim Biophys Acta. 2012 Jan; 1824(1): 133-45. In addition to proteases specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active. In many tissues, substances that inhibit active proteases exist, and their activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, active proteases may escape inhibition and become specifically activated. Active proteases can be measured using antibodies that recognize active proteases (PNAS 2013 Jan 2;110(1):93-98.) or by fluorescently labeling peptides that are quenched before cleavage but emit light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.).From one perspective, the term "target tissue-specific protease" can refer to any of the following: (i) a protease that is expressed at a higher level in a target tissue than in normal tissue; (ii) a protease that has a higher activity in a target tissue than in normal tissue; (iii) a protease that is expressed at a higher level in a target cell than in normal cells; or (iv) a protease that has a higher activity in a target cell than in normal cells.

[0162] Specific proteases include, but are not limited to, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matriptase (MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptophan, etc.), and the like. metalloproteases (including membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28); proteases A disintegrin and metalloprotease (ADAM), metalloproteases with A disintegrin or thrombospondin motifs (ADAMTS); meprin (meprin α alpha), meprin beta), CD10 (CALLA), as well as prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), granzyme B, guanidinobenzoatase (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otubain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, Gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-LikeProteases include AMSH, gamma secretase, antiplasmin-cleaving enzyme A (APCE), decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0163] From another perspective, the target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease. Examples of the cancer tissue-specific protease include proteases specifically expressed in cancer tissues, such as those disclosed in International Publication Nos. WO 2013 / 128194, WO 2010 / 081173, and WO 2009 / 025846.

[0164] The more specific the type of cancer tissue-specific protease is expressed in the cancer tissue to be treated, the more effective it is in reducing side effects. The concentration of the cancer tissue-specific protease in cancer tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the activity of the cancer tissue-specific protease in cancer tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, more preferably at least 4 times higher, more preferably at least 5 times higher, even more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the cancer tissue-specific protease may be bound to the cell membrane of the cancer cell, or may be secreted extracellularly without being bound to the cell membrane. When a cancer tissue-specific protease is not bound to the cell membrane of a cancer cell, it is preferable that the cancer tissue-specific protease be present inside or near the cancer tissue so that cytotoxicity by immune cells is specific to the cancer cell. As used herein, "near the cancer tissue" refers to a range within which the cancer tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts its antigen-binding activity. However, it is preferable that the range be such that normal cells are not harmed as much as possible. From another perspective, a cancer tissue-specific protease is any of the following: (i) a protease expressed at a higher level in cancer tissue than in normal tissue; (ii) a protease having higher activity in cancer tissue than in normal tissue; (iii) a protease expressed at a higher level in cancer cells than in normal cells; or (iv) a protease having higher activity in cancer cells than in normal cells. A single cancer tissue-specific protease may be used alone, or two or more types may be used in combination. The number of types 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.

[0165] From the above viewpoints, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matriptase (including MT-SP1), urokinase (uPA) and metalloproteases are more preferred, and MT-SP1, uPA, MMP2 and MMP9 are even more preferred.

[0166] The more specific the type of inflammatory tissue-specific protease is expressed in the inflammatory tissue to be treated, the more effective it is in reducing side effects. The concentration of the inflammatory tissue-specific protease in the inflammatory tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the activity of the inflammatory tissue-specific protease in the inflammatory tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, at least 4 times higher, at least 5 times higher, or even at least 10 times higher, more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammatory tissue-specific protease may be bound to the cell membrane of inflammatory cells, or may be secreted extracellularly without being bound to the cell membrane. When the inflammatory tissue-specific protease is not bound to the cell membrane of an inflammatory cell, it is preferable that the inflammatory tissue-specific protease be present inside or near the inflammatory tissue so that cytotoxicity by immune cells is specific to the inflammatory cell. As used herein, "near the inflammatory tissue" refers to a range within which the inflammatory tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts its antigen-binding activity. However, it is preferable that the range be within which normal cells are not harmed as much as possible. From another perspective, the inflammatory tissue-specific protease is any of the following: (i) a protease expressed at a higher level in inflammatory tissue than in normal tissue; (ii) a protease having higher activity in inflammatory tissue than in normal tissue; (iii) a protease expressed at a higher level in inflammatory cells than in normal cells; or (iv) a protease having higher activity in inflammatory cells than in normal cells. The inflammatory tissue-specific protease may be used alone or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the pathology of the target to be treated.

[0167] From the above viewpoint, among the above-mentioned proteases, metalloproteases are preferred as the inflamed tissue-specific proteases, and among the metalloproteases, ADAMTS5, MMP2, MMP7, MMP9, and MMP13 are more preferred.

