Labeled polypeptide, modified polypeptide, production method for these polypeptides, reagent containing these polypeptides, and measurement method for target substance

Abstract

To provide means that makes it possible to bind a label to a polypeptide with high efficiency.SOLUTION: A linker comprising a polyethylene glycol chain with a molecular weight of 1100 or larger is used, and a glutamine residue in a polypeptide is bound to a label through the linker, thereby solving the foregoing problem.SELECTED DRAWING: Figure 8A

Description

[Technical field]

[0001] The present invention relates to a labeled polypeptide and a method for producing the same.The present invention relates to a modified polypeptide and a method for producing the same.The present invention relates to a reagent comprising a labeled polypeptide or a modified polypeptide.The present invention relates to a method for measuring a target substance. [Background technology]

[0002] Transglutaminase (TG) is an enzyme that catalyzes a reaction in which a glutamine residue in a polypeptide is used as a substrate to form an amide bond between the carboxamide side chain of the glutamine residue and the amino group of a primary amine. More specifically, in the reaction catalyzed by TG, the amino group in the carboxamide side chain of the glutamine residue in the polypeptide is condensed with the primary amine, and the substituent of the primary amine is transferred to the glutamine residue, generating ammonia. In recent years, a technique has been developed in which functional substances such as drugs and labels are bound to polypeptides containing glutamine residues using TG.

[0003] For example, Patent Document 1 describes that an antibody to which an anticancer drug is bound is obtained by reacting an antibody having a peptide tag containing a glutamine residue added to the C-terminus of the heavy chain with an anticancer drug linked to a linker having an amino group in the presence of TG. A bifunctional linker having functional groups at both ends is generally used to bind a functional substance to a polypeptide using TG. As the functional group of the bifunctional linker, an amino group (-NH2) as an amine donor in the reaction catalyzed by TG and any functional group for covalently binding a functional substance to the linker are selected. The glutamine residue in the polypeptide and the functional substance are bound via such a linker. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent Application No. 2018 / 0037921 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have attempted to bind a functional substance to a glutamine residue in a polypeptide using the linker described in Prior Art 1, but the binding did not always occur with sufficient efficiency. Therefore, an object of the present invention is to provide a means that enables a label to be bound to a polypeptide with high efficiency. [Means for solving the problem]

[0006] The present inventors discovered that by using a bifunctional linker in which a polyethylene glycol (PEG) chain having a thiol group (-SH) and an amino group has a PEG chain portion with a molecular weight of 1,100 or more, the linker and label can be efficiently bound to a glutamine residue in a polypeptide, and completed the present invention.

[0007] The present invention provides a labeled polypeptide comprising a glutamine residue having a side chain represented by the following formula (I):

[0008] [ka] (In the formula, (C) is the α carbon of a glutamine residue, X is a linear alkylene group, Y is a PEG chain, Z is a label, L is a spacer or a bond, and the molecular weight of the PEG chain is 1100 or more.)

[0009] The present invention provides modified polypeptides comprising a glutamine residue having a side chain of formula (II):

[0010] [ka] (In the formula, (C) is the α carbon of a glutamine residue, X is a linear alkylene group, and Y is a PEG chain, the molecular weight of the PEG chain being 1100 or more.)

[0011] The present invention provides a reagent comprising the above-mentioned labeled polypeptide or modified polypeptide.The present invention also provides a method for measuring a target substance, comprising the steps of forming an immune complex between the above-mentioned labeled polypeptide and a target substance, and detecting a signal generated by the label contained in the immune complex.

[0012] The present invention provides a method for producing a modified polypeptide, the method comprising the steps of: contacting a polypeptide containing a glutamine residue with a linker represented by the following formula (VI) in the presence of transglutaminase, thereby binding the linker to the carboxamide side chain of the glutamine residue; and obtaining a modified polypeptide produced by binding of the carboxamide side chain to the linker, wherein the side chain of the glutamine residue in the modified polypeptide is represented by the above formula (II).

[0013] NH2-XY-SH (VI) (In the formula, X is a linear alkylene group, Y is a PEG chain, and the molecular weight of the PEG chain is 1100 or more.)

[0014] The present invention provides a method for producing a labeled polypeptide, in which the side chain of the glutamine residue in the labeled polypeptide is represented by formula (I), comprising the steps of: contacting a polypeptide containing a glutamine residue with a linker represented by formula (IV) in the presence of transglutaminase, thereby binding the linker to the carboxamide side chain of the glutamine residue; obtaining a modified polypeptide produced by binding of the carboxamide side chain to the linker; contacting the modified polypeptide with a label having a maleimide group, thereby binding a label to the linker bound to the modified polypeptide; and obtaining a labeled polypeptide produced by binding of the modified polypeptide to the label, wherein the side chain of the glutamine residue in the labeled polypeptide is represented by formula (I). Effect of the Invention

[0015] According to the present invention, the use of a linker having an amino group and a thiol group enables the attachment of a label to a polypeptide with high efficiency. [Brief description of the drawings]

[0016] [Figure 1A] FIG. 2 is a diagram showing an example of a reagent according to the present embodiment. [Figure 1B] FIG. 2 is a diagram showing an example of a reagent kit according to the present embodiment. [Figure 2A] FIG. 1 shows the results of mass spectrometry (Infusion MS) using the infusion method for SH-PEG-NH2 linker (2K) (Sigma-Aldrich). [Figure 2B] FIG. 13 shows the results of Infusion MS for another lot of SH-PEG-NH2 linker (2K) (Sigma-Aldrich). [Figure 2C] FIG. 1 shows the results of Infusion MS of SH-PEG-NH2 linker (2K) (Biopharma PEG Scientific Inc.). [Figure 2D] FIG. 1 shows the results of Infusion MS of SH-PEG-NH2 linker (2K) (Creative PEGWorks). [Diagram 3] FIG. 13 shows the results of size-exclusion column chromatography (SEC) analysis of the product after reaction of csF001-5Fab-Qtag with SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG. [Figure 4] FIG. 13 shows the results of SEC analysis of the products after reaction of csF001-5Fab-Qtag with SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG at different pH. [Diagram 5] FIG. 13 shows the results of SEC analysis of the product after reaction of csF028-22Fab-Qtag with SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG. [Figure 6] FIG. 13 shows the results of SEC analysis of the product after reaction of csF001-5Fab-Qtag with SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) by TG. [Figure 7] FIG. 13 shows the results of SEC and non-reducing SDS-PAGE analysis of the coupling reaction solution of SH-PEG(2K)-Fab derived from csF001-5Fab-Qtag with maleimide-modified ALP. [Figure 8A] FIG. 13 shows the results of SEC analysis of the coupling reaction solution between SH-PEG(2K)-Fab derived from csF001-5Fab-Qtag and maleimide-modified ALP. [Figure 8B] FIG. 1 shows the results of non-reducing SDS-PAGE analysis of the collected fractions. [Figure 8C] FIG. 1 shows the results of SEC analysis of the collected fractions. [Figure 9] FIG. 13 shows the results of SEC analysis of the coupling reaction solution between SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag and maleimide-modified ALP. [Figure 10A] FIG. 13 shows the results of SEC analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag and maleimide-modified ALP. [Figure 10B] FIG. 13 shows the results of non-reducing SDS-PAGE analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag and maleimide-modified ALP. [Figure 11A] FIG. 1 shows the results of SEC analysis of the coupling reaction solution between sF001-5Fab′ and maleimide-modified ALP. [Figure 11B] FIG. 1 shows the results of non-reducing SDS-PAGE analysis of fractions collected from the coupling reaction solution of sF001-5Fab′ and maleimide-modified ALP. [Figure 12] FIG. 1 shows the results of SEC analysis of fractions collected from the coupling reaction solution of sF001-5Fab′ and maleimide-modified ALP. [Figure 13A] 1 is a graph showing signal values ​​when HIV-1 p24 was measured by enzyme-linked immunosorbent assay (ELISA) using Fab-PEG-ALP and (Fab')n-ALP, respectively. [Figure 13B] 1 is a graph showing noise values ​​when HIV-1 p24 was measured by ELISA using Fab-PEG-ALP and (Fab')n-ALP, respectively. [Figure 13C] 1 is a graph showing the signal / noise (S / N) ratio when HIV-1 p24 was measured by ELISA using Fab-PEG-ALP and (Fab')n-ALP, respectively. [Figure 14] 1 is a graph showing the background when human serum was measured by ELISA using Fab-PEG-ALP and (Fab')n-ALP, respectively. [Figure 15] FIG. 1 shows the results of SEC analysis of the product after reaction of HBs628Fab-Qtag with SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG. [Figure 16A] FIG. 13 shows the results of SEC analysis of the coupling reaction solution of SH-PEG(2K)-Fab derived from HBs628Fab-Qtag with maleimide-modified biotin. [Figure 16B] FIG. 13 shows the results of non-reducing SDS-PAGE analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab from HBs628Fab-Qtag with maleimide-modified biotin. [Figure 16C] FIG. 1 shows the results of SEC analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab from HBs628Fab-Qtag and maleimide-modified biotin. [Figure 17A] FIG. 1 shows the results of liquid chromatography mass spectrometry (LC-MS) of a non-reduced sample of HBs628Fab-Qtag. [Figure 17B] FIG. 1 shows the results of LC-MS of a reduced sample of HBs628Fab-Qtag. [Figure 18] FIG. 13 shows the results of LC-MS of non-reduced samples of HBs628Fab-Qtag, its SH-PEG-Fab, and biotin-PEG-Fab. [Figure 19]FIG. 1 shows the results of LC-MS of reduced samples of HBs628Fab-Qtag and its SH-PEG-Fab. [Figure 20] FIG. 1 shows the results of SEC analysis of the coupling reaction solution between SH-PEG(2K)-Fab derived from HBs628Fab-Qtag and maleimide-modified ALP. [Figure 21] FIG. 1 shows the results of non-reducing SDS-PAGE analysis of the coupling reaction solution of SH-PEG(2K)-Fab derived from HBs628Fab-Qtag with maleimide-modified ALP and each starting material. [Figure 22] FIG. 13 shows the results of SEC analysis of the product after reaction of csF001-25Fab-Qtag with SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG. [Figure 23A] FIG. 13 shows the results of SEC analysis of the coupling reaction solution of SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag with Alexa488-maleimide. [Figure 23B] FIG. 13 shows the results of confirming UV and fluorescence absorption by SEC of a fraction obtained by desalting and purifying the coupling reaction solution of SH-PEG(2K)-Fab from csF001-25Fab-Qtag and Alexa488-maleimide. [Figure 24A] FIG. 13 shows the results of LC-MS of reduced samples of csF001-25Fab-Qtag, its SH-PEG-Fab, and Alexa488-PEG-Fab. [Figure 24B] FIG. 24B is a magnified view of the analysis results shown in FIG. 24A. [Diagram 25] FIG. 13 shows the results of SEC analysis of the product after reaction of anti-CD20 Fab-Qtag with another lot of SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by TG. [Figure 26A] FIG. 1 shows the results of SEC analysis of a reaction solution of R-phycoerythrin (R-PE) and an EMCS reagent. [Figure 26B] FIG. 1 shows the results of reverse-phase HPLC analysis of a reaction solution of R-PE and an EMCS reagent. [Figure 27] This is a diagram showing the results of SEC analysis of the coupling reaction solution of SH-PEG(2K)-Fab from csF001-25Fab-Qtag with maleimide-modified R-PE. The left panel is a chromatogram at UV (280 nm). The right panel is a chromatogram at UV (280 nm) and fluorescence (excitation wavelength 565 nm / fluorescence wavelength 574 nm) for the coupling reaction solution. [Figure 28] This is a diagram showing the results of SEC analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab from sF001-25Fab-Qtag and maleimide-modified ALP. The left panel is a chromatogram at UV (280 nm). The right panel is a chromatogram at fluorescence (excitation wavelength 565 nm / fluorescence wavelength 574 nm). [Figure 29] SEC analysis of the coupling reaction solution of SH-PEG(2K)-Fab from anti-CD20 Fab-Qtag with maleimide-modified R-PE. The left panel shows a chromatogram at UV (280 nm). The right panel shows a chromatogram at UV (280 nm) and fluorescence (excitation wavelength 565 nm / fluorescence wavelength 574 nm) for the coupling reaction solution. [Diagram 30] This is a diagram showing the results of SEC analysis of fractions collected from the coupling reaction solution of SH-PEG(2K)-Fab from anti-CD20 Fab-Qtag and maleimide-modified ALP. The left panel is a chromatogram at UV (280 nm). The right panel is a chromatogram at fluorescence (excitation wavelength 565 nm / fluorescence wavelength 574 nm). [Figure 31A] FIG. 1 shows the results of measuring the interaction between csF001-25Fab-Qtag and an antigen using Biacore™ T200 (Cytiva). [Figure 31B] FIG. 1 shows the results of the interaction between SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag and an antigen, measured using Biacore™ T200. [Figure 31C]This is a diagram showing the results of measuring the interaction between csF001-25Fab-Qtag and an antigen using Biacore (trademark) T200. The measurement date is different from that in Figure 31A. [Figure 31D] FIG. 1 shows the results of measuring the interaction between Alexa488-PEG-Fab derived from csF001-25Fab-Qtag and an antigen using Biacore™ T200. [Figure 32A] FIG. 1 shows the results of ELISA measurements (without adding antigen) using Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from csF001-25Fab-Qtag. [Figure 32B] FIG. 1 shows the results of ELISA measurements (with antigen addition) using Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from csF001-25Fab-Qtag. [Diagram 33] FIG. 1 shows the results of measuring CD20-expressing cells by FCM using Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from anti-CD20 Fab-Qtag. [Figure 34A] FIG. 1 shows the results of Infusion MS of SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich). [Figure 34B] FIG. 1 shows the results of Infusion MS of SH-PEG-NH2 linker (5K) (Sigma-Aldrich). [Figure 35A] FIG. 13 shows the results of SEC analysis of the product after reaction of csF001-25Fab-Qtag with SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) by TG. [Figure 35B] FIG. 13 shows the results of SEC analysis of the product after reaction of csF001-25Fab-Qtag with SH-PEG-NH2 linker (5K) (Sigma-Aldrich) by TG. [Figure 35C]FIG. 1 shows the results of non-reducing SDS-PAGE analysis of the products after reaction of csF001-25Fab-Qtag with SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) and SH-PEG-NH2 linker (5K) (Sigma-Aldrich) by TG. [Figure 36A] FIG. 13 shows the results of SEC analysis of the coupling reaction solution of SH-PEG(3.5K)-Fab derived from csF001-25Fab-Qtag with Alexa488-maleimide. [Figure 36B] FIG. 13 shows the results of confirming UV and fluorescence absorption by SEC of a fraction obtained by desalting and purifying the coupling reaction solution of SH-PEG(3.5K)-Fab from csF001-25Fab-Qtag and Alexa488-maleimide. [Figure 37A] FIG. 13 shows the results of SEC analysis of the coupling reaction solution of SH-PEG(5K)-Fab derived from csF001-25Fab-Qtag with Alexa488-maleimide. [Figure 37B] FIG. 13 shows the results of confirming UV and fluorescence absorption by SEC of a fraction obtained by desalting and purifying the coupling reaction solution of SH-PEG(5K)-Fab from csF001-25Fab-Qtag with Alexa488-maleimide. [Figure 38] FIG. 1 shows the results of SEC analysis of the product after reaction of csF001-5Fab-Qtag with SH-PEG-NH2 linker (400 Da) (Nanocs Inc.) by TG. [Figure 39] FIG. 13 shows the results of SEC analysis of the coupling reaction solution between SH-PEG(400 Da)-Fab derived from csF001-5Fab-Qtag and maleimide-modified ALP. [Diagram 40] FIG. 1 shows the results of SEC analysis of the product after reaction of csF001-5Fab-Qtag with SH-PEG-NH2 linker (1K) (Creative PEGWorks) by TG. [Diagram 41]FIG. 13 shows the results of SEC analysis of the coupling reaction solution between SH-PEG(1K)-Fab derived from csF001-5Fab-Qtag and maleimide-modified ALP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The labeled polypeptide of this embodiment contains a glutamine residue having a side chain represented by the above formula (I). In the labeled polypeptide, the glutamine residue in the polypeptide before the label is added is bound to the label via a bifunctional linker having a PEG chain. Specifically, the labeled polypeptide can be obtained by binding a PEG chain having a thiol group and an amino group as a bifunctional linker to the glutamine residue of the polypeptide using a reaction catalyzed by TG, and then reacting the thiol group of the linker with a label having a maleimide group (for details, see the explanation of the method for producing the labeled polypeptide described below).

[0018] In formula (I), (C) is the α carbon of the glutamine residue in the labeled polypeptide. The side chain represented by formula (I) has a structure in which a linker and a label are bound to the side chain of the glutamine residue. The labeled polypeptide of this embodiment is a polypeptide in which a glutamine residue that can be a substrate for TG in a polypeptide before the label is added is modified to a glutamine residue having a side chain represented by the above formula (I). That is, the amino acid sequence of the labeled polypeptide is the same as that of the polypeptide before the label is added, except that the labeled polypeptide contains a glutamine residue having a side chain represented by the above formula (I). In the labeled polypeptide, the type of polypeptide is not particularly limited. For example, the labeled polypeptide may be any protein such as an antibody, an antigen, a ligand, or a receptor. Among them, an antibody is preferable.

[0019] In the present specification, the term "antibody" also includes antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, domain antibodies (dAb), reduced IgG (rIgG), light chain, heavy chain antibodies, variable regions of heavy chain antibodies (VHH), diabodies, triabodies, and the like. The antibody may be either a monoclonal antibody or a polyclonal antibody. The origin of the antibody is not particularly limited, and it may be an antibody derived from an animal such as a mouse, rat, hamster, rabbit, goat, horse, camel, alpaca, chicken, ostrich, or shark. The antibody isotype may be any of IgG, IgM, IgE, IgA, and the like, but is preferably IgG. The antibody that specifically binds to the tag may be a commercially available antibody or an antibody prepared by a method known in the art.

[0020] In a polypeptide before being labeled, the glutamine residue that can be a substrate for TG can be, for example, a glutamine residue present on the surface of the polypeptide with a carboxamide side chain facing outward from the polypeptide. Alternatively, a peptide tag containing a glutamine residue (hereinafter also referred to as a "Q tag") may be added to a polypeptide before being labeled. The Q tag contains a glutamine residue in a predetermined amino acid sequence, so that the glutamine residue can be a substrate for TG. In general, it is preferable to add a Q tag, since not all glutamine residues in a polypeptide are substrates for TG. The Q tag itself is publicly known, and is described, for example, in Patent Document 1. The number of amino acid residues contained in the Q tag is preferably 3 or more, more preferably 4 or more. The number of amino acid residues contained in the Q tag is preferably 20 or less, more preferably 15 or less, and most preferably 10 or less. One or two of the amino acid residues in the amino acid sequence of the Q tag are glutamine residues. From the viewpoint of protecting the Q tag from cleavage by peptidase, it is preferable that the amino acid residue at the C-terminus of the Q tag is a proline residue. Examples of Q tags include peptide tags having amino acid sequences such as GVLNLAQSP (SEQ ID NO: 1), GLLQGP (SEQ ID NO: 2), and LLQGP (SEQ ID NO: 3).

[0021] The position of the Q tag in the polypeptide before the label is added is not particularly limited, but is preferably the N-terminus or C-terminus of the polypeptide, more preferably the C-terminus of the polypeptide. When the polypeptide is, for example, a full-length antibody, Fab, Fab' or F(ab')2, the Q tag can be added to the C-terminus of the heavy or light chain. The method of adding the Q tag to the polypeptide is not particularly limited. For example, the Q tag may be covalently bonded to the polypeptide using a crosslinker or linker. Alternatively, a fusion polypeptide of the polypeptide and the Q tag may be prepared by a known gene recombination method.

[0022] Preferably, the labeled polypeptide is a fusion polypeptide between a polypeptide and a peptide tag containing a glutamine residue having a side chain represented by the above formula (I). That is, the labeled polypeptide may be a polypeptide to which a Q tag has been added, in which a glutamine residue capable of serving as a TG substrate in the Q tag has been modified to a glutamine residue having a side chain represented by the above formula (I). More preferably, the labeled polypeptide is a fusion polypeptide between an antibody and a peptide tag containing a glutamine residue having a side chain represented by the above formula (I).