[0168] A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution. From the viewpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in the target tissue / cells to be treated or more specifically activated in the target tissue / cells to be treated. Specific protease cleavage sequences include target sequences that are specifically hydrolyzed by the proteases specifically expressed in cancer tissues and inflammatory tissue-specific proteases disclosed in International Publication Nos. WO 2013 / 128194, WO 2010 / 081173, WO 2009 / 025846, etc., as exemplified above. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences specifically hydrolyzed by known proteases, can also be used. The protease cleavage sequence may be one identified by a method known to those skilled in the art, such as that described in Nature Biotechnology 19, 661-667 (2001). Furthermore, a naturally occurring protease cleavage sequence may be used. For example, a protease cleavage sequence in a protein whose molecular shape changes upon protease cleavage can also be used, just as TGF-β is converted to its latent form upon protease cleavage.

[0169] Examples of protease cleavage sequences include, but are not limited to, those described in International Publication No. WO2015 / 116933, International Publication No. WO2015 / 048329, International Publication No. WO2016 / 118629, International Publication No. WO2016 / 179257, International Publication No. WO2016 / 179285, International Publication No. WO2016 / 179335, International Publication No. WO2016 / 179003, International Publication No. WO2016 / 046778, International Publication No. WO2016 / 014974, U.S. Patent Publication No. US2016 / 0289324, U.S. Patent Publication No. US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228., Beilstein J Nanotechnol. (2016) 7: 364-373. The protease cleavage sequence is preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease, as described above.

[0170] In one embodiment, the protease cleavage sequence further comprises a flexible linker attached to either or both ends. The flexible linker at one end of the protease cleavage sequence can be referred to as the first flexible linker, and the flexible linker at the other end can be referred to as the second flexible linker. In certain embodiments, the protease cleavage sequence and flexible linker have one of the following formulas: (protease cleavage sequence) (first flexible linker)-(protease cleavage sequence) (protease cleavage sequence)-(second flexible linker) (first flexible linker)-(protease cleavage sequence)-(second flexible linker). In this embodiment, the flexible linker is preferably a peptide linker. The first flexible linker and the second flexible linker are each independently and optionally present and may be the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly). For example, the protease cleavage sequence includes a sufficient number of residues to provide the desired protease accessibility (such as amino acids selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, especially Gly and Ser, particularly Gly, etc.).

[0171] Flexible linkers suitable for use on either end of a protease cleavage sequence typically improve protease access to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be readily selected and can range in length from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, or 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. In some embodiments, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0172] Examples of flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (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 linkers known in the art. Of these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and tend to function as neutral tethers between moieties.

[0173] As used herein, "association" can be rephrased as, for example, a state in which two or more polypeptide regions interact with each other. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the target polypeptide regions to form an association. As a commonly observed example of an association, in antibodies, such as natural antibodies, it is known that the heavy chain variable region (VH) and the light chain variable region (VL) maintain a pairing structure through non-covalent bonds between them.

[0174] In some embodiments, the repression domain of the delivery moiety associates with the antigen-binding domain. The repression domain may be a portion of the delivery moiety or the entire delivery moiety. From another perspective, the portion of the delivery moiety that associates with the antigen-binding domain can be referred to as the repression domain. In a more specific embodiment, the antigen-binding domain of a single-domain antibody and the repression domain of VL, VH, or VHH form an association similar to that of antibody VH and antibody VL. In an even more specific embodiment, the antigen-binding domain of a single-domain antibody and the repression domain of VL, VH, or VHH form an association similar to that of antibody VH and antibody VL. In this state, the repression domain conformationally inhibits binding between the antigen-binding domain and the antigen or changes the conformation of the antigen-binding site of the antigen-binding domain, thereby inhibiting the antigen-binding activity of the single-domain antibody by the VL, VH, or VHH. In embodiments in which VHH is used as a single-domain antibody, if the CDR3, the main antigen-binding site of VHH, or a site nearby it, is present at the interface where it associates with the repression domain, it is believed that the repression domain will conformationally inhibit binding of VHH to the antigen. Furthermore, the association between the repression domain and the antigen-binding domain can be eliminated, for example, by cleaving the cleavage site. Elimination of the association can be expressed, for example, as the elimination of the interaction between two or more polypeptide regions. The interaction between the two or more polypeptide regions may be completely eliminated, or only a portion of the interaction between the two or more polypeptide regions may be eliminated.

[0175] As used herein, the term "interface" generally refers to the surface at which association (interaction) occurs, and the amino acid residues that form the interface generally refer to one or more amino acid residues contained in the polypeptide region involved in the association, more preferably amino acid residues that come close to each other during association and are involved in the interaction. Specific examples of such interactions include non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges formed between amino acid residues that come close to each other during association.

[0176] As used herein, "amino acid residues forming an interface" refers, more specifically, to amino acid residues contained in a polypeptide region that constitutes the interface. The polypeptide region that constitutes the interface refers, for example, to a polypeptide region that is responsible for selective intramolecular or intermolecular binding in an antibody, ligand, receptor, substrate, etc. Specific examples of such polypeptide regions in antibodies include heavy chain variable regions and light chain variable regions, and in some embodiments, antigen-binding domains and inhibitory domains. Examples of amino acid residues that form an interface include, but are not limited to, amino acid residues that come into close proximity during association. Amino acid residues that come into close proximity during association can be identified, for example, by analyzing the three-dimensional structure of a polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during association of the polypeptide.

[0177] In some embodiments, to promote the association of the antigen-binding domain with the repression domain, amino acid residues in the antigen-binding domain involved in the association or amino acid residues in the repression domain involved in the association can be modified. In a more specific embodiment, amino acid residues in the antigen-binding domain that form an interface with the repression domain or amino acid residues in the repression domain that form an interface with the antigen-binding domain can be modified. In a preferred embodiment, the modification of amino acid residues that form the interface involves introducing mutations into the interface so that two or more amino acid residues that form the interface have different charges. Modifications of amino acid residues that result in different charges include modifying a positively charged amino acid residue to a negatively charged amino acid residue or an uncharged amino acid residue, modifying a negatively charged amino acid residue to a positively charged amino acid residue or an uncharged amino acid residue, and modifying an uncharged amino acid residue to a positively or negatively charged amino acid residue. Such amino acid modifications are intended to promote association, and the position and type of amino acid modified are not limited as long as the purpose of promoting association is achieved. Modifications include, but are not limited to, substitutions.

[0178] In some embodiments, the antigen-binding domain, VHH, associates with the inhibitory domain, VL. Examples of amino acid residues in VHH involved in the association with VL include amino acid residues that form the interface between VHH and VL. Examples of amino acid residues in VHH involved in the association with VL include, but are not limited to, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). Promotion of the association between VHH and VL inhibits the activity of VHH. Similarly, examples of amino acid residues in VL involved in the association with VHH include amino acid residues that form the interface between VHH and VL.

[0179] To promote the association of VHH and VL, amino acid residues in VHH involved in the association with VL can be modified. 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. Furthermore, a VHH that originally contains amino acid residues 37V, 44G, 45L, and / or 47W can be used without modifying any residues in the VHH. Furthermore, as long as the goal of promoting the association of VHH and VL is achieved, amino acid residues in VL involved in the association with VHH can be modified rather than amino acids in VHH. Furthermore, amino acid modifications can be introduced into both VHH and VL.

[0180] In some other embodiments, a VHH is used as the antigen-binding domain, and a VH or VHH is used as the repression domain, allowing the antigen-binding domain and the repression domain to associate. To promote the association between the VHH antigen-binding domain and the VH or VHH repression domain, amino acid residues in the VHH antigen-binding domain that are involved in the association with the VH or VHH repression domain can be identified and modified. Alternatively, amino acid residues in the VH or VHH repression domain that are involved in the association with the VHH antigen-binding domain can be identified and modified.

[0181] Furthermore, when using a single-domain antibody other than VHH as the antigen-binding domain, it is also possible to identify amino acid residues in the antigen-binding domain or the inhibitory domain that are involved in the association and modify those amino acid residues.

[0182] In some embodiments, the delivery moiety and the antigen-binding domain are fused via a linker. In more specific embodiments, the delivery moiety and the antigen-binding domain are fused via a linker that contains a cleavage site. In another specific embodiment, the delivery moiety and the antigen-binding domain are fused via a linker, and the resulting fusion protein contains a cleavage site.

[0183] In another embodiment, the delivery moiety and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino acid bond is formed between the N-terminal amino acid of the delivery moiety and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The resulting fusion protein contains a cleavage site. In a specific embodiment, one to several amino acids at the N-terminus of the delivery moiety and / or one to several amino acids at the C-terminus of the antigen-binding domain are modified to fuse the N-terminus of the delivery moiety to the C-terminus of the antigen-binding domain, thereby forming a cleavage site near the fusion site. More specifically, for example, the four C-terminal amino acids of the antigen-binding domain can be substituted with the LSGR (SEQ ID NO: 1836) sequence and the four N-terminal amino acids of the delivery moiety can be substituted with the SDNH (SEQ ID NO: 1837) sequence to form a cleavage site.

[0184] In some embodiments, the cleavage site of a polypeptide comprising a transport moiety and an antigen-binding domain comprises a protease cleavage sequence, which may be located anywhere in the polypeptide as long as it releases the antigen-binding domain upon cleavage by a protease and does not abolish the antigen-binding activity of the released antigen-binding domain.

[0185] In some embodiments, the delivery moiety comprises an antibody constant region, and the N-terminus of the antibody constant region is fused to the C-terminus of the antigen-binding domain with or without a linker. In certain embodiments, the protease cleavage sequence is located within the antibody constant region comprised in the delivery moiety. In this case, the protease cleavage sequence may be located within the antibody constant region so that the antigen-binding domain can be released upon protease cleavage. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region comprised in the delivery moiety, more specifically, on the antigen-binding domain side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably on the antigen-binding domain side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located within the antibody light chain constant region comprised in the delivery moiety, more specifically, closer to the antigen-binding domain than amino acid 130 (EU numbering) (Kabat numbering 130) in the antibody light chain constant region, preferably closer to the antigen-binding domain than amino acid 113 (EU numbering) (Kabat numbering 113) in the antibody light chain constant region.