[0023] In the above formula (I), "(C)-(CH2)2-(C=O)-" is derived from the carboxamide side chain of the glutamine residue in the polypeptide, and "-NH-XYS-" is derived from a PEG chain having a thiol group and an amino group, which are bifunctional linkers. Here, X is a linear alkylene group, and the number of carbon atoms in the linear alkylene group is preferably 2 to 10, more preferably 2 to 8, and most preferably 2 to 6. With respect to X, it is preferred that the linear alkylene group does not have a substituent. More preferably, X is an unsubstituted linear alkylene group having a carbon number of 2 to 6.

[0024] In the above formula (I), Y is a PEG chain having a molecular weight of 1100 or more. Since PEG is a polymer, the PEG having a thiol group and an amino group used in the preparation of the labeled polypeptide can be an aggregate of a plurality of molecules having a range of molecular weights. Therefore, the molecular weight of the labeled polypeptide prepared from such a PEG has a range. In this specification, "the molecular weight of the PEG chain is 1100 or more" means that the minimum molecular weight of the PEG chain portion in the labeled polypeptide is 1100. With respect to Y, the lower limit of the molecular weight of the PEG chain is preferably 1200, 1300, 1400 or 1500. With respect to Y, the upper limit of the molecular weight of the PEG chain is, for example, 20000, 10000, 7500, 7000, 6500, 6000 or 5500. The molecular weight of the PEG chain can be determined, for example, by analyzing the PEG chain having a thiol group and an amino group as a bifunctional linker by mass spectrometry before binding to the polypeptide. Examples of mass spectrometry include infusion MS, LC-MS, time-of-flight mass spectrometry (TOF-MS), and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). Mass spectrometry conditions can be appropriately determined. For example, when analyzing by infusion MS, the detailed conditions are as described in the Examples below.

[0025] The most abundant molecular weight among the results of the molecular weight analysis of the PEG chain measured by mass spectrometry is called the "most abundant molecular weight". The lower limit of the most abundant molecular weight of the PEG chain for Y is, for example, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, or 2050. The upper limit of the most abundant molecular weight of the PEG chain for Y is, for example, 18000, 15000, 12000, 9000, 8000, 7000, 6000, 5000, 4000, 3500, or 3000. The weight average molecular weight of the PEG chain can also be calculated based on the results of mass spectrometry. For example, when a PEG chain having a thiol group and an amino group as a bifunctional linker is analyzed by Infusion MS before binding to a polypeptide, the weight-average molecular weight of the PEG chain can be calculated from the molecular weight value and intensity of the most intense isotope molecular peak. Details of the conditions for analysis by Infusion MS are as described in the Examples below. With respect to Y, the weight-average molecular weight of the PEG chain is preferably 1300 or more, more preferably 1700 or more. With respect to Y, the lower limit of the weight-average molecular weight of the PEG chain is, for example, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or 2050. With respect to Y, the upper limit of the weight average molecular weight of the PEG chain is preferably lower than 20,000, for example 15,000, 12,000, 10,000, 8,000, 7,000, 6,000, 5,000, 4,500, 4,000, 3,500, 3,000 or 2,500.

[0026] The structure of Y is represented by the following formula (III): In formula (III), n is an integer of 25 or more, and preferably an integer of 39 or more.

[0027] -(OCH2CH2) n -or-(CH2CH2O) n - (III)

[0028] In the above formula (III), the lower limit of n is preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41. More preferably, the lower limit of n is 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. The upper limit of n is, for example, 455, 228, 171, 159, 147, 136, 125, 113, 102, 90, 79, 68, or 56.

[0029] In the side chain represented by the above formula (I), the portion of the structure represented by the following formula (IV) is derived from a label having a maleimide group. Z is a label, and L is a spacer or a bond that bonds the nitrogen atom of the imide ring to Z. With respect to L, the bond means that the nitrogen atom of the imide ring is directly bonded to Z without any other atom therebetween.

[0030] [ka]

[0031] A label is a substance that can be added to a polypeptide and is synonymous with a "labeling substance." A label can be a substance capable of specific detection, or a substance that generates a signal by itself (hereinafter also referred to as a "signal generating substance"), or a substance that catalyzes the reaction of another substance to generate a signal. The substance capable of specific detection is not particularly limited, as long as a substance capable of specifically binding to the substance exists or can be obtained. Examples of the substance capable of specific detection include biotins, haptens, and oligonucleotides. Biotins specifically bind to avidins. Examples of haptens include 2,4-dinitrophenyl (DNP) groups. DNP specifically binds to anti-DNP antibodies. An oligonucleotide specifically binds to an oligonucleotide having a sequence complementary to its base sequence.

[0032] As used herein, "biotins" includes biotin and its analogues. Examples of biotin analogues include desthiobiotin and biocytin. As used herein, "avidins" includes avidin and its analogues. Examples of avidin analogues include streptavidin, avidin-like protein derived from Pleurotus cornucopiae (Tamavidin (registered trademark)), bladdavidin, and rhizavidin.

[0033] Examples of signal generating substances include fluorescent substances, radioisotopes, chemiluminescent substances, etc. Examples of substances that catalyze the reaction of other substances to generate a detectable signal include enzymes. Enzymes generate signals such as light, color, etc. by reacting with an appropriate substrate. Examples of enzymes include alkaline phosphatase (ALP), peroxidase, β-galactosidase, luciferase, etc. Examples of fluorescent substances include fluorescent dyes such as Alexa Fluor (registered trademark), fluorescein isothiocyanate (FITC), rhodamine, etc., fluorescent proteins such as GFP, etc. Examples of radioisotopes include 125 I, 14 C. 32 P, 99m Tc, 225 Examples of chemiluminescent substances include ruthenium pyridine complexes and acridinium esters. Since it is difficult to bind a radioisotope itself as a label, a compound containing a radioisotope and having a maleimide group may be used as a label. For example, 125 I, 14 C or 32 Examples of the nucleic acid include nucleic acids, sugars, and oligopeptides that contain P and have a maleimide group added thereto. 99m Tc, 225 A maleimide derivative in which a maleimide group is added to a chelating agent capable of coordinating a metal element such as Ac may be used. 99m Tc, 225 The maleimide derivative can be used as a signal generating substance by coordinating Ac etc. An example of a chelating agent is deferoxamine.

[0034] Preferred labels are biotins, enzymes, fluorescent dyes, fluorescent proteins and haptens. As an enzyme, ALP is particularly preferred.

[0035] In the above formula (I), when L is a spacer connecting the nitrogen atom of the imide ring and Z, L is, for example, -(CH2) n -R-(C=O)-NH-, -(CH2) n -R-NH-(C=O)-, -(CH2) n -R-(C=O)-, -(CH2) n -R-(C=O)-O-, -(CH2) n -RO-(C=O)-, -(CH2) n -R-(C=S)-NH-, -(CH2) n -R-NH-(C=S)-, -(CH2) n -RO-, -(CH2) n -OR-, -(CH2) n -RS- or -(CH2) n It is represented by -SR-, where n is an integer of 1 or more and 10 or less.

[0036] Each R is independently a bond, an alkylene group having from 1 to 10 carbon atoms which may have a substituent, an arylene group or heteroarylene group having from 6 to 12 carbon atoms which may have a substituent, a cycloalkylene group or heterocycloalkylene group having from 3 to 8 carbon atoms which may have a substituent, or a combination thereof. With regard to R, a bond means that it is directly bonded without any other atom therebetween.

[0037] When R is an alkylene group having 1 to 10 carbon atoms, examples of such an alkylene group include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene, 2-ethylhexylene, nonylene, and decylene. Among these, an alkylene group having 1 to 4 carbon atoms is preferred. When R is an alkylene group having a substituent, the number of carbon atoms in the above does not include the number of carbon atoms of the substituent.

[0038] When R is an arylene group or a heteroarylene group, such a group may be an aromatic ring having 6 to 12 carbon atoms, which may contain one or more heteroatoms selected from N, S, O, and P. Examples of such groups include phenylene, naphthylene, biphenylylene, furanylene, pyrrolene, thiophenylene, triazolene, oxadiazolene, pyridylene, and pyrimidylene. When R is an arylene group or a heteroarylene group having a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.

[0039] When R is a cycloalkylene group or a heterocycloalkylene group, such a group may be a non-aromatic ring having 3 to 8 carbon atoms, which may contain one or more heteroatoms selected from N, S, O, and P. Examples of such groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, pyrrolidinylene, piperidinylene, piperazinylene, and morpholinylene. When R is a cycloalkylene group or a heterocycloalkylene group having a substituent, the number of carbon atoms in the above does not include the number of carbon atoms of the substituent.

[0040] Examples of the substituent in R include hydroxy, cyano, alkoxy, nitro, =O, =S, halogen, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether. R may have a plurality of substituents. Alkoxy refers to an -O-alkyl group, and this alkyl group is a linear or branched saturated aliphatic hydrocarbon group having 1 to 5 carbon atoms, preferably 1 or 2 carbon atoms.

[0041] In one molecule of the labeled polypeptide, the glutamine residue having the side chain represented by the above formula (I) may be one or more. In addition, multiple molecules of the polypeptide may share one molecule of the label, and each polypeptide may form a side chain represented by the above formula (I). In this case, the labeled polypeptide of formula (I) contains multiple structures in which "(C)-(CH2)2-(C=O)-NH-XYS-" and the above formula (IV) are linked to one Z. One example is a labeled polypeptide represented by the following formula (V). In formula (V), the definitions of (C), X, Y, Z, and L are the same as those in formula (I).

[0042] [ka]

[0043] The complex represented by formula (V) is formed by binding two molecules of the modified polypeptide described below to one molecule of a label having two maleimide groups. The complex represented by formula (V) is an example of a labeled polypeptide represented by formula (I). The labeled polypeptide may be, for example, a complex formed by binding three or more molecules of the modified polypeptide to one molecule of a label having three or more maleimide groups. Such a complex is prepared by binding one molecule of a label having multiple maleimide groups to multiple molecules of the polypeptide via a PEG chain having a thiol group and an amino group.

[0044] In the labeled polypeptide of this embodiment, the polypeptide and the label are separated by a PEG chain having a molecular weight of 1100 or more. This has the advantage that, for example, when the polypeptide is an antibody, the label is prevented from binding nonspecifically to the antibody. In addition, since the PEG chain having a molecular weight of 1100 or more has a flexible structure, when the polypeptide is an antibody, the antibody swings, which has the advantage that the target substance, the antigen, is easily captured. In addition, as shown in Experimental Example 8 and its results described below, when the labeled polypeptide of this embodiment, in which the polypeptide is an antibody, is used to detect an antigen in serum, the background is suppressed. This is thought to be due to the fact that the PEG chain having a molecular weight of 1100 or more is hydrated in a sample containing water, and therefore nonspecific binding of serum-derived impurities to the labeled polypeptide is suppressed.

[0045] The modified polypeptide of this embodiment contains a glutamine residue having a side chain represented by the above formula (II). In the modified polypeptide, the glutamine residue of the polypeptide is bound to a PEG chain having a thiol group. Specifically, the modified polypeptide can be obtained by binding a PEG chain having a thiol group and an amino group as a bifunctional linker to the glutamine residue of the polypeptide using a reaction catalyzed by TG (for details, see the description of the method for producing the modified polypeptide described below).

[0046] When the modified polypeptide is contacted with a label having a maleimide group, the thiol group of the modified polypeptide reacts with the maleimide group of the label to produce a labeled polypeptide. In other words, the modified polypeptide is an intermediate for obtaining the labeled polypeptide of the present embodiment, which is the final product.

[0047] In formula (II), (C) is the α carbon of the glutamine residue in the modified polypeptide. The side chain represented by formula (II) has a structure in which a bifunctional linker having a PEG chain structure having a thiol group and an amino group is bound to the side chain of the glutamine residue. The modified polypeptide of this embodiment is a polypeptide in which a glutamine residue that can be a substrate for TG in a polypeptide before the linker is bound is modified to a glutamine residue having a side chain represented by the above formula (II). That is, the amino acid sequence of the modified polypeptide is the same as that of the polypeptide before the linker is bound, except that it contains a glutamine residue having a side chain represented by the above formula (II). In the modified polypeptide, the type of polypeptide is not particularly limited, and is the same as that described for the labeled polypeptide. That is, the modified polypeptide can be any protein such as an antibody, an antigen, a ligand, or a receptor. Among them, an antibody is preferable.

[0048] The details of X and Y in formula (II) are the same as those described for the labeled polypeptide. In one molecule of the modified polypeptide, the number of glutamine residues having a side chain represented by the above formula (II) may be one or more.

[0049] As described above, thiol groups are generally easily oxidized, and disulfide bonds are easily formed between molecules having thiol groups. Therefore, when preparing a labeled polypeptide, the formation of disulfide bonds can be suppressed by adding a protecting group such as an acetyl group to the thiol group of the modified polypeptide. On the other hand, modified polypeptides have the advantage that the thiol groups at the very ends are not easily oxidized, and disulfide bonds are hardly formed between the modified polypeptides. This is thought to be because the hydration of the PEG chains having a molecular weight of 1100 or more in the modified polypeptide improves the dispersibility of the modified polypeptide, making it difficult for the thiol groups to come into contact with each other. Due to the above advantages, it is not necessarily necessary to protect the thiol groups in the modified polypeptide in advance. In other words, when the modified polypeptide is used, operations such as thiol group protection and removal of protecting groups are not required, and the preparation work of the labeled polypeptide can be simplified.

[0050] A further embodiment of the present invention relates to a method for producing a modified polypeptide, which comprises contacting a polypeptide containing a glutamine residue with a linker represented by formula (VI) above in the presence of TG, thereby attaching the linker to the carboxamide side chain of the glutamine residue.

[0051] TG itself is a known enzyme and is commercially available. TG may be a natural enzyme extracted and purified from a living organism or a biological sample, or an enzyme obtained by a genetic recombination method. The origin of TG is not particularly limited, and TG derived from any organism may be used. Preferably, TG derived from a microorganism (e.g., Streptomyces mobaraensis) is used.

[0052] The polypeptide is not particularly limited as long as it contains a glutamine residue that can be a substrate for TG. For example, the polypeptide can be selected from any protein such as an antibody, an antigen, a ligand, or a receptor. Among them, an antibody is preferred. The polypeptide containing a glutamine residue that can be a substrate for TG can be a polypeptide with the above-mentioned Q tag added. A preferred polypeptide is a fusion polypeptide of an antibody and a Q tag.

[0053] The linker represented by the above formula (VI) is a bifunctional linker having a structure of a PEG chain having a thiol group and an amino group. Details of X and Y in formula (VI) are the same as those described for X and Y in formula (I) for the labeled polypeptide of this embodiment. The linker represented by formula (VI) itself is known and commercially available. The molecular weight of the PEG chain can be examined by mass spectrometry. Details of the mass spectrometry and its conditions are as described above. When a commercially available linker is used, the molecular weight of the PEG chain may be a value disclosed by the manufacturer or supplier. In that case, the molecular weight of the disclosed linker is preferably 2000 or more.

[0054] It is considered that the linker represented by the above formula (VI) has improved dispersibility due to hydration of the PEG chain having a molecular weight of 1100 or more, and the thiol groups are unlikely to come into contact with each other. Therefore, the terminal thiol group of the linker is unlikely to be oxidized, and disulfide bonds are hardly formed between modified polypeptides. Therefore, in the step of binding the above polypeptide to the linker represented by formula (VI), it is not necessary to protect the thiol group of the linker in advance. In addition, since there is almost no loss of the thiol group due to the formation of a disulfide bond, the reaction between the modified polypeptide and the label having a maleimide group can be carried out quantitatively and efficiently.

[0055] The linker represented by the above formula (VI) may be in a free form or a salt with an inorganic acid (e.g., hydrochloric acid). Preferably, the linker represented by the above formula (VI) is a salt with an inorganic acid (e.g., hydrochloric acid). As described above, since the linker has a PEG chain with a molecular weight of 1100 or more, disulfide bonds between linkers are unlikely to occur even in a free form. When the linker is a salt with an inorganic acid, the dispersibility of the linker can be improved not only by hydration of the PEG chain but also by electrostatic interaction.

[0056] The contact of the polypeptide containing glutamine residues with the linker represented by the above formula (VI) in the presence of TG is preferably carried out in a suitable aqueous medium. For example, a solution of TG, a solution of the polypeptide, and a solution of the linker can be mixed to bring them into contact. The order of mixing is not particularly limited. Examples of the aqueous medium include water, physiological saline, and buffer solutions. Examples of the buffer solution include phosphate buffered saline (PBS), Tris-HCl, and Good's buffer. When TG derived from a microorganism is used, the pH of the aqueous medium is preferably near neutral (pH 6.5 to 8.5), more preferably pH 8 to 8.5. When dimethyl ester is added to the aqueous solvent, the pH of the aqueous medium is preferably near neutral (pH 6.5 to 8.5), more preferably pH 8 to 8.5. The concentration of DMSO in the aqueous solvent containing TG is preferably 20 w / v % or less, more preferably 10 w / v % or less.

[0057] In the above contact, it is preferable to incubate the solution containing the TG, the polypeptide, and the linker obtained by the above mixing under conditions in which the reaction catalyzed by the TG proceeds. Such conditions themselves are known. For example, the temperature is 4°C or higher and 37°C or lower, preferably 20°C or higher and 37°C or lower. The incubation time can be appropriately determined depending on the temperature, for example, 1 hour or higher and 24 hours or lower. When the contact is performed at a low temperature of 10°C or lower, it is preferable to extend the time (for example, 8 hours or more). In the above contact, the amino group of the carboxamide side chain of the glutamine residue in the polypeptide is exchanged with the amino group in the linker by the action of the TG to form an amide bond, and the linker is bound to the side chain. This produces the modified polypeptide of this embodiment.

[0058] The modified polypeptide produced by binding of the carboxamide side chain to the linker is then obtained. For example, the solution containing the TG, polypeptide, and linker after the enzymatic reaction may be purified by SEC or affinity chromatography using an appropriate column to remove the enzyme and unreacted components to obtain the modified polypeptide. The unreacted components are, for example, the polypeptide that has not been bound to the linker and the linker that has not been bound to the polypeptide. If necessary, the solution containing the obtained modified polypeptide may be desalted and concentrated.

[0059] A further embodiment of the present invention relates to a method for producing a labeled polypeptide. In this method, a polypeptide containing a glutamine residue is first contacted with a linker represented by formula (VI) in the presence of TG, thereby binding the linker to the carboxamide side chain of the glutamine residue. Then, a modified polypeptide produced by binding the carboxamide side chain to the linker is obtained. Details of these steps are the same as those described for the method for producing a modified polypeptide. Details of the labeling are the same as those described for the labeled polypeptide.

[0060] The obtained modified polypeptide is contacted with a label having a maleimide group, and the label is bound to the linker bound to the modified polypeptide. Since the modified polypeptide has a side chain represented by the above formula (II), the thiol group of the side chain reacts with the maleimide group of the label. This forms a thioether bond, and the label is bound to the end of the linker bound to the modified polypeptide.

[0061] The label having a maleimide group preferably has a structure represented by the following formula (VII): In formula (VII), Z is a label, L is a spacer or a bond, and R 1 and R 2 are the same or different and are a hydrogen atom or a bromine atom. 1 and R 2 are both hydrogen atoms or both bromine atoms. The details of L and Z are the same as those described for formula (I) for the labeled polypeptide of this embodiment. 1 and / or R 2 is a bromine atom, the label having the structure represented by the following formula (VII) is a label having a 3,4-dibromomaleimide group, which is a maleimide derivative, or a 3-bromomaleimide group. In this specification, the term "maleimide group" also includes a 3,4-dibromomaleimide group and a 3-bromomaleimide group.

[0062] [ka]

[0063] The label having the structure represented by the above formula (VII) can be prepared by binding the label to a bifunctional linker having a maleimide group and a specific reactive group. Such bifunctional linkers themselves are known and commercially available. The specific reactive group can be appropriately determined depending on the functional group of the label. For example, when the label has an amino group, a bifunctional linker having an N-hydroxysuccinimide (NHS) ester and a maleimide group can be used. In this case, the amino group of the label reacts with the NHS ester of the bifunctional linker, and the linker is bonded to the label to obtain a label having the structure represented by formula (VII). Alternatively, the label having the structure represented by formula (VII) may be a commercially available label.

[0064] A label having a 3,4-dibromomaleimide group as a maleimide group can be prepared, for example, according to the following reaction scheme (see Morais M. et al., Bioconjugatin, Methods and Protocols (Methods Mol Biol, 2019, 2033 SpringerLink), Chapter 2, pp.15-24). In the following reaction scheme, a label having a structure represented by formula (VIII) is obtained as a label having a maleimide group. In the following reaction scheme and formula (VIII), AcOH represents acetic acid, DBM-C2-acid represents an intermediate, EEDQ represents 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as a condensing agent, MeCN represents acetonitrile, and Z is a label.

[0065] [ka]

[0066] One 3,4-dibromomaleimide group can react with each of two thiol groups to form a thioether bond. Therefore, by using a label having a 3,4-dibromomaleimide group, for example, two molecules of modified polypeptide can be bound to one molecule of label. That is, the modified polypeptide is dimerized via the 3,4-dibromomaleimide group to obtain a complex of two molecules of labeled polypeptide. For example, when two molecules of modified polypeptide are bound to one molecule of a label having a structure represented by formula (VIII), a complex of labeled polypeptide represented by the following formula (IX) is obtained. In formula (IX), the definitions of (C), X, Y, Z, and L are the same as those in formula (I).

[0067] [ka]

[0068] The modified polypeptide and the label having a maleimide group are preferably contacted in a suitable aqueous medium. For example, the modified polypeptide and the label having a maleimide group can be contacted by mixing the solutions. The aqueous medium is preferably a buffer solution, such as triethanolamine buffer, PBS, Tris-HCl, Good's buffer, etc. The pH of the aqueous medium is preferably 6.5 or more and 7.5 or less.

[0069] In the above contact, it is preferable to incubate the solution containing the modified polypeptide and the label obtained by the above mixing under conditions in which the reaction between the thiol group and the maleimide group proceeds. Such conditions themselves are known. For example, the temperature is 5°C or higher and 37°C or lower, preferably 20°C or higher and 30°C or lower. The incubation time can be appropriately determined depending on the temperature, for example, 2 hours or higher and 6 hours or lower. When the contact is performed at a low temperature of 10°C or lower, it is preferable to extend the time (for example, 16 hours or longer). By the above contact, the thiol group of the modified polypeptide and the maleimide group of the label form a thioether bond to produce a labeled polypeptide.

[0070] Then, the labeled polypeptide produced by binding between the modified polypeptide and the label is obtained. For example, the solution containing the modified polypeptide and the label after the reaction may be purified by SEC using an appropriate column to remove unreacted components to obtain the labeled polypeptide. The unreacted components are, for example, the label that has not bound to the modified polypeptide and the modified polypeptide that has not bound to the label. If necessary, the solution containing the obtained labeled polypeptide may be desalted and concentrated.

[0071] In a further embodiment, a label having a haloacetyl group may be used instead of a label having a maleimide group to bind the label to the modified polypeptide (see Greg T. Hermanson, Bioconjugate Techniques Third Edition, Academic Press, Chapter 3 The reaction of Bioconjugation, pp.240-241, 2. Thiol reactions, 2.1 Haloacetyl and alkyl halide derivatives). For example, as shown in the following reaction scheme, a modified polypeptide and a label having a haloacetyl group represented by formula (X) (wherein R 3 is a bromine atom or an iodine atom), a labeled polypeptide represented by the following formula (XI) is obtained. In the following reaction scheme, the definitions of (C), X, Y, Z and L are the same as in formula (I).

[0072] [ka]

[0073] A further embodiment of the present invention relates to a reagent comprising a labeled polypeptide or a modified polypeptide (hereinafter also referred to as the "reagent of this embodiment"). When the polypeptide is an antibody, the reagent of this embodiment comprising a labeled polypeptide is used in the measurement method of this embodiment described below. The reagent of this embodiment comprising a modified polypeptide is used in a reaction to bind with any label having a maleimide group to obtain a labeled polypeptide. Details of the labeled polypeptide and the modified polypeptide are as described above.

[0074] The reagent of this embodiment may be provided to a user with the labeled polypeptide or modified polypeptide contained in a container. An example of the reagent of this embodiment is shown in FIG. 1A. Referring to FIG. 1A, 10 shows the reagent of this embodiment contained in a container. The labeled polypeptide or modified polypeptide in the reagent may be a solid (e.g., a powder, a crystal, a lyophilized product, etc.) or a liquid (e.g., a solution, a suspension, an emulsion, etc.). When the labeled polypeptide or modified polypeptide is contained in the reagent in a liquid form, examples of the solvent include the above-mentioned aqueous media. If necessary, a stabilizer such as casein or BSA may be added to the aqueous medium.

[0075] A further embodiment of the present invention relates to a reagent kit (hereinafter also referred to as "the reagent kit of this embodiment") comprising a reagent containing a labeled polypeptide or a modified polypeptide. When the polypeptide is an antibody, the reagent kit of this embodiment comprising a reagent containing a labeled polypeptide is used in the measurement method of this embodiment described below. The reagent of this embodiment comprising a reagent containing a modified polypeptide is bound to any label having a maleimide group and used in a reaction to obtain a labeled polypeptide. Details of the labeled polypeptide and the modified polypeptide are as described above.

[0076] The reagent kit of this embodiment may be provided to a user, for example, by packaging a container containing a reagent including a labeled polypeptide or a modified polypeptide in a box. A package insert may be included in the box. The package insert may describe the composition of the reagent, the structure of the labeled polypeptide or the modified polypeptide, how to use the reagent, how to store the reagent, and the like. An example of the reagent kit of this embodiment is shown in FIG. 1B. With reference to FIG. 1B, 11 indicates the reagent kit of this embodiment, 12 indicates a container containing a reagent including a labeled polypeptide or a modified polypeptide, 13 indicates a packaging box, and 14 indicates the package insert.

[0077] A further embodiment of the present invention relates to a method for measuring a target substance using the labeled polypeptide of this embodiment (hereinafter, also referred to as the "measurement method of this embodiment"). The labeled polypeptide used in the measurement method of this embodiment is a fusion polypeptide of an antibody and a peptide tag containing a glutamine residue having a side chain represented by the above formula (I) (hereinafter, also referred to as the "labeled antibody of this embodiment"). The labeled antibody of this embodiment is an antibody that has a label and specifically binds to a target substance, and is used as a detection antibody in the measurement method of this embodiment. The detection antibody refers to an antibody that binds to a target substance and provides a detectable signal via the label. The label is preferably a substance that catalyzes the reaction of a signal generating substance or other substance to generate a detectable signal. More preferably, the label is an enzyme, a fluorescent dye, or a fluorescent protein. The details of the labeled polypeptide of this embodiment are as described above.

[0078] In the measurement method of the present embodiment, first, an immune complex is formed between a labeled antibody and a target substance. The target substance may be a substance recognized by the labeled antibody, or may be a solid component containing such a substance. Examples of the substance recognized by the labeled antibody include proteins, oligopeptides, nucleic acids, lipids, sugar chains, and haptens. Examples of the solid components containing the substance recognized by the labeled antibody include cells, extracellular vesicles, microorganisms, viruses, and fragments thereof. Examples of the extracellular vesicles include exosomes, ectosomes, microvesicles, microparticles, and apoptotic bodies. Examples of the microorganisms include bacteria and fungi.

[0079] The immune complex can be formed by mixing a sample that may contain a target substance with a labeled antibody. The type of sample is not particularly limited, and examples include biological samples such as blood, plasma, serum, lymph, and saliva, excrement such as urine and feces, and environmental samples such as river water, seawater, and soil. The formation of the immune complex is preferably carried out in a solution. Therefore, the sample that may contain the target substance is preferably in a liquid state. The liquid sample is not limited to a solution, and includes a suspension, a sol, and the like. When the sample is not in a liquid state, for example, a suitable aqueous medium may be added to the sample to make it liquid. When the liquid sample contains insoluble impurities, the impurities may be removed from the sample by known means such as centrifugation and filtration. If necessary, the liquid sample may be diluted with the above-mentioned aqueous medium. It is preferable that the labeled antibody is also in a liquid form using a suitable aqueous medium. The aqueous medium is as described above.

[0080] Preferably, the immune complex is formed by forming an immune complex between a labeled antibody and a target substance on a solid phase. For example, a sample that may contain a target substance is mixed with a labeled antibody to form an immune complex, and then a solution containing the immune complex is brought into contact with a solid phase capable of immobilizing the labeled antibody or the target substance. This allows the immune complex to be formed on the solid phase. Alternatively, the target substance may be immobilized on a solid phase in advance, and the immune complex may be formed on the solid phase by contacting the solid phase with the labeled antibody.

[0081] The temperature and incubation time conditions for mixing are not particularly limited as long as they are suitable for the antigen-antibody reaction. Such conditions themselves are known. For example, the temperature is 4°C or higher and 40°C or lower, preferably 20°C or higher and 37°C or lower. The incubation time can be appropriately determined depending on the temperature, and is, for example, 1 minute or longer and 24 hours or shorter. When contact is performed at a low temperature of 10°C or lower, it is preferable to extend the time (for example, 1 hour or longer).

[0082] In the measurement method of this embodiment, in addition to the labeled antibody, a capture antibody that specifically binds to the target substance may be used. The capture antibody refers to an antibody that captures the target substance on the solid phase by being fixed to the solid phase. The capture antibody preferably recognizes a different epitope from the labeled antibody. When the target substance has a plurality of the same epitopes, the epitopes recognized by the capture antibody and the labeled antibody may be the same. When the capture antibody is further used, a sandwich immune complex is formed between the labeled antibody, the target substance, and the capture antibody. The sandwich immune complex refers to a complex containing the capture antibody, the target substance, and the labeled antibody, in which the capture antibody and the labeled antibody are bound to different sites on the target substance.

[0083] The sandwich immune complex can be formed by mixing a capture antibody, a sample that may contain a target substance, and a labeled antibody. In a preferred embodiment, a sandwich immune complex of the capture antibody, the target substance, and the labeled antibody is formed on a solid phase. For example, a sample that may contain a target substance, a capture antibody, and a labeled antibody are mixed to form a sandwich immune complex, and then a solution containing the immune complex is contacted with a solid phase that can immobilize the capture antibody. This allows the sandwich immune complex to be formed on the solid phase. Alternatively, a capture antibody that has been immobilized on a solid phase in advance may be used. That is, a sandwich immune complex can be formed on the solid phase by mixing a capture antibody immobilized on a solid phase, a sample that may contain a target substance, and a labeled antibody. The temperature and incubation time conditions for mixing are as described above.

[0084] The solid phase may be an insoluble carrier capable of immobilizing a target substance or a capture antibody. Preferably, the solid phase is an insoluble carrier capable of immobilizing a capture antibody. For example, the capture antibody can be immobilized on the solid phase by directly or indirectly binding the solid phase to the capture antibody. Examples of direct binding between the solid phase and the capture antibody include adsorption or covalent binding to the solid phase surface by hydrophobic interaction. For example, when the solid phase is an ELISA microplate, the capture antibody can be immobilized in the well of the plate by adsorption. In addition, when the solid phase has a functional group on its surface, the capture antibody can be immobilized on the solid phase surface by covalent binding using the functional group. For example, when the solid phase is a particle having a carboxyl group, the carboxyl group on the particle surface is activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC) and then reacted with NHS to form an NHS ester. When the particles having the NHS ester are brought into contact with the capturing antibody, the NHS ester reacts with the amino group of the capturing antibody, and the capturing antibody is immobilized on the particle surface by a covalent bond.

[0085] An example of the indirect binding between the solid phase and the capture antibody is a bond via a molecule that specifically binds to the capture antibody. The capture antibody can be immobilized on the solid phase by immobilizing such a molecule on the surface of the solid phase in advance. Examples of molecules that specifically bind to the capture antibody include protein A, protein G, and an antibody (secondary antibody) that specifically recognizes the capture antibody. The capture antibody and the solid phase can also be bound to each other using a combination of substances that are present between the capture antibody and the solid phase. Examples of such combinations of substances include combinations of biotins and avidins, haptens and anti-hapten antibodies, etc. For example, when the capture antibody is modified with DNP in advance, the capture antibody can be immobilized on the solid phase by the solid phase on which the anti-DNP antibody is immobilized.

[0086] The material of the solid phase can be selected from organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile butadiene styrene copolymer, vinyl chloride-acrylic acid ester copolymer, polyvinyl acetate acrylate, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, cobalt, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. Two or more of these may be used in combination.

[0087] The shape of the solid phase is not particularly limited, and examples thereof include particles, microplates, microtubes, and test tubes. Among these, particles and microplates are preferred, and magnetic particles are particularly preferred. When the solid phase is in the form of particles, a suspension of particles can be used as the solid phase for forming the immune complex. When the solid phase is in the form of a container such as a microplate, the immune complex can be formed in the container as the solid phase. When magnetic particles capable of immobilizing a capture antibody are used, the measurement method of this embodiment may be performed using a commercially available fully automated immunoassay device such as the HISCL (registered trademark) series (Sysmex Corporation).

[0088] Next, a signal generated by the label contained in the immune complex is detected. The label is a label possessed by the labeled antibody bound to the target substance. Therefore, the signal generated by the label in the immune complex reflects the amount of the target substance. In this specification, "detecting a signal" includes qualitatively detecting the presence or absence of a signal, quantifying the intensity of the signal, and semi-quantitatively detecting the intensity of the signal. "Semi-quantitatively detecting the intensity of the signal" means detecting the signal intensity in multiple stages such as "no signal generated," "weak," and "strong." Preferably, the intensity of the signal generated by the label contained in the immune complex is quantified to obtain a measurement value.

[0089] The detection result of the signal can be used as a measurement result of the target substance in the sample. For example, when quantifying the intensity of the signal, the measured value of the signal intensity itself or a value obtained from the measured value can be used as the measured value of the target substance. Examples of the value obtained from the measured value of the signal intensity include a value obtained by subtracting the measured value of the negative control or the background value from the measured value. The negative control may be appropriately selected, and examples thereof include a buffer solution not containing the target substance.

[0090] The measured values ​​of the signal intensity may be applied to the calibration curve to determine the amount or concentration of the target substance in the sample. The calibration curve may be created from the measured values ​​of a plurality of calibrators. The measured values ​​of the calibrators are obtained by measuring the calibrators, as well as the samples, using the measurement method of this embodiment. The calibration curve may be created by plotting the measured values ​​of a plurality of calibrators on an XY plane, with the concentration of the target substance in the calibrator on the X-axis and the measured values ​​(e.g., signal intensity) on the Y-axis, and obtaining a straight line or curve by a known method such as the least squares method. The calibrator may be prepared, for example, by adding an isolated or synthesized target substance at an arbitrary concentration to a buffer solution not containing the target substance. The buffer solution not containing the target substance itself may be used as the calibrator not containing the target substance.

[0091] The measurement method of this embodiment may be performed by a commercially available fully automated immunoassay device. A fully automated immunoassay device is a device that, when a user sets a sample and inputs an instruction to start measurement, automatically prepares a measurement sample and performs an immunoassay on the sample, and outputs the measurement result of the target substance. Examples of such fully automated immunoassay devices include the HISCL series (Sysmex Corporation), such as HISCL (registered trademark)-5000 and HISCL-2000i. The HISCL series devices perform measurement by a sandwich ELISA method using magnetic particles as a solid phase.

[0092] In this embodiment, between the formation of the immune complex and the detection of the signal, B / F (Bound / Free) separation may be performed to remove unreacted free components. The unreacted free components refer to components that do not constitute an immune complex. Examples include excess capture antibodies that have not bound to the target substance and the labeled antibodies of this embodiment. There is no particular limitation on the means of B / F separation. If the solid phase is a particle, B / F separation can be performed by recovering only the solid phase that has captured the immune complex by centrifugation. If the solid phase is a container such as a microplate or a microtube, B / F separation can be performed by removing the liquid containing the unreacted free components. In addition, if the solid phase is a magnetic particle, B / F separation can be performed by sucking and removing the liquid containing the unreacted free components with a nozzle while the magnetic particles are magnetically restrained by a magnet, which is preferable from the viewpoint of automation. After removing the unreacted free components, the solid phase that has captured the immune complex may be washed with an appropriate aqueous medium such as PBS.

[0093] When the label of the labeled antibody is a fluorescent dye or a fluorescent protein, the measurement method of this embodiment may be performed by a flow cytometer. In this specification, "flow cytometer" includes imaging flow cytometers (IFCs). An IFC is a flow cytometer equipped with an imaging unit such as a CCD camera. A flow cytometer without an imaging unit is an apparatus that irradiates light onto individual formed elements in a liquid flowing through a flow cell, and detects the scattered light and / or fluorescence emitted from each formed element as an optical signal. When the formed elements are cells, the size of each cell, the distribution of the amount of molecules on the cell surface or intracellular molecules, etc. can be measured. An IFC is, It is a device that can acquire images of individual formed elements in a liquid flowing through a flow cell. IFC can acquire and quantitatively measure fluorescent signals, scattered light signals, fluorescent images, and bright-field images (also called transmitted light images) from thousands to millions of formed elements in a short time of, for example, a few seconds to a few minutes. In addition, information on each formed element can be extracted by image processing.

[0094] When the measurement method of this embodiment is performed using a flow cytometer, the measurement target may be a formed component containing a substance recognized by a labeled antibody. Even when measuring using a flow cytometer, an immune complex may be formed by mixing a sample that may contain a target substance with a labeled antibody. As a result, the labeled antibody binds to the target substance present on the formed component, forming an immune complex on the formed component. The temperature and incubation time conditions for mixing are as described above.

[0095] The detection of the signal generated by the label contained in the immune complex is performed by a flow cytometer. That is, the immune complex is introduced into a flow cell of the flow cytometer, and the signal is detected by the flow cytometer. In the flow cytometer, when each of the formed components in which the immune complex is formed passes through the flow cell, the formed components are irradiated with light. Then, the fluorescent signal emitted from the formed components is detected. In the measurement method of the present embodiment using a flow cytometer, a labeled antibody having a fluorescent dye or a fluorescent protein is used as a label, so that the fluorescent signal emitted from the fluorescent dye or the fluorescent protein is detected. Then, the fluorescent information is obtained based on the detected fluorescent signal. If necessary, a scattered light signal from the formed components may be detected, and the scattered light information may be obtained based on the scattered light signal. Examples of scattered light include forward scattered light (for example, scattered light with a light receiving angle of 0 degrees to about 20 degrees) and side scattered light (for example, scattered light with a light receiving angle of about 20 degrees to about 90 degrees).

[0096] The light source of the flow cytometer is not particularly limited, and can be appropriately selected from light sources having wavelengths suitable for exciting fluorescent dyes or fluorescent proteins, such as semiconductor laser light sources, argon laser light sources, He-Ne laser light sources, and mercury lamps.

[0097] Examples of the fluorescence information include the pulse peak, pulse width, pulse area, transmittance, Stokes shift, ratio, change over time, and values ​​correlated thereto, etc. Examples of the scattered light information include the pulse peak, pulse width, pulse area, transmittance, Stokes shift, ratio, change over time, and values ​​correlated thereto, etc.

[0098] When the flow cytometer is an IFC, the fluorescence information may be, for example, a value based on a fluorescence signal obtained from an image captured by the IFC. In a fluorescence image of a particle captured by the IFC, each pixel constituting an area showing a fluorescence signal has a pixel value according to the fluorescence signal intensity. Therefore, the value based on an optical signal obtained from an image may be, for example, a value based on pixels constituting an area showing a fluorescence signal. Examples of such values ​​include fluorescence intensity, maximum fluorescence intensity, total fluorescence signal intensity, and fluorescence signal area value. "Fluorescence intensity" is the average value of pixel values ​​of pixels constituting an area showing a fluorescence signal in a fluorescence image of a solid component containing an immune complex. "Maximum fluorescence intensity" is the maximum value of pixel values ​​of pixels constituting an area showing a fluorescence signal in a fluorescence image of a solid component containing an immune complex. "Total fluorescence signal intensity" is the sum of pixel values ​​of pixels constituting an area showing a fluorescence signal in a fluorescence image of a solid component containing an immune complex. "Fluorescence signal area value" is the number of pixels constituting an area showing a fluorescence signal in a fluorescence image of a solid component containing an immune complex.

[0099] Based on the acquired fluorescence information, the solid component containing the immune complex may be detected. When the fluorescence information is information indicated by a numerical value such as a pulse peak of fluorescence or a fluorescence intensity acquired from a fluorescence image, the value of the fluorescence information of the solid component containing the immune complex is larger than the value of the fluorescence information of the solid component that does not form an immune complex. Therefore, the value of the acquired fluorescence information is compared with a predetermined threshold, and based on the comparison result, the measurement data of the solid component containing the immune complex can be extracted. For example, a solid component whose value of the acquired fluorescence information is equal to or greater than a predetermined threshold can be detected as a solid component containing an immune complex. The predetermined threshold is not particularly limited and can be appropriately determined. For example, a buffer solution not containing a target substance or a sample containing a solid component that is known not to contain a target substance is measured by the measurement method of this embodiment using a flow cytometer, and the value of the fluorescence information is acquired. The obtained value may be the predetermined threshold.