[0186] In some embodiments, the antigen-binding domain is a single-domain antibody, and the C-terminus of the single-domain antibody is fused to the N-terminus of the transport moiety with or without a linker. In certain embodiments, the protease cleavage sequence is located within the single-domain antibody. In more specific embodiments, the single-domain antibody is a single-domain antibody or VHH constructed from a VH, and the protease cleavage sequence is located closer to the transport moiety than amino acid 35b (Kabat numbering) of the single-domain antibody, preferably closer to the transport moiety than amino acid 95 (Kabat numbering) of the single-domain antibody, and more preferably closer to the transport moiety than amino acid 109 (Kabat numbering) of the single-domain antibody. In another specific embodiment, the single domain antibody is a single domain antibody generated from a VL, and the protease cleavage sequence is located closer to the transport moiety than amino acid 32 (Kabat numbering) of the single domain antibody, preferably closer to the transport moiety than amino acid 91 (Kabat numbering) of the single domain antibody, and more preferably closer to the transport moiety than amino acid 104 (Kabat numbering) of the single domain antibody.

[0187] In some embodiments, the delivery moiety comprises an antibody constant region, and the antigen-binding domain is a single-domain antibody, with the antibody constant region and the single-domain antibody fused with or without a linker. In a more specific embodiment, the N-terminus of the antibody constant region is fused with or without a linker to the C-terminus of the single-domain antibody. In another specific embodiment, the C-terminus of the antibody constant region is fused with or without a linker to the N-terminus of the single-domain antibody. In certain embodiments, a protease cleavage sequence is located within the antibody constant region comprised in the delivery moiety. In a more specific embodiment, the protease cleavage sequence is located closer to the single-domain antibody than amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably closer to the single-domain antibody than amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located closer to the antigen-binding domain than amino acid 130 (EU numbering) (Kabat numbering 130) in the antibody light chain constant region, preferably closer to the antigen-binding domain than amino acid 113 (EU numbering) (Kabat numbering 113) in the antibody light chain constant region. In a particular embodiment, the protease cleavage sequence is located within a single domain. In a more specific embodiment, the single domain antibody is a single domain antibody or VHH constructed from a VH, and the protease cleavage sequence is located closer to the antibody constant region than amino acid 35b (Kabat numbering) of the single domain antibody, preferably closer to the antibody constant region than amino acid 95 (Kabat numbering) of the single domain antibody, and more preferably closer to the antibody constant region than amino acid 109 (Kabat numbering) of the single domain antibody. In another specific embodiment, the single domain antibody is a single domain antibody constructed from a VL, and the protease cleavage sequence is located closer to the antibody constant region of the single domain antibody than amino acid 32 (Kabat numbering), preferably closer to the antibody constant region of the single domain antibody than amino acid 91 (Kabat numbering), and more preferably closer to the antibody constant region of the single domain antibody than amino acid 104 (Kabat numbering).In certain embodiments, the protease cleavage sequence is located near the boundary between the antigen-binding domain and the transport moiety. Near the boundary between the antigen-binding domain and the transport moiety refers to a region before or after the site where the antigen-binding domain and the transport moiety are linked that does not significantly affect the secondary structure of the antigen-binding domain. In more specific embodiments, the antigen-binding domain is linked to an antibody constant region contained in the transport moiety, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. Near the boundary between the antigen-binding domain and the antibody constant region can refer to near the boundary between the antigen-binding domain and the antibody heavy chain constant region, or near the boundary between the antigen-binding domain and the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody or VHH constructed from VH and is linked to an antibody heavy chain constant region, the vicinity of the boundary between the antigen-binding domain and the antibody constant region refers to the region between amino acid 101 (Kabat numbering) in the case of a single-domain antibody and amino acid 140 (EU numbering) in the case of an antibody heavy chain constant region, and preferably refers to the region between amino acid 109 (Kabat numbering) in the case of a single-domain antibody and amino acid 122 (EU numbering) in the case of an antibody heavy chain constant region. When the antigen-binding domain is a single-domain antibody or VHH constructed from VH and is linked to an antibody light-chain constant region, the vicinity of the boundary between the antigen-binding domain and the antibody light-chain constant region refers to the region between amino acid 101 (Kabat numbering) for single-domain antibodies and amino acid 130 (EU numbering) (Kabat numbering 130) for antibody light-chain constant regions, and preferably refers to the region between amino acid 109 (Kabat numbering) for single-domain antibodies and amino acid 113 (EU numbering) (Kabat numbering 113) for antibody light-chain constant regions. When the antigen-binding domain is a single-domain antibody constructed from VL, the vicinity of the boundary between the antigen-binding domain and the antibody constant region refers to the region from amino acid 96 (Kabat numbering) for single-domain antibodies, preferably from amino acid 104 (Kabat numbering) for single-domain antibodies.