[0100] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0101] Materials and Methods The materials and methods used in the experimental examples described below are described below. Details of the methods are also described in the individual experimental examples.

[0102] 1. Antibodies (1.1) Preparation of Q-tagged anti-p24 Fab antibody (1.1.1) Establishment of hybridomas producing anti-p24 antibodies (sF001-5, sF028-22, and sF001-25) Hybridomas producing anti-p24 antibodies (sF001-5, sF028-22, sF001-25) were established using mice (ddY; 5-week-old, female) as hosts and recombinant HIV p24 protein as antigen. Specifically, mice (ddY; 5-week-old, female) were immunized with recombinant HIV p24 protein using FCA (Freund's complete adjuvant) and FIA (Freund's incomplete adjuvant) as adjuvants. Mice with an increase in antibody titer in the blood were euthanized, and the spleens were removed at autopsy. The removed spleen cells were fused with mouse myeloma cells by the PEG method, and hybridomas were selected in HAT-containing medium. Furthermore, the antibodies secreted into the culture supernatant of each hybridoma were evaluated by ELISA, and hybridomas producing anti-p24 antibodies sF001-5, sF028-22, and sF001-25, respectively, were established.

[0103] (1.1.2) Preparation of sF001-5, sF028-22, and sF001-25 Mouse hybridoma-derived sF001-5, sF028-22, and sF001-25 were prepared. That is, the sF001-5, sF028-22, and sF001-25-producing hybridoma cells established above were cultured in SFM medium to secrete sF001-5, sF028-22, and sF001-25 into the medium, respectively. The culture supernatant from which the cells were removed using a filtration filter was subjected to column chromatography using Protein G carrier to purify sF001-5, sF028-22, and sF001-25 to obtain each antibody.

[0104] (1.1.3) Preparation of Q-tagged anti-p24 Fab antibody The Fab antibodies chimera sF001-5 (hereinafter abbreviated as "csF001-5"), chimera sF028-22 (hereinafter abbreviated as "csF028-22"), and chimera sF001-25 (hereinafter abbreviated as "csF001-25") were obtained by linking the hypervariable regions of sF001-5, sF028-22, and sF001-25 derived from mouse hybridomas with the constant regions of Fab of human IgG1, κ isotype. A Q tag sequence was inserted into the C-terminus of the heavy chain of these chimeric Fab antibodies to obtain Q-tagged anti-p24 Fab antibodies (hereinafter also referred to as "csF001-5Fab-Qtag", "csF028-22Fab-Qtag", and "csF001-25Fab-Qtag"). The amino acid sequence of the constant region containing the inserted Q tag sequence and the nucleotide sequence encoding it are shown below (the underlined part is the Q tag sequence).

[0105] Light chain amino acid sequence TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:4)

[0106] Light chain sequence ACCGTCGCGGCGCCGTCGGTCTTTATCTTCCCGCCGAGCGATGAACAACTGAAATCTGGTACCGCGAGTGTGGTTTGTCTGCTGAACAATTTTTATCCGCGTGAAGCGAAAGTCCAGTGGAAGGTGGACAACGCCCTGCAGTCTGGCAATAGTCAAGAATCCGTGACCGAACAAGATTCAAAAGACTCGACGTACAGCCTGAGTTCCACCCTGACGCTGAGCAAGGCAGATTATGAAAAACATAAGGTGTACGCTTGCGAAGTTACCCACCAGGGTCTGTCCAGTCCGGTTACGAAATCATTCAACCGCGGCGAATGT (SEQ ID NO: 5)

[0107] Amino acid sequence of the heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC GVLNLAQSP (SEQ ID NO: 6)

[0108] Base sequence of the heavy chain GCTAGCACAAAAGGGCCCAGCGTGTTTCCACTGGCTCCAAGCTCCAAATCTACTTCTGGGGGCACCGCCGCACTGGGCTGTCTGGTCAAGGACTACTTCCCAGAGCCCGTCACCGTGTCATGGAACTCCGGCGCACTGACTTCCGGAGTCCACACCTTTCCAGCCGTGCTGCAGTCCAGCGGACTGTACAGCCTGTCTAGTGTGGTCACAGTGCCTTCATCCAGCCTGGGAACTCAGACCTATATCTGCAACGTGAATCACAAGCCATCAAATACTAAAGTCGACAAGAAAGTGGAACCCAAGAGCTGTGGCGTCCTTAACCTCGCACAAAGTCCA (SEQ ID NO: 7)

[0109] Polynucleotides encoding the light chains of csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag were obtained by amplifying the light chain variable regions of sF001-5, sF028-22, and sF001-25 and the light chain κ constant region of human IgG by PCR, and then linking the fragments by overlap PCR. Polynucleotides encoding the heavy chains were obtained by linking a fragment with a Q tag sequence added to the C-terminus by a primer and a PCR-amplified fragment of the heavy chain variable region of sF001-5, sF028-22, and sF001-25 by overlap PCR, using the CH1 region of human IgG1 as a template. The prepared PCR products were introduced into pcDNA3.4 (Thermo Fisher Scientific) by TA cloning method to construct plasmids encoding csF001-5_LC (light chain in chimerized sF001-5), csF028-22_LC, and csF001-25_LC. Furthermore, plasmids encoding csF001-5_HC_Fab-Qtag (Qtag added to the C-terminus of the Fab of the heavy chain in chimerized sF001-5), csF028-22_HC_Fab-Qtag, and csF001-25_HC_Fab-Qtag were constructed. Next, the plasmids encoding the heavy and light chains were cotransfected into Expi-293cell (Thermo Fisher Scientific) to express csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag as recombinant proteins in the culture supernatant. The csF001-5Fab-Qtag, csF028-22 Fab-Qtag and csF001-25Fab-Qtag expressed in the supernatant were each purified using a Protein G column.

[0110] (1.2) Preparation of Q-tagged anti-HBs628 Fab antibody (1.2.1) Establishment of anti-HBs628 antibody-producing hybridomas Hybridomas producing mouse anti-HBs628 antibodies were prepared using HBs628 antigen according to the method of KOHLER and Milstein (see KOHLER G. and Milstein C., Nature, 256, 495-497 (1975)).

[0111] (1.2.2) Q-tagged HBs628Fab antibody The Q-tagged HBs628Fab antibody (hereinafter referred to as "HBs628Fab-Qtag") was prepared by inserting a Q-tag sequence into the C-terminus of the heavy chain of the Fab portion of mouse hybridoma-derived HBs628. The amino acid sequence of the constant region containing the inserted Q-tag sequence and the base sequence encoding it are shown below (the underlined portion is the Q-tag sequence).

[0112] Light chain amino acid sequence QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS (SEQ ID NO:8)

[0113] Light chain sequence CAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCACACTGGTGTGTACGATCACTGATTTCTACCCAGGTGTGGTGACAGTGGACTGGAAGGTAGATGGTACCCCTGTCACTCAGGGTATGG AGACAACCCAGCCTTCCAAACAGAGCAACAACAAGTACATGGCTAGCAGCTACCTGACCCTGACAGCAAGAGCATGGGAAAGGCATAGCAGTTACAGCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGAAGAGTTTGTCCCGTGCTGACTGTTCC (Sequence number 9)

[0114] Heavy chain amino acid sequence AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GLLQGP (SEQ ID NO: 10)

[0115] Base sequence of the heavy chain GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGCCTGCTCCAGGGCCCCTGATAA (SEQ ID NO: 11)

[0116] The polynucleotide encoding the light chain of HBs628Fab-Qtag was obtained by cloning from the RNA of the hybridoma by the 5'-RACE method and amplifying it by the PCR method. The polynucleotide encoding the heavy chain of HBs628Fab-Qtag was obtained by cloning from the RNA of the hybridoma by the 5'-RACE method and amplifying the fragment with the Q tag sequence added to the C-terminus by the primer by the PCR method. The prepared PCR product was introduced into pcDNA3.4 (Thermo Fisher Scientific) by the TA cloning method to construct a plasmid encoding HBs628Fab-Qtag. Next, the plasmid was transfected into Expi-293cell (Thermo Fisher Scientific) to express HBs628Fab-Qtag as a recombinant protein in the culture supernatant. The HBs628Fab-Qtag expressed in the supernatant was purified using a Capture Select LC-Lambda(mur) affinity column (Thermo Fisher Scientific).

[0117] (1.3) Preparation of Q-tagged anti-CD20 Fab antibody The Q-tagged anti-CD20 Fab antibody (hereinafter referred to as "anti-CD20Fab-Qtag") was prepared by inserting a Q-tag sequence into the C-terminus of the heavy chain of a human-derived anti-CD20 Fab. The amino acid sequences of the hypervariable region and the constant region containing the inserted Q-tag sequence, as well as the base sequences encoding them, are shown below (the underlined portion is the Q-tag sequence).

[0118] Light chain amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12)

[0119] Base sequence of the light chain CAAATCGTGCTTAGCCAGTCACCAGCGATATTGTCCGCTAGTCCTGGAGAGAAGGTGACCATGACCTGTAGGGCATCTTCCAGCGTTTCCTACATTCACTGGTTTCAGCAGAAGCCAGGGTCCAGCCCTAAACCCTGGATCTATGCCACCAGTAACCTTGCCTCAGGGGTACCTGTGCGATTCAGCGGAAGCGGTAGTGGCACCTCATACTCCCTGACCATTAGCCGCGTTGAAGCTGAAGATGCGGCTACCTACTACTGCCAACAGTGGACGTCTAACCCACCCACATTTGGTGGCGGCACTAAACTGGAGATCAAGCGGACTGTGGCTGCACCATCCGTATTCATCTTTCCGCCCTCTGATGAGCAGCTGAAATCTGGCACAGCTAGCGTCGTGTGCCTGCTGAACAACTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGACAATGCCCTGCAGTCTGGCAATTCACAGGAATCAGTCACCGAACAGGACTCTAAGGATAGCACGTACTCCTTGAGCAGCACACTCACTCTCTCTAAAGCCGACTATGAGAAGCACAAGGTCTATGCCTGTGAGGTGACTCATCAAGGGCTGAGTAGTCCCGTCACAAAGTCCTTCAATCGTGGAGAA (SEQ ID NO: 13)

[0120] Amino acid sequence of the heavy chain QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHT GLLQG(Sequence number 14)

[0121] Base sequence of the heavy chain CAGGTGCAGTTGCAGCAACCTGGGGCAGAACTCGTGAAGCCTGGGGCATCCGTTAAGATGTCCTGTAAAGCAAGCGGCTATACTTTCACTTCCTACAACATGCATTGGGTGAAACAGACTCCTGGCAGAGGCTTGGAGTGGATAGGAGCCATCTACCCAGGGAATGGTGACACATCTTACAACCAGAAGTTCAAGGGCAAAGCCACCCTCACTGCCGACAAAAGTAGCTCTACAGCCTATATGCAGCTGTCATCCCTGACGAGCGAGGATTCAGCCGTGTATTATTGCGCTAGGTCTACCTACTATGGAGGCGATTGGTACTTCAATGTGTGGGGTGCCGGAACTACCGTGACTGTGTCTGCTGCGAGCACCAAGGGTCCAAGTGTCTTTCCCCTTGCACCGTCCAGCAAATCCACAAGCGGAGGTACTGCTGCTCTTGGCTGTCTGGTGAAGGACTATTTTCCAGAGCCAGTGACGGTTAGCTGGAATTCAGGCGCCCTTACATCTGGGGTACACACGTTTCCTGCCGTTCTGCAAAGCTCAGGACTGTACAGCCTGTCTAGTGTGGTCACAGTCCCCTCAAGCAGTCTCGGCACCCAGACATACATCTGCAATGTCAACCACAAACCCTCCAACACCAAGGTCGACAAGAAGGCAGAACCCAAAAGTTGCGATAAGACCCATACAGGGCTGCTCCAA (Sequence number 15)

[0122] A polynucleotide encoding a light chain Fab and a polynucleotide having a sequence in which a Q tag sequence was inserted at the C-terminus of a heavy chain Fab were inserted into pcDNA3.4 (Thermo Fisher Scientific), and expression plasmids encoding anti-CD20 Fab-LC and anti-CD20 Fab-HC-Qtag were constructed by total synthesis. Next, this plasmid was transfected into Expi-293cell (Thermo Fisher Scientific) to express anti-CD20 Fab-Qtag as a recombinant protein in the culture supernatant. The anti-CD20 Fab-Qtag expressed in the supernatant was purified using a CaptureSelect™ Kappa XL Pre-packed Column (Thermo Fisher Scientific).

[0123] 2. PEG linker having an amino group and a thiol group (SH-PEG-NH2 linker) The following SH-PEG-NH2 linkers were purchased: (a) HS-PEG2K-NH2, HCl Salt, average Mn 2000 (Sigma-Aldrich), (b) SH-PEG-NH2, MW 2K (Biopharma PEG Scientific Inc.), (c) HS-PEG-NH2, MW 2K (Creative PEGWorks), (d) HS-PEG-NH2, HCl Salt, average Mn 3500 (Sigma-Aldrich), (e) HS-PEG-NH2, HCl Salt, average Mn 5000 (Sigma-Aldrich), (f) Thiol PEG-NH2, average Mn 400Da (Nanocs Inc.), and (g) HS-PEG-NH2, MW 1kDa (Creative PEGWorks). In addition, a different lot of HS-PEG2K-NH2, HCl Salt, average Mn 2000 (Sigma-Aldrich) (hereinafter also referred to as linker (a')) from the linker (a) above was purchased. Linkers (a), (a'), (b) and (c) were sold as products with an average molecular weight of 2000. Hereinafter, each of linkers (a), (a'), (b) and (c) is also referred to as "SH-PEG-NH2 linker (2K)". Linker (d) was sold as a product with an average molecular weight of 3500. Hereinafter, linker (d) is also referred to as "SH-PEG-NH2 linker (3.5K)". Linker (e) was sold as a product with an average molecular weight of 5000. Hereinafter, linker (e) is also referred to as "SH-PEG-NH2 linker (5K)". Linker (f) was sold as a product with an average molecular weight of 400. Hereinafter, linker (f) is also referred to as "SH-PEG-NH2 linker (400 Da)". Linker (g) was sold as a product with an average molecular weight of 1000. Hereinafter, linker (g) is also referred to as "SH-PEG-NH2 linker (1K)". Linkers (a), (a'), (b), (d), and (e) were hydrochloride salts, and linkers (c), (f), and (g) were free forms. The structures of each linker were as follows:

[0124] [ka]

[0125] Hereinafter, the Fab antibody bound to the SH-PEG-NH2 linker is also referred to as "SH-PEG-Fab". In particular, each Fab antibody bound to any of the above SH-PEG-NH2 linkers (a), (a'), (b) and (c) is also referred to as "SH-PEG(2K)-Fab". In addition, each Fab antibody bound to the above SH-PEG-NH2 linkers (d), (e), (f) and (g) is also referred to as "SH-PEG(3.5K)-Fab", "SH-PEG(5K)-Fab", "SH-PEG(400Da)-Fab" and "SH-PEG(1K)-Fab", respectively.

[0126] 3. Crosslinkers with maleimide groups and NHS esters (EMCS reagents) EMCS reagent (product name: EMCS, CAS number: 55750-62-5) was purchased from Dojindo Laboratories, Inc. The molecular formula and molecular weight are 14 H 16 N2O6 = 308.29 and the structure was as follows:

[0127] [ka]

[0128] 4.Signs (4.1) Biotin with a maleimide group Biotin-PEAC5-maleimide (product name: Biotin-PEAC5-maleimide, CAS number: 374592-98-0) was purchased from Dojindo Laboratories. The molecular formula and molecular weight are: 26 H 41 ClN6O5S = 585.16 and the structure was as follows:

[0129] [ka]

[0130] (4.2) Fluorescent dyes with maleimide groups Alexa488-maleimide (product name: Alexa Fluor™ 488 C5 Maleimide, CAS number: 500004-82-0) was purchased from Thermo Fisher Scientific. The molecular formula and molecular weight are 30 H 25 N4NaO 12 S2 = 720.66 and the structure was as follows:

[0131] [ka]

[0132] (4.3) R-Phycoerythrin (R-PE) R-PE (product name: OB1) was purchased from One Biotech.

[0133] (4.4) Alkaline phosphatase (ALP) Bovine small intestine-derived ALP (product name: ALP-55) was purchased from Oriental Yeast Co., Ltd.

[0134] 5. Transglutaminase (TG) As the TG, ACTIVA (registered trademark) KS-CT (Ajinomoto Co., Inc.), a microbial transglutaminase, was purified and used. Hereinafter, the purified enzyme is also referred to as "BTGase."

[0135] 6. Infusion Mass Spectrometry (Infusion MS) In order to confirm the molecular weight distribution and the presence or absence of association of each of the above SH-PEG-NH2 linkers (a), (a'), (b), (c), (d) and (e), these linkers were analyzed by Infusion MS. A Q Exactive (Thermo Fisher Scientific) was used as the MS device. The ionization mode was positive. The measurement samples were prepared by dissolving each of the above SH-PEG-NH2 linkers in a BTGase reaction solution (50 mM Tris, 2 mM EDTA, pH 8.2 or 20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2) and then diluting 100 times with a 30% acetonitrile solution containing 0.1% formic acid. From the results of mass spectrometry of each SH-PEG-NH2 linker having a repeating structure, the weight average molecular weight was calculated from the molecular weight and intensity of the most intense isotopic peak for peaks with an S / N of 5 or more. In addition, the molecular weight width was confirmed from the MS peak.

[0136] 7. Quantitation of target protein The amounts of various Fab antibodies and their derivatives were determined by calculation from the Abs280 peak area values ​​of SEC analysis. Recombinant human interleukin 6 (rhIL-6) (extinction coefficient 0.43) was used as a reference.

[0137] 8. Non-reducing SDS-PAGE Analysis Various Fab antibodies and their derivatives were analyzed by non-reducing SDS-PAGE under the following conditions.

[0138] 10-20% polyacrylamide gel: e-PAGEL (ATTO Corporation) Electrophoresis device: PAGERUN (ATTO Corporation) Electrophoresis conditions: 20 mA, 80 min. Sample buffer: NuPAGE LDS Sample Buffer (Invitrogen)

[0139] Experimental Example 1: Modification of Fab antibody with SH-PEG-NH2 linker (2K) using BTGase (1) Binding of SH-PEG-NH2 linker (2K) to Fab antibody Using BTGase, the SH-PEG-NH2 linker (2K) (Sigma-Aldrich) described above in (a) was bound to each of csF001-5Fab-Qtag, csF028-22Fab-Qtag, csF001-25Fab-Qtag, and HBs628Fab-Qtag. The SH-PEG-NH2 linker (2K) (Sigma-Aldrich) described above in (a') was bound to anti-CD20Fab-Qtag. Specifically, the procedure was as follows. csF001-5Fab-Qtag or HBs628Fab-Qtag was dissolved in MES buffer (50 mM MES, 2 mM EDTA, pH 7.0) to a concentration of 10 μM, and incubated with 50 equivalents of SH-PEG-NH2 linker (2K) and 0.1 U / mL BTGase at 25° C. for 3 hours. The activity of BTGase was measured by the hydroxamic method (Folk JE and Cole PW, J. Biol. Chem. 241, 5518-5525 (1966)). The progress of the reaction was analyzed by SEC.

[0140] To study the efficiency of introducing the SH-PEG-NH2 linker (2K), 50 equivalents of the linker were added to 10 μM csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag in Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2 or pH 8.5) and reacted at room temperature for 5 hours. The results of SEC analysis of the reaction products at pH 8.2 and pH 8.5 were compared with those at pH 7.0.

[0141] The above reaction conditions (pH 8.2 or 8.5) that improved the introduction efficiency were applied to all reactions between Fab-Qtag and SH-PEG-NH2 linker. The reaction solution of csF001-5Fab-Qtag was desalted and concentrated using Amicon 10K (Merch) to remove the SH-PEG-NH2 linker. The reaction solution of csF028-22Fab-Qtag was fractionated using Superdex 200 Increase 10 / 300 and concentrated using Amicon 10K (Merch). The reaction solution of HBs628Fab-Qtag was fractionated using Superdex 75 Increase 10×300 mm and concentrated using Amicon 10K (Merch). The reaction solutions of csF001-25Fab-Qtag and anti-CD20 Fab-Qtag were desalted and purified using a Protein G column, and then concentrated with Amicon 10K (Merck).

[0142] (2) SEC analysis The reaction mixture of CSF001-5Fab-Qtag, csF028-22Fab-Qtag, csF001-25Fab-Qatg and their SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was analyzed by SEC. Fractions of csF028-22Fab-Qtag and SH-PEG-NH2 linker (2K) were also collected. The conditions were as follows.

[0143] Column: Superdex 200 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0144] (3) SEC analysis and fractionation The reaction solution of HBs628Fab-Qtag and SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was analyzed and separated under the following conditions.

[0145] Column: Superdex 75 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Strong, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0146] (4) Separation of SH-PEG-Fab using Protein G SH-PEG(2K)-Fab was isolated from csF001-25Fab-Qtag and anti-CD20 Fab-Qtag under the following conditions.

[0147] Column: HiTrap™ Protein G HP Column (1 mL, Cytiva) Solvent A: 20 mM phosphate buffer, 150 mM NaCl, pH 7.0 Solvent B: 0.1M glycine, pH 2.7 Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0148] Experimental Example 2: Biotin labeling of SH-PEG-Fab from HBs628Fab-Qtag (1) Biotin labeling of SH-PEG-Fab The SH-PEG(2K)-Fab from HBs628Fab-Qtag was labeled with biotin as follows. The reaction solution of HBs628Fab-Qtag and the above-mentioned (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) using BTGase was desalted and concentrated with Amicon 10K (Merck) to obtain a concentrated solution containing SH-PEG-Fab. Biotin-PEAC5-maleimide was added to 100 equivalents of SH-PEG-Fab in 50 mM MES, 2 mM EDTA buffer (pH 7.0) to react overnight at 5°C. The reaction solution was purified by SEC. The reaction product of SH-PEG-Fab and maleimide-labeled biotin is also referred to as "biotin-PEG-Fab" below. Biotin-PEG-Fab had the structure of formula (I) (wherein X is ethylene, Y is PEG chain, Z is biotin, and L is spacer).

[0149] (2) SEC analysis and fractionation of biotin-PEG-Fab Biotin-PEG-Fab from HBs628Fab-Qtag was analyzed and separated under the following conditions.

[0150] Column: Superdex 75 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Strong, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0151] (3) Analysis by Western blot (WB) HBs628Fab-Qtag, its SH-PEG(2K)-Fab and biotin-PEG-Fab were separated by non-reducing SDS-PAGE as described above and then analyzed by WB as follows. Fab (5-10 ng equivalent) was separated by SDS-PAGE (10-20% polyacrylamide gel) and transferred to a PVDF membrane (Invitrogen) using an iBlot 2 dry blotting apparatus (Thermo Fisher Scientific). The PVDF membrane after transfer was blocked with 1% skim milk-containing TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween20, pH 7.4) for 1 hour at room temperature. The blocked PVDF membrane was washed with TBST (10 min x 3 times). HRP-Conjugated Streptavidin (Thermo Fisher Scientific) diluted 1:20,000 with TBST containing 1% skim milk was added to the PVDF membrane and incubated at room temperature for 1 hour. The PVDF membrane was washed with TBST (10 min x 3 times). ECL Prime Western Blotting Detection Reagent (Cytiva) was added to the PVDF membrane, and the presence or absence of biotin labeling was detected using an Amersham Imager 680 (Cytiva).

[0152] (4) LC-MS analysis MS analysis of SH-PEG-Fab from HBs628Fab-Qtag was performed by two methods. That is, non-reduced and reduced samples were analyzed. The reduction conditions were as follows: 85 mM TCEP (tris(2-carboxyethyl)phosphine) was added in excess to 10 μg of Fab, and the mixture was left at 5°C overnight, followed by LC-MS analysis. The analysis conditions were as follows:

[0153] Column: Develosil C18 (3 μm, 2 mm ID × 100 mm L, Nomura Chemical Co., Ltd.) Analysis temperature: 25℃ Solvent A: 0.1% formic acid Solvent B: 0.1% formic acid, 100% acetonitrile Gradient: 1 min gradient from 10% B to 60% B Flow rate: 100μL / min LC equipment: LC-20A (Shimadzu Corporation) MS instrument: Q Exactive (Thermo Fisher Scientific) Ionization mode: positive

[0154] Experimental Example 3: ALP labeling of SH-PEG-Fab (1) Maleimide modification of ALP To label the SH-PEG(2K)-Fab from csF001-5Fab-Qtag, csF028-22Fab-Qtag, and HBs628Fab-Qtag, ALP was modified with maleimide as follows. ALP was dissolved in 25 M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0) to a concentration of 10 μM. 20 equivalents of EMCS reagent was added to the ALP solution and incubated at 37°C for 1 hour. The reaction solution was desalted using a PD10 column (Cytiva) and then concentrated using Amicon 10K.

[0155] (2) ALP labeling of SH-PEG(2K)-Fab (2.1) Labeling of SH-PEG(2K)-Fab from csF001-5Fab-Qtag SH-PEG(2K)-Fab from csF001-5Fab-Qtag was labeled with ALP as follows: 20 μM SH-PEG(2K)-Fab and 10 μM maleimide-modified ALP were incubated overnight at 5° C. in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The reaction solution was analyzed by HPLC using Superdex 200 Increase (Cytiva), and the following were purified and separated: ALP-labeled Fab in which Fab was bound to one maleimide group of one molecule of ALP (hereinafter referred to as "Fab-PEG-ALP"), ALP-labeled Fab in which Fab was bound to each of two maleimide groups of one molecule of ALP (hereinafter referred to as "(Fab-PEG)2-ALP"), and ALP-labeled Fab in which Fab was bound to each of three maleimide groups of one molecule of ALP (hereinafter referred to as "(Fab-PEG)3-ALP"). Fab-PEG-ALP is a complex of one molecule of ALP and one molecule of Fab, (Fab-PEG)2-ALP is a complex of one molecule of ALP and two molecules of Fab, and (Fab-PEG)3-ALP is a complex of one molecule of ALP and three molecules of Fab. Fab-PEG-ALP, (Fab-PEG)2-ALP and (Fab-PEG)3-ALP each had the structure of formula (I) (wherein X is ethylene, Y is a PEG chain, Z is ALP and L is a spacer).

[0156] (2.2) Labeling of SH-PEG(2K)-Fab from csF028-22Fab-Qtag SH-PEG(2K)-Fab from csF028-22Fab-Qtag was labeled with ALP as follows. SH-PEG(2K)-Fab was concentrated in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0) using Amicon 10K (Merck) and buffer exchanged. The resulting 20 μM reaction solution and 6.7 μM maleimide-modified ALP were incubated overnight at 5° C. in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The reaction solution was analyzed by HPLC using Superdex 200 Increase (Cytiva), and Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP were purified and separated, respectively. The quantification of each ALP-labeled Fab was carried out by the above-mentioned quantitative method for the target protein. BSA (Proliant) was added to each ALP-labeled Fab solution to a final concentration of 0.1%.

[0157] (2.3) Labeling of SH-PEG(2K)-Fab from HBs628Fab-Qtag The SH-PEG(2K)-Fab derived from HBs628Fab-Qtag was labeled with ALP as follows. SH-PEG(2K)-Fab and maleimide-modified ALP (hereinafter referred to as "Mal") n The mixture was incubated overnight at 5°C in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0) at the molar ratios shown in Table 1. The reaction mixture was analyzed by HPLC using a Superdex 200 Increase (Cytiva), and Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP were purified and separated. n -ALP represents one molecule of ALP to which n (n is an integer of 1 or more) maleimide groups are added.

[0158] [Table 1]

[0159] (3) SEC analysis and fractionation of Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP (3.1) Analysis conditions for ALP-PEG-Fab from csF001-5Fab-Qtag and HBs628Fab-Qtag Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0160] (3.2) Analytical and preparative conditions for Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0161] Experimental Example 4: ALP labeling of Fab' by random labeling method (1) Maleimide modification of ALP To label ALP of Fab' without a Q tag, ALP was modified with maleimide as follows. ALP was dissolved in D-PBS(-) (pH 7.4) to a concentration of 20 μM. 30 equivalents of EMCS reagent was added to the ALP solution and incubated at 37°C for 1 hour. The reaction solution was desalted using a PD10 column (Cytiva) and buffer exchanged into 0.1M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). It was then concentrated using an Amicon 50K.

[0162] (2) Preparation of F(ab')2 from full-length antibodies and SEC separation sF001-5 was dissolved in McIlvaine buffer (pH 3.8) to a concentration of 6.67 μM, and pepsin (Sigma) was dissolved to a concentration of 0.95 μM, and incubated at 37°C for 3 hours. The reaction was then neutralized by adding 10v% 1M Tris-HCl (pH 8.5) to stop the reaction. The product was concentrated using Amicon 10K, and F(ab')2 was purified and separated using Superdex 200 Increase (Cytiva). The resulting F(ab')2 solution was concentrated again using Amicon 10K. It was confirmed that this operation did not decrease the reactivity of the antibody per substance amount. The separation conditions were as follows.

[0163] Column: Superdex 200 Increase 10×300 mm Purification equipment: AKTAgo (Cytiva) Analysis temperature: room temperature Solvent: 0.1M phosphate buffer (1mM EDTA·2Na, pH6.0) Flow rate: 0.5 ml / min Detection: UV 280nm

[0164] (3) Preparation of Fab' from F(ab')2 and SEC separation The F(ab')2 prepared in (2) above was dissolved in 0.1M phosphate buffer (1mM EDTA·2Na, pH 6.0) to a concentration of 20μM. 1500 equivalents of 2-mercaptoethylamine reagent (Nacalai Tesque, Inc.) was added to the F(ab')2 solution and incubated at 37℃ for 90 minutes. Then, using Superdex 200 Increase (Cytiva), only the fraction of sF001-5Fab' produced was separated and purified, and concentrated using Amicon 10K. The separation conditions were as follows:

[0165] Column: Superdex 200 Increase 10×300 mm Purification equipment: AKTAgo (Cytiva) Analysis temperature: room temperature Solvent: 0.1M phosphate buffer (1mM EDTA·2Na, pH6.0) Flow rate: 0.5 ml / min Detection: UV 280nm

[0166] (4) ALP labeling of Fab' (random labeling method) Fab' was labeled with ALP by coupling the cysteine ​​residue in Fab' with maleimide-modified ALP. Specifically, the procedure was as follows. 44 μM sF001-5Fab' and 8.8 μM maleimide-modified ALP were incubated overnight at 5° C. in 0.1 M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The resulting coupling product (hereinafter referred to as "(Fab') n The conditions for separation were as follows. n -ALP represents a complex in which Fab' is bound to each of n (n is an integer of 1 or more) maleimide groups possessed by ALP.

[0167] Column: Superdex 200 Increase 10×300 mm Purification equipment: AKTAgo (Cytiva) Analysis temperature: room temperature Solvent: 0.1M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0) Flow rate: 0.5 ml / min Detection: UV 280nm

[0168] Purified (Fab') n The samples containing -ALP were analyzed by non-reducing SDS-PEG and SEC. The SEC analysis conditions were as follows:

[0169] Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0170] Experimental Example 5: Fluorescent labeling of SH-PEG-Fab with Alexa488-maleimide (1) Conjugation of SH-PEG-Fab with Alexa488-maleimide The SH-PEG(2K)-Fab from csF001-25Fab-Qtag was labeled with a fluorescent dye as follows. The SH-PEG(2K)-Fab was concentrated with Amicon 10K (Merck) and buffer exchanged into 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). The 6 μM reaction solution and 120 μM Alexa488-maleimide were incubated overnight at 5° C. in 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). The Alexa488-labeled Fab (Alexa488-PEG-Fab) in the reaction solution was separated and analyzed by HPLC using a Superdex 200 Increase (Cytiva). The reaction product of SH-PEG-Fab and Alexa488-maleimide is hereinafter also referred to as "Alexa488-PEG-Fab". Alexa488-PEG-Fab had the structure of formula (I), where X is ethylene, Y is a PEG chain, Z is Alexa488, and L is a spacer.

[0171] (2) Conditions for SEC fractionation Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: 50 mM phosphate buffer, 2 mM EDTA, pH 7.0 Flow rate: 0.8 ml / min Detection: UV 280nm

[0172] (3) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm FL (Ex. 495 nm / Em. 519nm) PMT Super Low

[0173] (4) LC-MS analysis The SH-PEG(2K)-Fab and Alexa488-PEG-Fab from csF001-25Fab-Qtag were analyzed by LC-MS as follows: After reduction with 200 equivalents of TCEP in 1M Tris (pH 7.5) at 37°C for 2 hours, the samples were analyzed by LC-MS. The analytical conditions were as follows:

[0174] Column: PLRP-1000 column (2 μm × 100 mmL, PL laboratory) Analysis temperature: 25℃ Solvent A: 0.1% formic acid Solvent B: 0.1% formic acid, 100% acetonitrile Gradient: 1 min gradient from 10% B to 60% B Flow rate: 100μL / min LC equipment: LC-20A (Shimadzu Corporation) MS instrument: Q Exactive (Thermo Fisher Scientific) Ionization mode: positive

[0175] Experimental Example 6: Fluorescent labeling of SH-PEG-Fab with R-PE (1) Maleimide modification of R-PE To use R-PE for labeling SH-PEG(2K)-Fab from csF001-25Fab-Qtag and anti-CD20 Fab-Qtag, R-PE was modified with maleimide as follows. R-PE was dialyzed into 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). Then, 400 μM EMCS reagent was added to the 5 μM R-PE solution and incubated at 37° C. for 1 hour. The reaction solution was desalted using a PD10 column (Cytiva) and then concentrated using Amicon 3K. The obtained maleimide-modified R-PE is hereinafter referred to as "(Mal) n -R-PE" (Mal) n-R-PE represents one molecule of R-PE to which n (n is an integer of 1 or more) maleimide groups are added.

[0176] (2) Conjugation of SH-PEG-Fab with maleimide-modified R-PE The SH-PEG(2K)-Fab from csF001-25Fab-Qtag and anti-CD20 Fab-Qtag were labeled with fluorescent dye as follows. The SH-PEG(2K)-Fab was concentrated with Amicon 10K (Merck) and buffer exchanged into 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). 6 μM reaction solution and 2 μM (Mal) n -R-PE was incubated overnight at 5°C in 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). Unreacted maleimide groups were capped by adding L-cysteine ​​to the reaction solution to a final concentration of 0.1 mM. The mixture was then analyzed by HPLC using a Superdex 200 Increase (Cytiva). R-PE-labeled Fab in which Fab was bound to one maleimide group of one molecule of R-PE (hereinafter referred to as "Fab-PEG-R-PE"), R-PE-labeled Fab in which Fab was bound to each of two maleimide groups of one molecule of R-PE (hereinafter referred to as "(Fab-PEG)2-R-PE"), and R-PE-labeled Fab in which Fab was bound to each of three maleimide groups of one molecule of R-PE (hereinafter referred to as "(Fab-PEG)3-R-PE") were purified and separated. They were also analyzed by SEC separately. The separation and analysis conditions were as follows. Fab-PEG-R-PE is a complex of one molecule of R-PE and one molecule of Fab, (Fab-PEG)2-R-PE is a complex of one molecule of R-PE and two molecules of Fab, and (Fab-PEG)3-R-PE is a complex of one molecule of R-PE and three molecules of Fab. Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE each have the structure of formula (I) (wherein X is ethylene, Y is a PEG chain, Z is R-PE, and L is a spacer).

[0177] (3) Conditions for SEC fractionation Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: 50 mM phosphate buffer, 2 mM EDTA, pH 7.0 Flow rate: 0.8 ml / min Detection: UV 280nm

[0178] (4) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Detection: UV 280nm FL (Ex. 565 nm / Em. 574nm) PMT Super Low

[0179] Experimental Example 7: Measurement of ALP activity of ALP-labeled antibody The ALP activity of Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP from csF028-22Fab-Qtag prepared in Experimental Example 3 was measured as follows. ALP activity was measured using a calibrator (ALP-55 (Oriental Yeast Co., Ltd.) with activity values ​​given by Hitachi 7170 biochemical automatic analyzer) and each ALP-labeled antibody diluted to fall within the calibration curve range, with CDP-star (Thermo Fisher Scientific) as a substrate. The ALP activity value of each ALP-labeled antibody was calculated by multiplying the ALP activity of each measurement sample by the dilution factor, and the specific activity to unmodified ALP was compared.

[0180] Experimental Example 8: Fab-PEG-ALP and (Fab') n -Performance evaluation of reagents containing ALP Fab-PEG-ALP from csF001-5Fab-Qtag prepared in Experimental Example 3 and (Fab') prepared in Experimental Example 4 n The HIV p24 antigen was measured by an immunoassay using each of Fab-PEG-ALP and (Fab') as a detection antibody. n The performance of the reagents containing each of ALP and ALP was examined. Measurements were performed using a fully automated immunoassay device, HISCL (registered trademark)-2000i (Sysmex Corporation).

[0181] (1) Preparation of reagents Except for the R3 reagent, which is an antibody reagent for detection, the R1 reagent (biotin-labeled antibody), R2 reagent (streptavidin-immobilized magnetic particles), R4 reagent (chemiluminescent substrate dilution solution), and R5 reagent (chemiluminescent substrate) included in the HIV1 p24 antigen / HIV antibody kit "HISCL (registered trademark) HIV Ag+Ab Reagent" (Sysmex Corporation), or reagents prepared by a method similar to the manufacturing method of these reagents were used. The R3 reagent was Fab-PEG-ALP or (Fab') n Fab-PEG-ALP and (Fab') were prepared as follows. n Each antibody solution was diluted with 0.1M triethanolamine buffer (3% BSA, 0.5% sodium caseinate, 1mM MgCl2, 0.1mM ZnCl2, 150mM NaCl, pH 6.5) to the concentrations shown in Table 2. Each antibody solution was filtered through Millex-GS 0.22μm (Merck) to obtain R3 reagent. The ALP activity of each antibody was calculated in the same manner as in Experimental Example 7.

[0182] [Table 2]

[0183] (2) Sample preparation HIV p24 antigen (Abcam) was diluted with D-PBS (0.1% BSA, pH 7.4) and human pooled serum (Nissui Co., Ltd.) to prepare samples with antigen concentrations of 10 pg / mL, 100 pg / mL, and 1000 pg / mL. D-PBS (0.1% BSA, pH 7.4) and human pooled serum were used as samples containing no antigen (0 pg / mL).

[0184] (3) Measurement The R1 to R5 reagents described in (1) above were set in a HISCL-2000i (Sysmex Corporation), and the eight types of specimens prepared in (2) above were measured. The measurement procedure using the HISCL-2000i was as follows. After mixing the specimen (20 μL) with the R1 reagent (50 μL), the R2 reagent (30 μL) was added. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and the magnetic particles were washed by adding HISCL washing solution (300 μL). The supernatant was removed, and the R3 reagent (100 μL) was added to the magnetic particles and mixed. The magnetic particles in the resulting mixture were collected, the supernatant was removed, and the magnetic particles were washed by adding HISCL washing solution (300 μL). The supernatant was removed, and R4 reagent (50 μL) and R5 reagent (100 μL) were added to the magnetic particles, and the chemiluminescence intensity was measured.

[0185] Experimental Example 9: Evaluation of antigen-binding ability of Alexa488-labeled antibodies The antigen binding ability of SH-PEG(2K)-Fab and Alexa488-PEG-Fab from csF001-25Fab-Qtag prepared in Experimental Example 5 was measured by surface plasmon resonance (SPR) reaction using Biacore (trademark) T200 (Cytiva). For comparison, csF001-25Fab-Qtag was also measured. Specifically, it was as follows. Human Fab Binder attached to Human Fab Capture Kit (Cytiva) was immobilized on Sensor Chip CM5 (Cytiva) by amine coupling. SH-PEG(2K)-Fab, Alexa488-PEG-Fab, and unlabeled Fab (csF001-25Fab-Qtag) were bound as ligands, respectively. Recombinant protein HIV-1 p24 (Prospec) was reacted as an analyte. Interaction analysis was performed using a 1:1 Binding reaction model with Biacore T200 Evaluation software. The apparatus, reagents and reaction conditions used were as follows.