[0188] 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 carrier moiety comprises an IgG antibody constant region, and a single domain antibody that serves as the antigen-binding domain replaces the VH of the IgG antibody, and antigen-binding activity is inhibited by the VL; or embodiments in which the carrier moiety comprises an IgG antibody constant region, and a single domain antibody that serves as the antigen-binding domain replaces the VL of the IgG antibody, and antigen-binding activity is inhibited by the VH; or embodiments in which the carrier moiety comprises an IgG antibody constant region, and a single domain antibody that serves as the antigen-binding domain replaces one of the VH / VL of the IgG antibody, and another single domain antibody that inhibits the antigen-binding activity of the antigen-binding domain replaces the other of the VH / VL of the IgG antibody.

[0189] The term "IgG antibody-like molecule" as used herein is used to define a molecule that has a portion substantially similar to the structure of a constant domain or constant region like that of an IgG antibody and a portion substantially similar to the structure of a variable domain or variable region like that of an IgG antibody, and that has a three-dimensional structure substantially similar to that of an IgG antibody. However, the "IgG antibody-like molecule" as used herein is not limited to molecules that exhibit antigen-binding activity while retaining a structure similar to that of an IgG antibody.

[0190] The polypeptide may contain one or more antigen-binding domains. The repression domains that repress the antigen-binding activity of each of the multiple antigen-binding domains may also be one or more. Each of the multiple antigen-binding domains may be associated with a repression domain. Each of the multiple antigen-binding domains may be fused to a transport moiety. Each of the multiple antigen-binding domains may be releasable from the polypeptide. There may be multiple cleavage sites for releasing the multiple antigen-binding domains, each corresponding to one of the antigen-binding domains.

[0191] 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, a single-domain antibody, an antibody fragment, a module called an A domain of about 35 amino acids contained in an Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO 2004 / 044011 and WO 2005 / 040229), an Adnectin containing the 10Fn3 domain, which is a domain that binds to a protein in fibronectin, a glycoprotein expressed on the cell membrane (International Publication No. WO 2002 / 032925), an Affibody scaffolded with an IgG-binding domain consisting of a three-helix bundle of 58 amino acids from Protein A (International Publication No. WO 1995 / 001937), and a DARPins (Designed Ankyrin Repeat (AR)) region exposed on the molecular surface of an ankyrin repeat (AR) having a structure in which a 33-amino acid turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Examples of such a domain include an anticalin domain, which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich-repeat (LRR) modules are repeatedly stacked in the variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO 2008 / 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 linked antigen-binding domain and second antigen-binding domain is 60 kDa or less.In more specific embodiments, the antigen-binding domain and the second antigen-binding domain are single-domain antibodies each having a different antigen-binding specificity, and the linked antigen-binding domain and the second antigen-binding domain can be released from the polypeptide, and the released antigen-binding domain and the second antigen-binding domain form a bispecific antigen-binding molecule. Examples of such bispecific antigen-binding molecules include, but are not limited to, bispecific antigen-binding molecules in which the antigen-binding domain specifically binds to a target cell surface antigen and the second antigen-binding domain specifically binds to an immune cell surface antigen, bispecific antigen-binding molecules in which the antigen-binding domain and the second antigen-binding domain bind to different subunits of the same antigen, and bispecific antigen-binding molecules in which the antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same antigen. Such bispecific antigen-binding molecules are considered useful in treating diseases caused by target cells, as they can recruit immune cells to the vicinity of the target cells. The antigen-binding activity of the second antigen-binding domain may or may not be inhibited by the delivery moiety. Furthermore, the second antigen-binding domain may or may not be associated with a partial structure of the delivery moiety. In particular, when the antigen-binding domain and the second antigen-binding domain have different antigen-binding specificities, even if the antigen-binding activity of the second antigen-binding domain is not suppressed or even if the second antigen-binding domain is not associated with a partial structure of the delivery moiety, the antigen-binding activity of the antigen-binding domain cannot be exhibited in an unreleased state, and a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain are linked cannot exhibit the function of bispecifically binding to two types of antigens.

[0192] The antigen-binding molecules of the present application include, for example, the antigen-binding molecules described in WO2018 / 097308. In one aspect, the antigen-binding molecules of the present application relate to ligand-binding molecules that are capable of binding to a ligand, wherein the molecule is a polypeptide having at least one cleavage site, and the binding to the ligand is attenuated when the molecule is cleaved at the at least one cleavage site.

[0193] A ligand-binding molecule is a molecule capable of binding to a ligand, particularly a molecule capable of binding to a ligand in an uncleaved state. Here, "binding" typically refers to binding through interactions primarily based on non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Suitable examples of ligand-binding modes of ligand-binding molecules include, but are not limited to, antigen-antibody reactions in which antigen-binding regions, antigen-binding molecules, antibodies, and antibody fragments bind to antigens.