[0186] [Equipment and Reagents] Device: Biacore(TM) T200 (Cytiva) Sensor chip: Sensor chip CM5 (Cytiva) Capture kit: Human Fab capture kit (Cytiva) Coupling kit: Amine coupling kit (Cytiva) Buffer: HBS-EP+ buffer (Cytiva)

[0187] [Ligand capture] Ligands: SH-PEG(2K)-Fab, Alexa488-PEG-Fab and unmodified Fab (csF001-25Fab-Qtag) Flow rate: 30μL / min Addition time (Contact time): 60sec

[0188] [sample] Analyte: HIV-1 p24 (Prospec) (2.5-80 nM) Flow rate: 30μL / min Addition time (Contact time): 30sec Dissociation time: 300sec

[0189] [reproduction] Renaturation buffer: 10 mM glycine-HCl pH 2.1 Flow rate: 30μL / min Addition time (Contact time): 30sec

[0190] Experimental Example 10: Antigen binding ability of R-PE-labeled antibodies The antigen binding ability of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE from csF001-25Fab-Qtag prepared in Experimental Example 6 was measured by ELISA as follows. Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE were each diluted so that the fluorescence intensity of R-PE was approximately the same to prepare a detection reagent. A black 96-well microplate with immobilized anti-His tag antibody was reacted with His-tagged recombinant HIV-1 p24 (Prospec) as an antigen at room temperature for 1 hour. For comparison, an anti-His tag antibody-immobilized microplate to which no antigen was added was also prepared. Then, each well was washed with a washing solution (0.05% Tween 20-containing saline). Each detection reagent was provided to the wells of the microplate and reacted at room temperature for 1 hour. After each well was washed with a washing solution, the R-PE-labeled antibody in the microplate was detected using a fluorescent plate reader (excitation wavelength 488 nm, fluorescence wavelength 578 nm).

[0191] Experimental Example 11: Performance evaluation of reagents containing R-PE-labeled antibodies CD20-expressing cells were measured by flow cytometry (FCM) using Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE from anti-CD20 Fab-Qtag prepared in Experimental Example 6 as detection antibodies. The measurement results were compared to examine the performance of the reagents containing each R-PE-labeled antibody. The specific measurement procedure was as follows. Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE were each diluted with dilution buffer (2% FBS, 2 mM EDTA / D-PBS (pH 7.4)) so that the fluorescence intensity of R-PE was approximately the same, to prepare detection reagents. CD20-expressing Ramos cells were stirred in each detection reagent and reacted at 4°C for 30 minutes. The cells were pelleted by centrifugation and washed with dilution buffer. Each R-PE-labeled antibody bound to CD20 on the surface of Ramos cells was detected using a BD Accuri™ C6 Plus flow cytometer (Becton, Dickinson and Company).

[0192] Experimental Example 12: Modification of Fab antibody with SH-PEG-NH2 linker (3.5K) and SH-PEG-NH2 linker (5K) (1) Binding of SH-PEG-NH2 linker (3.5K) and SH-PEG-NH2 linker (5K) to Fab antibody Using BTGase, the SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) described above in (d) and the SH-PEG-NH2 linker (5K) (Sigma-Aldrich) described above in (e) were each bound to csF001-25Fab-Qtag. Specifically, the procedure was as follows. csF001-25Fab-Qtag was dissolved in Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2) to a concentration of 10 μM, and incubated overnight at 5° C. with 100 equivalents of SH-PEG-NH2 linker (3.5K) or SH-PEG-NH2 linker (5K) and 0.1 U / mL BTGase. The activity of BTGase was measured by the hydroxamic method. The progress of the reaction was analyzed by SEC. The reaction solution for each linker was desalted and purified using a Protein G column, and then concentrated using an Amicon 10K (Merck). The conditions for SEC analysis were as follows.

[0193] (2) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm, FL (Ex:295nm / Em:335nm)

[0194] Experimental Example 13: Fluorescent labeling of SH-PEG-Fab with Alexa488-maleimide (1) Fluorescent labeling of SH-PEG-Fab SH-PEG(3.5K)-Fab and SH-PEG(5K)-Fab from csF001-25Fab-Qtag were labeled with fluorescent dyes as follows. Each SH-PEG-Fab was concentrated with Amicon 10K (Merck) and buffer exchanged into 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). 6 μM reaction solution and 120 μM Alexa488-maleimide were incubated overnight at 5°C in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). Alexa488-PEG-Fab in the reaction solution was analyzed by HPLC using Superdex 200 Increase (Cytiva). The SEC analysis conditions were as follows.

[0195] (2) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm FL (Ex. 565 nm / Em. 574nm) PMT Super Low

[0196] Experimental Example 14: Modification of Fab antibody with SH-PEG-NH2 linker (400 Da) (1) Binding of SH-PEG-NH2 linker (400 Da) to Fab antibody The SH-PEG-NH2 linker (400 Da) (Nanocs Inc.) described above in (f) was bound to csF001-5Fab-Qtag using BTGase. Specifically, the procedure was as follows. csF001-5Fab-Qtag was dissolved in Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2) to a concentration of 10 μM, and incubated overnight at 5° C. with 50 equivalents of SH-PEG-NH2 linker (400 Da) and 0.1 U / mL BTGase. The activity of BTGase was measured by the hydroxamic method. The reaction solution was desalted and purified using Superdex 200 Increase (Cytiva), and then concentrated using Amicon 10K (Merch).

[0197] (2) ALP labeling of SH-PEG(400Da)-Fab 10 μM SH-PEG(400Da)-Fab and 5 μM maleimide-modified ALP were incubated overnight at 5° C. in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The reaction solution was analyzed by HPLC using Superdex 200 Increase (Cytiva). The SEC analysis conditions were as follows.

[0198] (3) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm FL (Ex. 295 nm / Em. 335 nm)

[0199] Experimental Example 15: Modification of Fab antibody with SH-PEG-NH2 linker (1K) (1) Binding of SH-PEG-NH2 linker (1K) to Fab antibody The SH-PEG-NH2 linker (1K) (Creative PEGWorks) described above in (g) was bound to HBs628Fab-Qtag using BTGase. Specifically, the procedure was as follows. HBs628Fab-Qtag was dissolved in MES buffer (50 mM MES, 2 mM EDTA, pH 7.0) to a concentration of 10 μM, and incubated with 50 equivalents of SH-PEG-NH2 linker (1K) and 0.1 U / mL BTGase at 25°C for 3 hours. The progress of the reaction was analyzed by SEC. The activity of BTGase was measured by the hydroxamic method. The reaction solution was desalted, concentrated, and purified using Amicon 10K (Merck).

[0200] (2) ALP labeling of SH-PEG(1K)-Fab 10 μM SH-PEG(1K)-Fab and 5 μM maleimide-modified ALP were incubated overnight at 5° C. in 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The reaction solution was analyzed by HPLC using Superdex 200 Increase (Cytiva). The SEC analysis conditions were as follows.

[0201] (3) Conditions of the SEC Analysis Column: Superdex 200 Increase 10×300 mm (Cytiva) HPLC equipment: Chromaster (registered trademark) (Hitachi High-Tech Science Corporation) Analysis temperature: room temperature Solvent: Arg-SEC Mobile Phase (Standard, Nacalai Tesque, Inc.) Flow rate: 0.8 ml / min Detection: UV 280nm FL (Ex. 295 nm / Em. 335 nm)

[0202] [result] The results of each of the above experiments are described below.

[0203] 1. Results of infusion MS analysis of SH-PEG-NH2 linker (2K) The results of the analysis of each of the above SH-PEG-NH2 linkers (2K) (a), (a'), (b) and (c) by Infusion MS are shown in Figures 2A, B, C and D. In Figures 2A, B and D, the arrows indicate the peak showing the minimum molecular weight, the peak showing the mode molecular weight, and the peak showing the maximum molecular weight. The mode molecular weights of the above linkers (a), (a'), (b) and (c) were 2014.2, 2058.2, 1397.8 and 1617.9, respectively. In addition, the weight average molecular weight of each linker was calculated based on the analysis results. As can be seen from Figures 2A, B, C and D, the weight average molecular weight and molecular weight distribution of each linker were different. Specifically, they were as follows. The weight average molecular weight was calculated from MS peaks with an S / N of 5 or more for linkers other than (a'), and was calculated from MS peaks with an S / N of 1.5 or more for linker (a').

[0204] The above linker (a): weight average molecular weight 2095.2, molecular weight distribution 1838.1 to 2410.4 The above (a') linker: weight average molecular weight 2029.5, molecular weight distribution 1794.1 to 2454.4 The linker (b) above: weight average molecular weight 1460.0, molecular weight distribution 1177.7 to 1794.1 The linker (c) above: weight average molecular weight 1611.9, molecular weight distribution 1221.7 to 2058.2

[0205] For all linkers, the molecular weight of the PEG chain portion excluding the functional groups (SH-CH2CH2- and -NH2) was 1100 or more. In addition, the weight-average molecular weight of the PEG chain portion excluding the functional groups was 1300 or more. After the addition of the BTGase reaction solution (NaCl-free, pH 8.2), the SH-PEG-NH2 linker (2K) did not show dimerization via disulfide (SS) bonds, and only the MS spectrum of the monomer correlated with the molecular weight distribution of the PEG chain was detected. Therefore, it was shown that none of the SH-PEG-NH2 linkers (2K) in (a), (a'), (b), and (c) above were associated in the BTGase reaction solution.

[0206] 2. Modification of csF001-5Fab-Qtag with SH-PEG-NH2 linker (2K) using BTGase (Experimental Example 1) The results of SEC analysis of the product after the reaction of csF001-5Fab-Qtag with the above-mentioned SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by BTGase are shown in FIG. 3. Specifically, it is a chromatogram of the desalted and concentrated reaction solution. In the figure, "+SH-PEG-NH2" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (2K) was added, and "-SH-PEG-NH2" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (2K) was not added. As shown in FIG. 3, when the linker was not added, no change was observed in the Fab peak. In other words, no intermolecular association between the Fabs occurred between the Gln residue in the Q tag and the Lys residue on the Fab. On the other hand, when the linker was added, the peak of unmodified Fab at around retention time 18.5 minutes decreased, and one peak shifted to the high molecular weight side at around retention time 17.5 minutes was newly confirmed. This indicates that the apparent size of SH-PEG-Fab, in which one molecule of the linear SH-PEG-NH2 linker is bound via a Q-tag, is increased and observed as a separate peak. In fact, the peak is broader than the unmodified Fab peak because the PEG chain in the linker has a wide molecular weight distribution as shown in Figures 2A-D. The reaction efficiency calculated from the peak area was about 73%. This was a similar trend to the previous case of modifying a polymeric PEG linker with BTGase (see Sato H. et al., Biochemistry, 35(40) 13072-13080 (1996)).

[0207] Furthermore, in order to increase the introduction efficiency with BTGase, the analysis results when the reaction pH was changed from 7.5 to 8.2 and 8.5 are shown in FIG. 4. In the figure, "SH-PEG" represents the SH-PEG-NH2 linker (2K) (Sigma-Aldrich) in (a) above. As shown in FIG. 4, the nucleophilicity of the amino group of the SH-PEG-NH2 linker to the thioether bond intermediate between the active SH group in BTGase and the Gln side chain in Fab-Qtag was increased, and the reaction efficiency was improved from 73% to about 95% and about 96%, respectively, confirming that quantitative introduction is possible. At pH 7.0, the reaction efficiency was about 73%, as in the past, and it was shown that the introduction of the SH-PEG-NH2 linker can be performed quantitatively by setting the pH of the BTGase reaction to around 8 to 8.5.

[0208] 3. Modification of csF028-22Fab-Qtag with SH-PEG-NH2 linker (2K) using BTGase (Experimental Example 1) The results of SEC analysis of the product after the reaction of csF028-22Fab-Qtag with the above-mentioned SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by BTGase under reaction conditions (pH 8.2 or 8.5) that increased the introduction efficiency are shown in FIG. 5. In the figure, "SH linker +" is the analysis result of the reaction solution to which SH-PEG-NH2 linker (2K) was added, and "SH linker -" is the analysis result of the reaction solution to which SH-PEG-NH2 linker (2K) was not added. As shown in FIG. 5, when the linker was not added, no change was observed in the Fab peak, as with csF001-5Fab-Qtag. In other words, no intermolecular association between Fabs occurred between the Gln residue in the Q tag and the Lys residue on the Fab. On the other hand, when the linker was added, the peak of unmodified Fab at around retention time 19.3 minutes decreased, and a new broad peak shifted to the high molecular weight side at around retention time 18.2 minutes was confirmed. This indicates that in the SH-PEG-Fab in which one molecule of the linear SH-PEG-NH2 linker is bound via a Q-tag, the apparent size increased and it was observed as a separated peak, although it was not completely separated. The peak was broader than the unmodified Fab peak because the PEG chain in the linker has a wide molecular weight distribution. The reaction efficiency was 96% because the peak was not completely separated from the unmodified form. In fact, the peak after fractionation was a single broad peak.

[0209] 4. Modification of csF001-5Fab-Qtag with SH-PEG-NH2 linker (3.5K) using BTGase (Experimental Example 12) Figure 6 shows the results of SEC analysis of the product after the reaction of csF001-5Fab-Qtag with the above-mentioned SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) by BTGase under the reaction conditions that improved the introduction efficiency. In the figure, "SH linker +" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (3.5K) was added, and "SH linker -" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (3.5K) was not added. When the linker was not added, no change was observed in the Fab peak, as with csF001-5Fab-Qtag. In other words, no intermolecular association between Fabs occurred between the Gln residue in the Q tag and the Lys residue on the Fab. On the other hand, when the linker was added, the peak of unmodified Fab at around retention time 19.3 minutes decreased, and a new broad peak shifted to the high molecular weight side at around retention time 17.8 minutes was confirmed. This indicates that the apparent size of SH-PEG-Fab, in which one molecule of the linear SH-PEG-NH2 linker is bound via a Q-tag, is increased, and although not completely separated, it is observed as a separated peak. The peak is broader than the unmodified Fab peak because the PEG chain in the linker has a wide molecular weight distribution. The reaction efficiency was about 84%, which was slightly lower than that of the reaction using the SH-PEG-NH2 linker (2K). This is thought to be because the molecular chain length of the SH-PEG-NH2 linker (3.5K) is longer than that of the SH-PEG-NH2 linker (2K), which somewhat reduced the substrate reactivity of BTGase. In fact, the peak after separation was a single broad peak.

[0210] 5. ALP labeling of SH-PEG-Fab from csF001-5Fab-Qtag (Experimental Example 3) (1) SEC analysis of coupling reaction mixture SH-PEG(2K)-Fab from csF001-5Fab-Qtag, maleimide-modified ALP ((Mal) nFigure 7 shows the results of SEC analysis of the SH-PEG(2K)-Fab solution and its coupling reaction solution, as well as the results of its non-reducing SDS-PAGE. In the figure, "Fab+SH-PEG" refers to the analysis result of the SH-PEG(2K)-Fab solution, "ALP+EMCS" refers to the analysis result of the solution containing maleimide-modified ALP, and "Fab:ALP (1:1) coupling" and "coupling" refer to the analysis result of the coupling reaction solution of SH-PEG(2K)-Fab and maleimide-modified ALP. Despite the fact that the mixture ratio of Fab and ALP was only 1:1, only about 20% of the peak derived from SH-PEG(2K)-Fab (retention time approximately 17.5 minutes) remained, and (Mal) n The peak derived from -ALP (retention time: about 15.3 min) also decreased. A main peak (Fab-PEG-ALP, retention time: about 13.5 min) estimated to be one Fab molecule bound to one ALP molecule, and a sub-peak ((Fab-PEG)2-ALP, retention time: about 12.5 min) estimated to be two Fab molecules bound to one ALP molecule were confirmed. In fact, taking into account the results of non-reducing SDS-PAGE (corresponding to the product band of the dimeric ALP monomer (ALP(m))) of each raw material and reaction solution, the peak separation of the Fab-coupled product was very high compared to unmodified ALP. This was due to the apparent increase in size caused by the binding of the Fab molecule to ALP via the linear PEG molecule.

[0211] (2) SEC fractionation of the coupling reaction peak and SEC analysis of the obtained fractions The coupling reaction solution of SH-PEG(2K)-Fab from csF001-5Fab-Qtag and maleimide-modified ALP was fractionated by SEC, and the results of non-reducing SDS-PAGE and SEC analysis of each fraction obtained are shown in Figures 8A to 8C. In Figure 8A, the two peaks enclosed by dashed lines were concluded to be ALP-labeled Fab ((Fab-PEG)2-ALP) in which ALP and two molecules of Fab are bound, and ALP-labeled Fab (Fab-PEG-ALP) in which one molecule of ALP and one molecule of Fab are bound, in order of the earliest retention time. In fact, in Figure 8B, for Fab-PEG-ALP, a band of ALP(m) (molecular weight about 60,000) and two bands at a molecular weight of about 100,000 to 130,000, which are thought to be the binding of one molecule of Fab, were detected. Regarding the detection of two bands for ALP(m), it was considered that the maleimide modification of the ALP molecule was a random modification of the side chain of the Lys residue in the ALP molecule, and therefore two kinds of Fab-ALP monomer conjugate species of apparent size were generated by the binding site of SH-PEG-Fab to ALP via SH-PEG. In fact, a case in which a random single molecule conjugate of a linear polymeric PEG chain was confirmed as two kinds of bands in electrophoresis has been reported in the past (see Sato H., Advanced Drug Delivery Reviews, 54, 487-504 (2002)). It was considered that this was due to the effect of the EMCS linker on ALP, which is bound to Fab via SH-PEG, being randomly modified to the ALP molecule. For (Fab-PEG)2-ALP, almost no band of unmodified ALP was observed in the electrophoresis. The main one was Fab-PEG-ALP, which has the above-mentioned molecular weight of about 100,000 to 130,000, which is two bands. Furthermore, a band thought to be derived from (Fab-PEG)2-ALP was detected at a molecular weight of about 200,000. These results were reasonable. Of these, we decided to evaluate the performance of Fab-PEG-ALP. As shown in Figure 8C, both purified products had one peak in the SEC analysis, and the purity was high.The molecular weight estimated from the migration band of the purified product was detected on the higher molecular weight side than the sum of the molecular weights of each protein. This was thought to be because the apparent size of the conjugated molecule increased due to the conjugation via the linear polymer PEG (2K).

[0212] 6. ALP labeling of SH-PEG-Fab from csF028-22Fab-Qtag (Experimental Example 3) (1) SEC analysis of coupling reaction mixture SH-PEG(2K)-Fab from csF028-22Fab-Qtag, maleimide-modified ALP ((Mal) n The results of SEC analysis of SH-PEG(2K)-Fab and maleimide-modified ALP, as well as their coupling reaction solutions, are shown in Figure 9. In the figure, "coupling" refers to the analysis result of the coupling reaction solution of SH-PEG(2K)-Fab and maleimide-modified ALP. Even though the mixture ratio of Fab to ALP was only 3:1, only about 30% of the peak derived from SH-PEG(2K)-Fab (retention time approximately 18.4 minutes) remained, and (Mal) n Only about 10% of the peak derived from -ALP (retention time: approximately 15.3 min) remained. The reaction efficiency was high, similar to other ALP couplings. In addition to the sub-peak estimated to be Fab-PEG-ALP (retention time: approximately 14.8 min) and the sub-peak estimated to be (Fab-PEG)2-ALP (retention time: approximately 13.8 min), a main peak estimated to be one ALP molecule bound to three Fab molecules ((Fab-PEG)3-ALP, retention time: approximately 12.8 min) was confirmed.

[0213] (2) SEC fractionation of the coupling reaction peak and SEC analysis of the obtained fractions SH-PEG(2K)-Fab from csF028-22Fab-Qtag and maleimide-modified ALP ((Mal) nThe coupling reaction solution with (Fab-PEG)3-ALP was fractionated by SEC, and the results of non-reducing SDS-PAGE and SEC analysis of each fraction obtained are shown in Figures 10A and B. As shown in Figure 10A, bands corresponding to the ALP monomer modification profile according to the estimated number of Fabs bound were observed in each fraction. In Figure 10B, the peaks were concluded to be (Fab-PEG)3-ALP, (Fab-PEG)2-ALP, and Fab-PEG-ALP in order of the earliest retention time. The purity of (Fab-PEG)3-ALP and (Fab-PEG)2-ALP was high, while the purity of Fab-PEG-ALP was slightly lower. However, the main product was confirmed as the main peak in both cases.