[0194] Note that "capable of binding to a ligand" means that the ligand-binding molecule is capable of binding to the ligand, even if the ligand and the ligand are separate molecules, and does not mean that the ligand-binding molecule and the ligand are connected by a covalent bond. For example, the fact that a ligand and a ligand-binding molecule are covalently bonded via a linker does not mean that the ligand is capable of binding. Furthermore, "the binding to the ligand is weakened" means that the ability to bind is weakened. For example, when a ligand and a ligand-binding molecule are covalently bonded via a linker, cleavage of the linker is not considered to be a weakening of the binding to the ligand. Note that as long as the ligand-binding molecule is capable of binding to the ligand, the ligand-binding molecule may be connected to the ligand via a linker or the like.

[0195] The ligand-binding molecule is limited only by its ability to bind to a ligand in its uncleaved state, and any molecule of any structure can be used as long as it can bind to the target ligand in its uncleaved state. Examples of ligand-binding molecules include, but are not limited to, antibody heavy chain variable regions (VH) and antibody light chain variable regions (VL), single domain antibodies (sdAb), modules called A domains of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes (International Publication WO2002 / 032925), Affibodies using as a scaffold an IgG-binding domain that constitutes a bundle of three helices consisting of 58 amino acids of Protein A (International Publication WO1995 / 001937), and DARPins (Designed Ankyrin Repeats), which are regions exposed on the molecular surface of ankyrin repeats (AR) that have a structure in which a turn containing 33 amino acid residues, two antiparallel helices, and a loop subunit are repeatedly stacked. Examples include lipocalin molecules such as lipocalin proteins (International Publication WO 2002 / 020565), which are highly conserved eight antiparallel strands supporting one side of a barrel structure twisted toward the center, and anticalin (International Publication WO 2003 / 029462), which are found in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure formed by repeated stacking of leucine-rich-repeat (LRR) modules in the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO 2008 / 016854).

[0196] A ligand-binding molecule is a polypeptide that contains a cleavage site. The cleavage site can be cleaved, for example, by an enzyme, reduced by a reducing agent, or photolyzed. The cleavage site can be located anywhere in the polypeptide, as long as cleavage attenuates the binding of the ligand-binding molecule to the ligand. Furthermore, a polypeptide can contain one or more cleavage sites.

[0197] Furthermore, the ligand-binding molecule has weaker (i.e., attenuated) ligand binding in the cleaved state compared to the uncleaved state. In embodiments in which the binding between the ligand-binding molecule and the ligand is an antigen-antibody reaction, attenuation of ligand binding can be evaluated by the ligand binding activity of the ligand-binding molecule.

[0198] The binding activity of ligand-binding molecules and ligands can be evaluated using well-known methods, such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). ALPHA screen is performed using ALPHA technology, which uses two beads, donor and acceptor, based on the following principle: A molecule bound to the donor bead interacts with a molecule bound to the acceptor bead, and a luminescent signal is detected only when the two beads are in close proximity. A photosensitizer within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor beads, and when it reaches a nearby Abystar bead, it triggers the Beads chemiluminescence reaction, ultimately emitting light. If the molecules bound to the donor beads and the molecules bound to the acceptor beads do not interact, the singlet oxygen produced by the donor beads does not reach the acceptor beads, and the chemiluminescence reaction does not occur.

[0199] For example, biotin-labeled ligand-binding molecules are bound to donor beads, and glutathione S-transferase (GST)-tagged ligands are bound to acceptor beads. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecules interact with the ligand, generating a signal at 520-620 nm. The untagged ligand-binding molecules compete with the interaction between the tagged ligand-binding molecules and the ligand. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from competition. Biotinylation of ligand-binding molecules such as antibodies using sulfo-NHS-biotin or similar is well known. GST-tagging of ligands can be achieved by expressing the GST-fused ligand in cells harboring a vector capable of expressing a fusion gene in which a polynucleotide encoding the ligand and a polynucleotide encoding GST are fused in frame, followed by purification using a glutathione column. The resulting signals are suitably analyzed by fitting to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0200] One of the substances to be observed for interaction (ligand) is immobilized on a gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance to be observed for interaction (analyte) is passed over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The sensorgram curves provide kinetics: the association rate constant (ka) and dissociation rate constant (kd), and the ratio of these constants determines the dissociation constant (KD). Inhibition assays and equilibrium analysis are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and an example of an equilibrium value analysis is described in Methods Enzymol. 2000;323:325-40.

[0201] A ligand-binding molecule's ability to bind to a ligand is attenuated, for example, when, based on the above-described measurement method, the amount of ligand binding per test ligand-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to a control ligand-binding molecule. Any suitable indicator of binding activity may be used, for example, the dissociation constant (KD). When the dissociation constant (KD) is used as an indicator of binding activity, a larger dissociation constant (KD) for the test ligand-binding molecule relative to the dissociation constant (KD) for the control ligand-binding molecule indicates that the binding activity of the test ligand-binding molecule relative to the control ligand-binding molecule is weaker. The term "ligand-binding function is attenuated" means, for example, that the dissociation constant (KD) of the test ligand-binding molecule for the ligand is at least 2-fold, preferably at least 5-fold, at least 10-fold, and particularly preferably at least 100-fold, compared to the dissociation constant (KD) of the control ligand-binding molecule for the ligand. Examples of the control ligand-binding molecule include uncleaved ligand-binding molecules.

[0202] In one embodiment, the cleavage site of the ligand-binding molecule is cleaved, thereby releasing the ligand from the ligand-binding molecule. Here, if the ligand is bound to a portion of the ligand-binding molecule via a linker and the linker does not have a cleavage site, the ligand will be released while remaining connected to the portion of the ligand-binding molecule via the linker. Thus, even if the ligand is released together with a portion of the ligand-binding molecule, it can be said that the ligand has been released from the ligand-binding molecule as long as it is released from the majority of the ligand-binding molecules.

[0203] One method for detecting the release of a ligand from a ligand-binding molecule upon cleavage at the cleavage site is to detect the ligand using a ligand-detecting antibody that recognizes the ligand. When the ligand-binding molecule is an antibody fragment, the ligand-detecting antibody preferably binds to the same epitope as the ligand-binding molecule. Ligand detection using a ligand-detecting antibody can be confirmed by well-known methods, such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE using surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). For example, when detecting ligand release using Octet, a ligand-detecting antibody that recognizes the ligand can be biotinylated, contacted with a biosensor, and then measured for binding to the ligand in the sample to detect ligand release. Specifically, ligand release can be detected by measuring the amount of ligand using a ligand-detecting antibody in a sample containing a ligand-binding molecule and a ligand before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment. Alternatively, ligand release can be detected by measuring the amount of ligand using a ligand-detecting antibody in a sample containing a protease, a ligand-binding molecule, and a ligand, and in a sample containing a ligand-binding molecule and a ligand without protease, and comparing the amount of ligand detected in the sample with and without protease. More specifically, ligand release can be detected by the methods described in the Examples. When a ligand-binding molecule is fused with a ligand to form a fusion protein, ligand release can be detected by measuring the amount of ligand using a ligand-detecting antibody in a sample containing the fusion protein before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment.Furthermore, the amount of ligand can be measured using a ligand-detecting antibody for a sample containing a protease and a fusion protein and a sample containing the fusion protein but not the protease, and the amount of ligand detected in the samples with and without the protease can be compared to detect the release of the ligand. More specifically, the release of the ligand can be detected by the method described in the Examples of the present application.

[0204] In embodiments in which the physiological activity of a ligand is inhibited upon binding to a ligand-binding molecule, ligand release can be detected by measuring the physiological activity of the ligand in a sample. Specifically, ligand release can be detected by measuring and comparing the physiological activity of a sample containing a ligand-binding molecule and a ligand before or after protease treatment. Ligand release can also be detected by measuring and comparing the physiological activity of a sample containing a protease, a ligand-binding molecule, and a ligand, and a sample containing a ligand-binding molecule and a ligand without protease. When a ligand-binding molecule is fused to a ligand to form a fusion protein, ligand release can be detected by measuring and comparing the physiological activity of a sample containing the fusion protein before or after protease treatment. Ligand release can also be detected by measuring and comparing the physiological activity of a sample containing a protease and the fusion protein, and a sample co...

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 of 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 according to the EU index in the non-natural human IgG CH1 domain has any one of the following characteristics: (1) the amino acid at any one or more sites from position 122 to position 128 and from position 130 to position 132 is Ala; (2) the amino acid at any one or more sites from position 122 to position 132 is Ile; (3) the amino acid at any one or more sites from position 122 to position 124 and from position 126 to position 132 is Val; (4) the amino acid at any one or more sites from position 122 to position 127 and from position 129 to position 132 is Leu; (5) the amino acid at any one or more sites from position 122 to position 125 and from position 127 to position 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 is inserted at one or more sites selected from the sites between position 121 and position 122, between position 122 and position 123, between position 123 and position 124, between position 124 and position 125, between position 125 and position 126, between position 126 and position 127, between position 127 and position 128, between position 128 and position 129, between position 129 and position 130, between position 130 and position 131, between position 131 and position 132, and between position 132 and position 133.

3. A method for detecting and / or quantifying antigen-binding molecules in a biological sample, comprising the following steps: (a) treating a biological sample containing antigen-binding molecules with a digestive enzyme to generate peptides from the antigen-binding molecules, wherein the antigen-binding molecules comprise an antigen-binding domain, a CL domain, and a non-natural human IgG CH1 domain, and 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 the 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 according to the EU index in the non-natural human IgG CH1 domain; (b) mass-analyzing the biological sample treated in (a) to detect the peptide; and (c) detecting and / or quantifying the antigen-binding molecules based on the analysis results of (b).