[0214] 7. Coupling reaction of sF001-5Fab' with maleimide-modified ALP (Experimental Example 4) sF001-5Fab' and (Mal) n The chromatograms obtained by SEC fractionation of the coupling reaction solution with -ALP and the results of non-reducing SDS-PAGE analysis of each fraction obtained are shown in Figures 11A and B. In the ALP labeling of SH-PEG-Fab from csF001-5Fab (see Experimental Example 3), SH-PEG-Fab and (Mal) n The molar ratio of Fab':ALP was 1:1 in this reaction, whereas the coupling ratio of Fab':ALP was 5:1. As a result, as shown in Figure 11A, the unreacted (Mal) n The area of ​​the -ALP peak was larger than that of the coupled product. This is due to the SH group in the side chain of the Cys residue in sF001-5Fab' and (Mal) n The coupling efficiency of SH-PEG-Fab from csF001-5Fab with ALP was nThis meant that the coupling efficiency with SH-PEG-Fab and maleimide-modified ALP was lower than that with -ALP. In fact, the maleimide modification rate of ALP used in each of the SH-PEG linker method and random labeling method was higher, with the amount of EMCS reagent added being 20 equivalents for the former and 30 equivalents for the latter. In addition, since multiple bands were observed on SDS-PAGE due to the actual EMCS modification, it was considered that the maleimide modification rate of ALP in the coupling reaction in the random labeling method was also high. These results showed that the coupling efficiency of SH-PEG-Fab and maleimide-modified ALP by the SH-PEG linker method was significantly higher than that by the random labeling method.

[0215] ALP exists as a 150 kDa dimer consisting of two 75 kDa molecules, but as shown in Figure 11B, it was observed as a monomer in SDS-PAGE, and a band was observed around 70 kDa. On the other hand, multiple bands were observed at 100 kDa or higher in the coupled product. Unlike the conjugate using PEG-SH linker, the coupled product reflects the apparent size, and it is considered that bands of one Fab molecule conjugate, two Fab molecules conjugate, and even conjugates with multiple molecules bound are generated. Furthermore, in the high molecular weight coupled product, since no oxidation step was performed after reduction, it is possible that some Fab' molecules were generated in which the SS between the H chain and L chain of Fab' remained reduced and two ALP monomers were bound to the Fab' molecule. However, since almost no bands derived from unlabeled ALP were detected in the fractions, the (Fab) used in the following evaluation was not included. n -ALP was hardly contaminated with unlabeled ALP. n The SEC analysis results of the purified SH-PEG-Fab-ALP sample are shown in Figure 12. Unlike the results of the SEC analysis of the homogeneous complexes of SH-PEG-Fab and ALP from csF001-5Fab ((Fab-PEG)2-ALP and Fab-PEG-ALP) (see Figure 5), Figure 12 shows that (Fab) n The SEC peak of -ALP was broad, and the peak resolution for each number of Fab bonds was low. This is because the former has a larger apparent molecular weight due to the Fab and ALP bonded via the PEG chain, and the SEC resolution was improved, whereas the (Fab) nIn the case of -ALP, the apparent molecular weight was not large because the bond was via a short EMCS linker, and the separation was low. In addition, the individual peaks for each number of Fab bonds were broad for (Fab-PEG)2-ALP and Fab-PEG-ALP, respectively. This is because Fab-PEG is a bond only to the SH group at the end of the PEG chain, which is selective for the hinge region, whereas the randomly labeled coupling product has multiple Fab bond sites, such as the hinge region Cys residue and the interchain Cys residue, resulting in a more heterogeneous ALP bond structure.

[0216] 8. Measurement of ALP activity of ALP-labeled antibody (Experimental Example 7) Table 3 shows the ALP specific activity of the ALP molecule itself and Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP from csF028-22Fab-Qtag. As shown in Table 3, the specific activity of each ALP-labeled antibody did not decrease even when the number of Fab-PEG bonds to the ALP molecule increased. This shows that the production method of this embodiment can prepare an ALP-labeled polypeptide that retains ALP activity. The fact that the specific activity exceeded 100% was believed to be due to the influence of the purity of the fractionated sample.

[0217] [Table 3]

[0218] 9. Fab-PEG-ALP and (Fab') n -Performance evaluation of reagents containing ALP (Experimental Example 8) (1) Reactivity of R3 reagent Fab-PEG-ALP from csF001-5Fab-Qtag and (Fab') obtained by random labeling method nThe results of measuring samples based on D-PBS (0.1% BSA, pH 7.4) using each of the R3 reagents, and the results are shown in Figures 13A-C, divided into signal, noise (also called background), and signal / noise (S / N) ratio. In the figures, the antibody concentration on the horizontal axis represents the activity of ALP bound to each antibody. The measurement value (count) of the HISCL (registered trademark) HIV Ag+Ab reagent increases depending on the antigen concentration, so in the graph of Figure 13A, the higher the signal value, the higher the reactivity with the antigen. Conversely, in the graph of Figure 13B, the lower the noise value, the higher the specificity. (Fab') n In the case of -ALP, the signal was relatively large, but the noise was also large. As a result, as shown in Figure 13C, (Fab') n In the case of csF001-5Fab-Qtag, the signal-to-noise ratio (S / N), which indicates the sensitivity of the detection reagent, was low. On the other hand, in the case of Fab-PEG-ALP from csF001-5Fab-Qtag, both the signal and noise were (Fab') n -It is smaller than ALP. In particular, the noise is (Fab') n As a result, the signal-to-noise ratio (S / N) indicating sensitivity was 0.5 U / mL (Fab'). n The ALP activity of Fab-PEG-ALP was 4 times higher than that of Fab'-ALP. n The reason why -ALP showed a higher signal value was considered to be that, on average, multiple Fab molecules bound to one ALP molecule. From the above results, it is expected that the sensitivity of in vitro diagnostic agents will be improved by using the enzyme-labeled antibody obtained by the production method of this embodiment.

[0219] (2) Suppression of background increase due to serum-derived components To compare the background, Fab-PEG-ALP from csF001-5Fab-Qtag and (Fab') nThe results of measuring D-PBS (0.1% BSA, pH 7.4) and human pooled serum using each of the R3 reagents are shown in Figure 14. In the measurement of the PBS-based sample, Fab-PEG-ALP from csF001-5Fab-Qtag had a higher (Fab') n As mentioned above, the background was lower than that of -ALP, but as shown in FIG. 14, the difference was even more remarkable when the background was compared in human serum. n In the case of Fab-PEG-ALP, the background increased 5.6-fold in serum, whereas in the case of Fab-PEG-ALP, the increase was barely noticeable, at 1.26-fold. Since serum contains various proteins and lipids, the background is often higher than in buffer-based samples. In these results, the heterogeneous structure (Fab') n The reason why Fab-PEG-ALP via SH-PEG linker was able to suppress the serum-derived background increase compared to -ALP is thought to be that Fab-PEG-ALP is a molecular species composed of a linear PEG linker that is highly hydrophilic upon hydration in addition to having a homogeneous structure. Although not shown in the figure, the sensitivity of Fab-PEG-ALP increased over the entire range of p24 antigen concentrations due to the reduced background, and was improved by up to about 30 times.

[0220] 10. Preparation of SH-PEG-Fab from HBs628Fab-Qtag and its biotin labeling (Experimental Examples 1 and 2) (1) Modification of HBs628Fab-Qtag with SH-PEG-NH2 linker (2K) using BTGase The results of SEC analysis of the product after the reaction of HBs628Fab-Qtag with the above-mentioned SH-PEG-NH2 linker (2K) (Sigma-Aldrich) by BTGase are shown in FIG. 15. In the figure, "+SH-PEG-NH2" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (2K) was added, and "-SH-PEG-NH2" is the analysis result of the reaction solution to which the SH-PEG-NH2 linker (2K) was not added. As shown in FIG. 15, when the linker was not added, no change was observed in the Fab peak, as with csF001-5Fab-Qtag. In other words, no intermolecular association between Fab molecules occurred between the Gln residue in the Q tag and the Lys residue on the Fab. On the other hand, when the linker was added, the peak of unmodified Fab at around retention time 12.3 minutes decreased, and one peak shifted to the high molecular weight side at around retention time 11.5 minutes was newly confirmed. This indicates that the apparent size of SH-PEG-Fab, in which one molecule of the linear SH-PEG-NH2 linker is bound via a Q-tag, is increased and observed as a separate peak. The peak is broader than the unmodified Fab peak because the PEG chain in the linker has a wide molecular weight distribution. The reaction efficiency calculated from the peak area was approximately 72%, which was comparable to the results for csF001-5Fab-Qtag.

[0221] (2) Biotin labeling of SH-PEG-Fab from HBs628Fab-Qtag The results of the SEC analysis of the peak at a retention time of 11.5 minutes of the reaction solution of csF001-5Fab-Qtag and the above (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) and the results of the SEC analysis of the peak of the reaction solution of the concentrated solution containing SH-PEG-Fab and Biotin-PEAC5-maleimide are shown in Figures 16A and B. The peaks separated in the SEC analysis of each reaction solution were the peaks surrounded by the dashed line in Figure 16A. Referring to Figure 16B, the non-reducing SDS-PAGE analysis of the reaction solution of csF001-5Fab-Qtag and SH-PEG-NH2 linker (2K) and the separated sample showed that the product SH-PEG-Fab was slightly shifted to the high molecular weight side at around 40 KDa compared to the unreacted Fab (band indicated by →), suggesting that one molecule of SH-PEG-NH2 linker was bound. In addition, two bands were detected around 20 KDa in the electrophoresis due to the fact that the H chain and L chain were not partially linked. This was due to the change in the packing structure of the Fab due to the binding of the SH-PEG-NH2 linker (2K), and some of the bands were reduced by the SH groups of the linker. In fact, even if the Fab and the SH-PEG-NH2 linker (2K) were left in the reaction solution without the addition of BTGase, the SS bond between the H chain and L chain of the Fab was not reduced (Data not shown). Referring to Figure 16C, the purified sample had a high SEC purity and was one peak.

[0222] Referring to FIG. 16B, the non-reducing SDS-PAGE analysis of the reaction solution of SH-PEG-Fab with Biotin-PEAC5-maleimide and the fractionated sample showed a band shifted slightly from unmodified Fab at around 40 KDa, similar to SH-PEG-Fab. The band intensity reflected the peak intensity ratio of the modified fraction in the SEC fractionated chromatogram of the reaction solution. In addition, the bands derived from the H chain and L chain observed in SH-PEG-Fab were hardly detected, and it was confirmed that the SS bond of Fab was reconstituted. This was considered to be because the packing structure of SH-PEG-Fab was stabilized by the modification with Biotin-PEAC5-maleimide, and the interchain SS bond that had been partially broken was reconstituted. Furthermore, only the reaction solution of this biotin-modified product and its fraction showed a high intensity band at the position of the product in non-reducing SDS-PAGE, indicating that SH-PEG-Fab was labeled with biotin.

[0223] 11. LC-MS analysis of SH-PEG-Fab and biotin-PEG-Fab from HBs628Fab-Qtag (Experimental Example 2) (1) LC-MS analysis of HBs628Fab-Qtag LC-MS spectra of non-reduced and reduced samples of HBs628Fab-Qtag are shown in Figures 17A and B. Referring to Figure 17A, in the LC-MS of the non-reduced sample, multiply charged ions of +20 to +41 ions of HBs628Fab-Qtag were observed. Although some Cys residues may have been reduced, the spectrum reflected the measured molecular weight. On the other hand, referring to Figure 17B, in the LC-MS of the reduced sample, multiply charged ions of the L chain and H chain were observed. In addition, for the L chain, a cleavage product in which two residues at the N-terminus were cleaved was observed. Although the N-terminus of the L chain and H chain was pyroglutamylated, it was shown that they were the designed backbone structure.

[0224] (2) Results of LC-MS analysis of SH-PEG-Fab and biotin-PEG-Fab The LC-MS spectra of the non-reduced samples of HBs628Fab-Qtag, its SH-PEG-Fab, and biotin-PEG-Fab are shown in FIG. 18. For SH-PEG-Fab and biotin-PEG-Fab, respectively, multivalent ions indicated by ▼ were observed in the middle and lower spectra of FIG. 18. The signal reflected the molecular weight distribution of the PEG chain and was observed as a broad signal. The measured average molecular weights (number average molecular weights) of non-reduced SH-PEG-Fab and biotin-PEG-Fab were 48710 and 49223, respectively. The difference from the measured average molecular weight (number average molecular weight) of unmodified HBs628Fab-Qtag was 2141 and 2654, respectively. Therefore, it was suggested that SH-PEG-Fab and biotin-PEG-Fab were bound to one SH-PEG chain (2K) and one biotin-PEG chain (2K), respectively, on average.

[0225] The LC-MS spectra of the reduced samples of HBs628Fab-Qtag and its SH-PEG-Fab are shown in Figure 19. Multivalent ions of the L chain and H chain were observed upon reduction. The spectral pattern of the L chain of SH-PEG-Fab was not different from that of HBs628Fab-Qtag. On the other hand, the H chain of SH-PEG-Fab was observed as a broad signal reflecting the molecular weight of the PEG chain (see the part marked with an arc "⌒"). These results indicate that one molecule of the SH-PEG-NH2 linker (2K) (Sigma-Aldrich) in (a) above is bound to the H chain of HBs628Fab-Qtag by a reaction catalyzed by BTGase. Here, it is known that endogenous Gln residues in antibodies, except for Gln295 after deglycosylation of the Fc portion, are not substrates for BTGase (see International Publication No. 2012 / 059882 and Jeger S. et al., Angew. Chem. Int. Ed. Engl., 49, 9995-9997 (2010)). Therefore, it was suggested that one molecule of SH-PEG-NH2 linker (2K) was bound to the Gln residue in the Q tag of HBs628Fab-Qtag.

[0226] 12. ALP labeling of SH-PEG-Fab from HBs628Fab-Qtag (Experimental Example 3) SH-PEG(2K)-Fab from HBs628Fab-Qtag and maleimide-modified ALP ((Mal) n The coupling reaction solution with Fab and ALP) was fractionated by SEC, and the results of SEC analysis of each fraction obtained are shown in Figure 20. The coupling reaction solution and each raw material were analyzed by non-reducing SDS-PAGE, and the results are shown in Figure 21. Referring to Figure 20, there was no significant difference in the residual rate of SH-PEG-Fab calculated from the peak area ratio in the SEC analysis of reaction solutions with a mixing ratio of Fab and ALP of 2:1, 1:1, or 1:2, and approximately 80% had reacted. The (Mal) of SH-PEG-Fab n The higher the molar ratio of (Fab-PEG)2-ALP added, the higher the proportion of the peak area at approximately 12.3 minutes derived from (Fab-PEG)2-ALP. This indicates that, similar to the results for csF001-5Fab-Qtag, the reaction efficiency of SH-PEG-Fab with maleimide-modified ALP is increased by the PEG linker effect. Furthermore, it was shown that the binding ratio of Fab to ALP can be adjusted by controlling the reaction molar ratio. In addition, under the three reaction conditions, (Mal) n The residual rate of the -ALP peak decreased as the molar ratio of the added SH-PEG-Fab increased, which was reasonable. Referring to FIG. 21, the results of non-reducing SDS-PAGE (corresponding to the product band of the dimeric ALP monomer) of each raw material and reaction solution show that (Mal) n The higher the ratio of SH-PEG-Fab to -ALP, the more bands that were thought to be SH-PEG-Fab bound to ALP monomers were observed. The results of non-reducing SDS-PAGE also showed that the coupling ratio between proteins such as Fab and ALP can be adjusted, as the proportion of bands in the high molecular weight range increased.

[0227] 13. Preparation of SH-PEG-Fab from csF001-25Fab-Qtag and its fluorescent labeling (Experimental Examples 1 and 5) (1) Modification of csF001-25Fab-Qtag with SH-PEG-NH2 linker (2K) using BTGase Figure 22 shows the results of SEC analysis of the desalted and concentrated reaction solution after the reaction of csF001-25Fab-Qtag with the above-mentioned SH-PEG-NH2 linker (2K) (Sigma-Aldrich) using BTGase under reaction conditions that increased the introduction efficiency. In the figure, "Fab" refers to the analysis result of the reaction solution to which no SH-PEG-NH2 linker (2K) was added. The peak of unmodified Fab at a retention time of approximately 19.8 minutes almost disappeared, and a new peak shifted to the high molecular weight side at a retention time of approximately 18.9 minutes was confirmed. The reaction efficiency was 93% based on the peak area.

[0228] (2) Fluorescent labeling of SH-PEG-Fab with Alexa488-maleimide The results of SEC analysis of the reaction solution of SH-PEG(2K)-Fab from csF001-25Fab-Qtag and Alexa488-maleimide are shown in Figures 23A and B. In Figure 23A, the upper chromatogram is the analysis result of the reaction solution to which 20 equivalents of Alexa488-maleimide was added, and the lower chromatogram is the analysis result of the reaction solution to which Alexa488-maleimide was not added. Referring to Figure 23A, no difference was observed in the retention time, indicating that aggregation due to fluorophore labeling did not occur. Furthermore, in the reaction solution to which Alexa488-maleimide was not added, the lower chromatogram did not show any peaks derived from the association between SH-PEG chain linkers, indicating that no association occurred. The SEC analysis of the desalted and purified fraction of the reaction solution to which Alexa488-maleimide was added was confirmed by UV and fluorescence absorption, and as shown in Figure 23B, the product showed fluorescence absorption derived from Alexa488. This demonstrates that Alexa488-PEG-Fab in which SH-PEG-Fab and Alexa488-maleimide are bound in a one-to-one ratio was prepared.

[0229] 14. LC-MS analysis of SH-PEG-Fab and Alexa488-PEG-Fab from csF001-25Fab-Qtag (Experimental Example 5) LC-MS spectra of the reduced samples of csF001-25Fab-Qtag and its SH-PEG-Fab and Alexa488-PEG-Fab are shown in Figures 24A and B. Figure 24B is an enlarged view of Figure 24A. Due to the binding of the SH-PEG-NH2 linker (2K), the spectral pattern of the L chain of SH-PEG-Fab was not different from that of csF001-25Fab-Qtag. On the other hand, the multivalent ion of the H chain of SH-PEG-Fab (see the part marked with ↓) was observed as a broad signal reflecting the molecular weight of the PEG chain (see the part marked with ▼). This result indicated that one molecule of SH-PEG-NH2 linker (2K) was bound to the H chain of csF001-25Fab-Qtag by the reaction catalyzed by BTGase. As mentioned above, it is known that endogenous Gln residues in antibodies are not substrates for BTGase, except for Gln295 after removal of the glycan from the Fc portion. This suggests that one molecule of the SH-PEG-NH2 linker (2K) (Sigma-Aldrich) in (a) above is bound to the Gln residue in the Q tag of HBs628Fab-Qtag. Furthermore, the broad spectrum of the multivalent ions of the H chain originating from the PEG chain almost disappeared upon binding of Alexa488-maleimide, and a shift reflecting the binding of one molecule of Alexa488 was observed (see the part marked with a ▼). This result indicated that Alexa488-PEG-Fab is a Fab to which one molecule of Alexa488 is bound via the SH-PEG linker.

[0230] 15. Modification of anti-CD20 Fab-Qtag with SH-PEG-NH2 linker (2K) using BTGase (Experimental Example 1) The results of SEC analysis of the desalted and concentrated reaction solution after the reaction of anti-CD20 Fab-Qtag with the above (a') SH-PEG-NH2 linker (2K) (Sigma-Aldrich) using BTGase under reaction conditions with improved incorporation efficiency are shown in Figure 25. In the figure, "Fab-Qtag" refers to the analysis result of the reaction solution to which SH-PEG-NH2 linker (2K) was not added. The peak of unmodified Fab at retention time 20.2 minutes almost disappeared, and a new peak shifted to the high molecular weight side, at retention time 19.0 minutes, was confirmed. The reaction efficiency was 77% based on the peak area. Although the reaction was performed under conditions with improved incorporation efficiency, the reaction efficiency was lower than that of other Fab-Qtags. This is probably because the Q tag does not have a proline residue at its C-terminus, making it susceptible to the effects of peptidases, and the Fab-Qtag raw material contained Qtag that had been cleaved.

[0231] 16. R-PE labeling of SH-PEG-Fab (Experimental Example 6) (1) Maleimide modification of R-PE The results of SEC and reverse-phase HPLC analysis of the reaction solution between R-PE and EMCS reagent are shown in Figures 26A and B. Referring to Figure 26A, the peak of maleimide-modified R-PE obtained by reaction with EMCS reagent did not show a significant difference in retention time compared to unmodified R-PE. This suggests that the structure of R-PE was maintained. Meanwhile, in the reverse-phase HPLC analysis results, as shown in Figure 26B, the peaks presumed to be the α and β subunits, which are the main constituent units of R-PE, were broadened toward the rear of the retention time due to EMCS modification. This suggests that the maleimide-modified product (Mal) to which multiple highly hydrophobic EMCS reagents are bonded is a maleimide-modified product. n Based on the peak area values, the peaks were assigned to the β subunit with a larger molecular weight, and the front peak to the α subunit with a smaller molecular weight, but these assignments were tentative.