4. A method for detecting and / or quantifying each of multiple types of antigen-binding molecules in a biological sample, comprising the following steps: (a) treating a biological sample containing multiple types of antigen-binding molecules with a digestive enzyme to generate multiple types of peptides from the multiple types of antigen-binding molecules, wherein the multiple types of antigen-binding molecules comprise antigen-binding molecules comprising an antigen-binding domain, a CL domain, and a natural or non-natural human IgG CH1 domain, and the amino acid sequence between positions 121 and 133 according to the EU index in the human IgG CH1 domain of the multiple types of antigen-binding molecules is different among the multiple types of antigen-binding molecules, and the multiple types of peptides consist of the amino acid sequence between positions 121 and 133 according to the EU index in the natural or non-natural human IgG CH1 domain, and the amino acid sequence is different among the multiple types of peptides; (b) mass-analyzing the biological sample treated in (a) to detect each of the multiple types of peptides; and (c) detecting and / or quantifying each of the multiple types of antigen-binding molecules based on the analysis results of (b).

5. The method according to claim 4, wherein the plurality of types of peptides include (I) two or more types of peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in a non-natural human IgG CH1 domain, wherein the amino acid sequence is different 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 according to the EU index in a natural human IgG CH1 domain and one or more types of peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in a non-natural human IgG CH1 domain, wherein the amino acid sequence is different from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG.

6. A method for designing, selecting, or manufacturing a modified antigen-binding molecule comprising an artificially modified peptide moiety detectable by mass spectrometry, the method comprising the following steps: (I) identifying a peptide obtained after treating an antigen-binding molecule with a digestive enzyme, the peptide being detectable by mass spectrometry; (II) designing a peptide artificially modified by any of the following: (II-1) substituting or deleting one or more amino acid residues in the peptide identified in (I) with other amino acid residues; (II-2) inserting one or more amino acid residues into the peptide identified in (I); (II-3) adding amino acid residues to the N-terminus or C-terminus of the peptide identified in (I); (II-4) a combination of (II-1) and (II-2); (II-5) a combination of (II-1) and (II-3); (II-6) a combination of (II-2) and (II-3); (II-7) a combination of (II-1), (II-2), and (II-3); (III) performing the modification described in (II) on the peptide moiety identified in (I) in the antigen-binding molecule of (I) to produce a modified antigen-binding molecule; (IV) selecting the modified antigen-binding molecule if the modified antigen-binding molecule produced in (III) satisfies any of the following: (IV-1) the Tm value of the modified antigen-binding molecule is 50 °C or higher; (IV-2) the antigen-binding activity of the modified antigen-binding molecule is 10% or more of the antigen-binding activity of the antigen-binding molecule of (I); (IV-3) both of the above (IV-1) and (IV-2); (V) selecting the modified antigen-binding molecule if the portion corresponding to the peptide designed in (II) contained in the modified antigen-binding molecule selected in (IV) is detectable by mass spectrometry after treating a biological sample containing the modified antigen-binding molecule with the digestive enzyme of (I).

7. The method according to claim 6, wherein the amino acid residue to be inserted in (II-2) 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 produced by the method according to claim 6 or 7.

9. A peptide consisting of an amino acid sequence between positions 121 and 133 according to the EU index in a non-natural human IgG CH1 domain, wherein the amino acid sequence is different from the corresponding sequence in a natural human IgG of the same isotype as the non-natural human IgG.

10. A set comprising two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in a natural or non-natural human IgG CH1 domain, wherein the amino acid sequences are different between the two or more peptides.

11. The set according to claim 10, wherein the set comprises two or more peptides consisting of amino acid sequences between positions 121 and 133 according to the EU index in a non-natural human IgG CH1 domain, and the amino acid sequence between positions 121 and 133 according to the EU index in the non-natural human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 6 to 294 and 1565 to 1833.

12. The set includes a peptide consisting of an amino acid sequence between positions 121 and 133 according to the EU index in the natural human IgG CH1 domain, and one or more peptides consisting of an amino acid sequence between positions 121 and 133 according to the EU index in a non-natural human IgG CH1 domain. When the amino acid sequence between positions 121 and 133 according to the EU index in the natural human IgG CH1 domain is SEQ ID NO: 5, the amino acid sequence between positions 121 and 133 according to the EU index in the non-natural human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 6 to 294. When the amino acid sequence between positions 121 and 133 according to the EU index in the natural human IgG CH1 domain is SEQ ID NO: 945, the amino acid sequence between positions 121 and 133 according to the EU index in the non-natural human IgG CH1 domain is selected from the group consisting of SEQ ID NOs: 1565 to 1833. The set according to claim 10.

13. A method for detecting each of a plurality of peptides derived from a plurality of antigen-binding molecules in a biological sample by mass spectrometry, or the set according to claim 10 for use in a method for detecting and / or quantifying each of a plurality of antigen-binding molecules in a biological sample.

14. A method for administering a plurality of antigen-binding molecules each containing a peptide in the set according to any one of claims 10 to 13 to an individual or a subject in admixture.

15. A method for selecting an antigen-binding molecule, comprising the following steps: (a) detecting and / or quantifying each of the plurality of antigen-binding molecules in a biological sample derived from an individual or a subject administered with a plurality of antigen-binding molecules each containing a peptide in the set according to any one of claims 10 to 13 in admixture; and (b) selecting an antigen-binding molecule based on the detection and / or quantification results in (a).

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