[0232] (2) Coupling reaction of SH-PEG-Fab derived from csF001-25Fab-Qtag with maleimide-modified R-PE SH-PEG(2K)-Fab from sF001-25Fab-Qtag, (Mal) n The results of SEC analysis of SH-PEG(2K)-Fab, SH-PEG(2K)-R-PE, and their coupling reaction solutions are shown in Figure 27. Even though the mixture ratio of Fab and R-PE was only 3:1, the peak derived from SH-PEG(2K)-Fab (retention time: approximately 19.2 minutes) decreased, and (Mal) n Only about 7% of the peak derived from -R-PE (retention time: approximately 16.0 min) remained. In other words, the reaction efficiency was high. Based on the retention time assumed to be the PEG effect (increasing apparent molecular weight), in addition to the peak estimated to be Fab-PEG-R-PE (retention time: approximately 14.8 min), peaks estimated to be (Fab-PEG)2-R-PE (retention time: approximately 13.4 min) and (Fab-PEG)3-R-PE (retention time: approximately 12.8 min) were confirmed.

[0233] (3) SEC fractionation of the coupling reaction peak and SEC analysis of the obtained fractions The coupling reaction solution of SH-PEG(2K)-Fab from sF001-25Fab-Qtag and maleimide-modified ALP was fractionated by SEC, and the results of SEC analysis of each fraction obtained are shown in Figure 28. From Figure 28, it was concluded that each peak was (Fab-PEG)3-R-PE, (Fab-PEG)2-R-PE, and Fab-PEG-R-PE in order of the earliest retention time. The fluorescence intensity per UV peak area tended to decrease as the number of Fab-PEG bonds increased, reflecting the predicted bond structure. The purity of (Fab-PEG)3-ALP and (Fab-PEG)2-ALP was high, while the purity of Fab-PEG-ALP was slightly lower. However, the main product was confirmed as the main peak in both cases. The product peak was confirmed to have PE-derived fluorescence absorption due to the reaction with R-PE.

[0234] (4) Coupling reaction of SH-PEG-Fab from anti-CD20 Fab-Qtag with maleimide-modified R-PE SH-PEG(2K)-Fab from anti-CD20 Fab-Qtag, (Mal) n The results of SEC analysis of SH-PEG(2K)-Fab, SH-PEG(2K)-R-PE, and their coupling reaction mixtures are shown in Figure 29. Even though the mixture ratio of Fab and R-PE was only 3:1, the peak derived from SH-PEG(2K)-Fab (retention time: approximately 19.2 minutes) decreased, and (Mal) n Almost no peak derived from -R-PE (retention time approximately 15.8 minutes) remained. In other words, the reaction efficiency was high. Based on the retention time assumed to be the PEG effect (increasing apparent molecular weight), in addition to a peak estimated to be Fab-PEG-R-PE (retention time approximately 14.8 minutes), peaks estimated to be (Fab-PEG)2-R-PE (retention time approximately 13.4 minutes) and (Fab-PEG)3-R-PE (retention time approximately 12.8 minutes) were confirmed. Fluorescence absorption derived from PE was confirmed in the product peak due to the reaction with R-PE.

[0235] (5) SEC fractionation of the coupling reaction peak and SEC analysis of the obtained fractions The coupling reaction solution of SH-PEG(2K)-Fab from anti-CD20 Fab-Qtag and maleimide-modified ALP was fractionated by SEC, and the results of SEC analysis of each fraction obtained are shown in Figure 30. From Figure 30, it was concluded that each peak was (Fab-PEG)3-R-PE, (Fab-PEG)2-R-PE, and Fab-PEG-R-PE in order of the earliest retention time. The fluorescence intensity per UV peak area tended to decrease as the number of Fab-PEG bonds increased, reflecting the predicted bond structure. The purity of (Fab-PEG)3-ALP and (Fab-PEG)2-ALP was high, while the purity of Fab-PEG-ALP was slightly lower. However, the main product was confirmed as the main peak in both cases. The product peak was confirmed to have PE-derived fluorescence absorption due to the reaction with R-PE.

[0236] 17. Evaluation of antigen binding ability of Alexa488-labeled antibodies (Experimental Example 9) The interaction of csF001-25Fab-Qtag with its SH-PEG(2K)-Fab and Alexa488-PEG-Fab with the antigen was measured using Biacore™ T200 (Cytiva), and the results are shown in Table 4 and Figures 31A-D. The measurement dates for SH-PEG(2K)-Fab and Alexa488-PEG-Fab were different, so the results are shown separately. As shown in Table 4, the KD values ​​of SH-PEG(2K)-Fab and Alexa488-PEG-Fab were not different from the KD value of unmodified Fab (csF001-25Fab-Qtag). In addition, as shown in Figures 31A-D, no significant difference was observed in the sensorgram patterns. Therefore, there was no effect on the antigen binding ability by this labeling method.

[0237] [Table 4]

[0238] 18. Antigen binding ability of R-PE-labeled antibody (Experimental Example 10) The results of measuring the antigen binding ability of Fab-PEG-R-PE, (Fab-PEG)2-R-PE and (Fab-PEG)3-R-PE from csF001-25Fab-Qtag by ELISA are shown in Figures 32A and B. Figure 32A shows the results when no antigen was added, and Figure 32B shows the results when antigen was added. Referring to Figure 32A, the fluorescence intensity of the detection reagent was made uniform (i.e., the molar concentration of the R-PE-labeled antibody was made uniform), and the fluorescence signal values ​​at each dilution ratio were compared. As a result, no significant difference was observed in the detected fluorescence signal value at any dilution ratio. On the other hand, referring to Figure 32B, when antigen was added, differences were observed in the detected fluorescence signal value between the R-PE-labeled antibodies. Specifically, at the same dilution ratio, the fluorescence signal value of Fab-PEG-R-PE was the lowest, the fluorescence signal value of (Fab-PEG)2-R-PE was the second highest, and the fluorescence signal value of (Fab-PEG)3-R-PE was the highest. That is, the higher the number of Fab-PEG molecules bound, the higher the fluorescent signal value. This is thought to be because the avidity effect in antigen binding is exerted by binding multiple Fab-PEG molecules to the R-PE molecule, and the antigen capture ability per R-PE molecule is increased. In fact, by making the concentrations of (Fab-PEG)2-R-PE and (Fab-PEG)3-R-PE in the detection reagent about 1 / 3 and about 1 / 5 of those of Fab-PEG-R-PE, respectively, the fluorescent signal value was shown to be about the same as that when Fab-PEG-R-PE was used. From this result, it was shown that the R-PE-labeled antibody in which multiple Fab-PEG molecules are bound to R-PE has an increased antigen binding ability and has the effect of increasing the fluorescent signal value of the antibody-R-PE conjugate converted to a molecule.

[0239] 19. Performance evaluation of reagents containing R-PE-labeled antibodies (Experimental Example 11) The results of measuring CD20-expressing cells (Ramos cells) by FCM using Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE from anti-CD20 Fab-Qtag as detection antibodies are shown in FIG. 33. The fluorescence signal values ​​at each dilution ratio were compared after the fluorescence intensity of the detection reagent was made uniform (i.e., the molar concentration of the R-PE-labeled antibody was made uniform). Referring to FIG. 33, differences were observed between the R-PE-labeled antibodies in the detected fluorescence signal values. Specifically, at the same dilution ratio, the fluorescence signal value of Fab-PEG-R-PE was the lowest, the fluorescence signal value of (Fab-PEG)2-R-PE was the second highest, and the fluorescence signal value of (Fab-PEG)3-R-PE was the highest. In other words, the higher the number of Fab-PEG molecules bound, the higher the fluorescence signal value. This is believed to be because the binding of multiple Fab-PEG molecules to the R-PE molecule exerts an avidity effect in binding to CD20 on cells, increasing the antigen capture ability per R-PE molecule. In fact, by reducing the concentrations of (Fab-PEG)2-R-PE and (Fab-PEG)3-R-PE in the detection reagent to about 1 / 3 and about 1 / 4 of that of Fab-PEG-R-PE, respectively, the fluorescence signal value was similar to that when Fab-PEG-R-PE was used. Therefore, it was shown that the R-PE-labeled antibody, in which multiple Fab-PEG molecules are bound to R-PE, can improve the performance of the detection reagent used in FCM due to the avidity effect.

[0240] 20. Results of Infusion MS analysis of SH-PEG-NH2 linker (3.5K) and SH-PEG-NH2 linker (5K) The results of the analysis of each of the SH-PEG-NH2 linkers (Sigma-Aldrich) (d) and (e) by Infusion MS are shown in Figures 34A and B. In the figures, the arrows indicate the peak showing the minimum molecular weight, the peak showing the mode molecular weight, and the peak showing the maximum molecular weight. The mode molecular weights of the linkers (d) and (e) were 3511.1 and 4699.8, respectively. In addition, the weight-average molecular weight of each linker was calculated based on the analysis results. As can be seen from Figures 34A and B, the weight-average molecular weight and molecular weight distribution of each linker had a profile close to the average molecular weight disclosed by the manufacturer. Specifically, it was as follows.

[0241] The linker (d) above: weight average molecular weight 3544.2, molecular weight distribution 2982.7 to 4171.5 The above (e) linker: weight average molecular weight 4586.6, molecular weight distribution 3951.3 to 5228.1

[0242] After addition of the BTGase reaction solution (NaCl-free, pH 8.2), almost no dimerization via disulfide (SS) bonds was observed for each SH-PEG-NH2 linker, and the MS spectrum of the monomer correlated with the molecular weight distribution of the PEG chain was detected. Therefore, it was shown that neither of the SH-PEG-NH2 linkers (d) and (e) above were associated.

[0243] 21. Fluorescent labeling of SH-PEG-Fab with Alexa488-maleimide (Experimental Examples 12 and 13) (1) Modification of csF001-5Fab-Qtag with SH-PEG-NH2 linker (3.5K / 5K) using BTGase The results of SEC analysis and non-reducing SDS-PAGE of the desalted and concentrated reaction solution after the reaction of csF001-25Fab-Qtag with the above-mentioned (d) SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) and (e) SH-PEG-NH2 linker (5K) (Sigma-Aldrich) under the reaction conditions with improved introduction efficiency by BTGase are shown in Figures 35A-C. Referring to Figure 35A, the peak of unmodified Fab at retention time 19.8 minutes almost disappeared, and new peaks shifted to the high molecular weight side at retention times of 18.4 minutes (3.5K) and 17.9 minutes (5K) were confirmed. These retention times were shifted to the high molecular weight side compared to SH-PEG(2K)-Fab. The reaction efficiency was 98% (3.5K) and 92% (5K) based on the peak area.

[0244] (2) Fluorescent labeling of SH-PEG-Fab with Alexa488-maleimide The results of SEC analysis of the reaction solution between SH-PEG(3.5K)-Fab derived from csF001-25Fab-Qtag and Alexa488-maleimide are shown in Figures 36A and B. The results of SEC analysis of the reaction solution between SH-PEG(5K)-Fab derived from csF001-25Fab-Qtag and Alexa488-maleimide are shown in Figures 37A and B. In Figures 36A and 37A, the upper chromatograms are the analysis results of the reaction solution to which 20 equivalents of Alexa488-maleimide was added, and the lower chromatograms are the analysis results of the reaction solution to which no Alexa488-maleimide was added.

[0245] Referring to Figure 36A, in the SEC analysis of the reaction solution, a slight difference in retention time was observed due to the binding of Alexa488 (SH-PEG-Fab: about 18.6 minutes, Alexa488-PEG-Fab: about 18.4 minutes), but it was shown that no aggregation due to the fluorophore labeling occurred. In addition, as shown in Figure 36B, in the SEC analysis of the reaction solution, the product showed fluorescence absorption derived from Alexa488 at a retention time of about 18.4 minutes. Therefore, it was shown that Alexa488-PEG-Fab in which SH-PEG(3.5K)-Fab and Alexa488-maleimide were bound in a one-to-one ratio was produced.

[0246] Referring to Figure 37A, in the SEC analysis of the reaction solution, a slight difference in retention time was observed due to the binding of Alexa488 (SH-PEG-Fab: about 18.2 minutes, Alexa488-PEG-Fab: about 17.9 minutes), but it was shown that no aggregation due to the fluorophore labeling occurred. In addition, as shown in Figure 37B, in the SEC analysis of the reaction solution, the product showed fluorescence absorption derived from Alexa488 at a retention time of about 18.0 minutes. Therefore, it was shown that Alexa488-PEG-Fab in which SH-PEG(5K)-Fab and Alexa488-maleimide were bound in a one-to-one ratio was produced.

[0247] 22. Modification of Fab antibody with SH-PEG-NH2 linker (400 Da) (Experimental Example 14) (1) Modification of csF001-5Fab-Qtag with SH-PEG-NH2 linker (400 Da) using BTGase The results of SEC analysis (fractionation) of the reaction solution of csF001-5Fab-Qtag and the above (f) SH-PEG-NH2 linker (400 Da) (Nanocs Inc.) with BTGase are shown in Figure 38. As shown in Figure 38, when SH-PEG-NH2 linker (400 Da) was added, the peak top shifted slightly forward in retention time. There was a possibility that the apparent size of the Fab bound to one molecule of the linker was no different from that of the unmodified Fab (csF001-5Fab-Qtag) on ​​the chromatogram. Therefore, the fractionation was used for the next coupling reaction with maleimide-modified ALP.

[0248] (2) ALP labeling of SH-PEG(400Da)-Fab SH-PEG(400Da)-Fab from csF001-5Fab-Qtag, maleimide-modified ALP ((Mal) n The results of SEC analysis of the reaction mixture, (Mal) Fab reaction product and (Mal) Fab reaction product, and their coupling reaction solution are shown in Figure 39. As shown in Figure 39, a new broad peak was detected at a retention time of about 13.8 minutes in the SEC analysis of the reaction mixture. n The decrease in the peak area of ​​the raw material derived from -ALP was hardly observed. n It was concluded that the coupling reaction with -ALP hardly proceeded, and even if a coupling product was formed, it was only in a small amount. Therefore, it was shown that the SH-PEG-NH2 linker (400 Da) is not suitable for the method for producing the labeled polypeptide of this embodiment.

[0249] 23. Modification of Fab antibody with SH-PEG-NH2 linker (1K) (Experimental Example 15) (1) Modification of HBs628Fab-Qtag with SH-PEG-NH2 linker (1K) using BTGase The results of SEC analysis of the reaction solution of HBs628Fab-Qtag and the above (g) SH-PEG-NH2 linker (1K) (Creative PEGWorks) with BTGase are shown in Figure 40. As shown in Figure 40, when the linker was not added, no change was observed in the Fab peak. In other words, no intermolecular association of Fabs occurred between the Gln residue in the Q tag and the Lys residue on the Fab. On the other hand, when the linker was added, the peak of unmodified Fab at a retention time of about 18.7 minutes decreased, and although the separation was not good, a new peak was confirmed on the high molecular weight side. Compared to the chain length of the SH-PEG-NH2 linker (2K), the chain length of the SH-PEG-NH2 linker (1K) was half that of the SH-PEG-NH2 linker (2K), so it was thought that the apparent size would not increase significantly even if it was bound to Fab-QTag. Therefore, the peak at a retention time of about 17.7 minutes was presumed to be SH-PEG (1K)-Fab. In addition to this peak, a higher molecular weight peak was observed at a retention time of approximately 16.9 minutes. This position was on the higher molecular weight side than the elution position by SEC of the fraction presumed to be SH-PEG(1K)-Fab. Therefore, it was speculated that the higher molecular weight fraction was PEG-SS-PEG-Fab generated by association via SS bonds between SH groups of the SH-PEG linker. In fact, Dickgiesser S. et al. (see Bioconjugate Chemistry, 31, 1070-1076 (2020)) attempted to selectively introduce a low molecular weight linker with an SH group, Cysteamine (SH-CH2-CH2-NH2), into Gln residues in an antibody as a TG substrate in a reaction with BTGase, but reported that a dimer in which the SH groups in the above cysteamine were associated with each other was introduced in the TG-modified antibody with cysteamine. Therefore, after the reaction, the reaction was completely reduced using the reducing agent TCEP, and then SS reconstitution was performed by reoxidation. Given these findings, it was believed that the dimer formation by the association of SS groups presumed to have been generated by the SH-PEG-NH2 linker (1K) was undoubted. Even if unreacted Fab-Qtag and PEG-SS-PEG-Fab were present in the coupling reaction solution, they did not have free SH groups and were (Mal)n Since it did not react with -ALP, the reaction solution was desalted and used in the subsequent coupling reaction with maleimide-modified ALP.

[0250] (2) ALP labeling of SH-PEG(1K)-Fab SH-PEG(1K)-Fab from HBs628Fab-Qtag, maleimide-modified ALP ((Mal) n The results of SEC analysis of the coupling reaction mixtures of Fab and ALP are shown in FIG. 41. As a result of the coupling reaction being carried out at a mixing ratio of Fab and ALP of 2:1, as shown in FIG. 41, (Mal) n The peak derived from -ALP (retention time approximately 15.2 min) reacted at approximately 43%, and among the SH-PEG(1K) reaction products, the peak component thought to be derived from SH-PEG-Fab (retention time approximately 17.8 min) disappeared, and a new peak was confirmed that was presumed to be Fab-PEG-ALP, in which one molecule of Fab is bound to ALP. However, the main product was the peak presumed to be Fab-PEG-ALP (retention time approximately 13.8 min), and only a small amount of (Fab-PEG)2-ALP (retention time approximately 12.8 min), in which two molecules of Fab are bound to ALP, was produced. Thus, when the SH-PEG-NH2 linker (1K) reacted with Fab-Qtag, the linkers dimerized, resulting in (Mal) n The coupling efficiency with -ALP was low. From these results, it was found that Fab-Qtag and (Mal) n The results showed that the coupling product with -ALP was poorly separable by SEC between SH-PEG(1K)-Fab and unmodified Fab-Qtag. In addition, the coupling efficiency between proteins was low due to side reactions caused by the association of linkers, and it was determined that the use of the SH-PEG-NH2 linker (1K) was not practical.

[0251] From the above experimental results, it was found that the present invention provides a higher efficiency of introducing a label into a polypeptide than when the SH-PEG-NH2 linker (400 Da) or the SH-PEG-NH2 linker (1K) is used. [Explanation of symbols]

[0252] 10: Reagents 11: Reagent kit 12: 1st container 13: Packing box 14: Attached Documents

Claims

1. A labeled polypeptide comprising a glutamine residue having a side chain represented by formula (I): 【Chemistry 1】 (In the formula, (C) is the α carbon of a glutamine residue, X is a linear alkylene group, Y is a polyethylene glycol chain, Z is a label, L is a spacer or a bond, and the molecular weight of the polyethylene glycol chain is 1100 or more.)

2. The tagged polypeptide according to claim 1, which is a fusion polypeptide between an antibody and a peptide tag containing a glutamine residue having the side chain.

3. A modified polypeptide comprising a glutamine residue having a side chain represented by formula (II): 【Chemistry 2】 (In the formula, (C) is the α carbon of the glutamine residue, X is a linear alkylene group, and Y is a polyethylene glycol chain, the molecular weight of the polyethylene glycol chain being 1100 or more.)

4. The modified polypeptide according to claim 3, which is a fusion polypeptide between an antibody and a peptide tag containing a glutamine residue having the side chain.

5. The polypeptide according to any one of claims 1 to 4, wherein the weight-average molecular weight of the polyethylene glycol chain is 1,300 or more.

6. The polypeptide according to any one of claims 1 to 4, wherein the weight-average molecular weight of the polyethylene glycol chain is 1700 or more.

7. The polyethylene glycol chain has the following formula (III): -(OCH 2 CH 2 ) n - or -(CH 2 CH 2 O) n -(III) (In the formula, n is an integer of 25 or more.) The polypeptide according to any one of claims 1 to 4, wherein the polypeptide is represented by the formula:

8. The polypeptide according to claim 7, wherein n is an integer of 39 or greater.

9. The polypeptide according to any one of claims 1 to 4, wherein the linear alkylene group has 2 or more and 10 or less carbon atoms.

10. 3. The labeled polypeptide according to claim 1, wherein the label is at least one selected from the group consisting of biotins, enzymes, fluorescent dyes, fluorescent proteins and haptens.

11. The polypeptide of claim 2 or 4, wherein the antibody is a Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, dAb, rIgG, light chain, heavy chain antibody, variable region of a heavy chain antibody, diabody or triabody.

12. A reagent comprising the polypeptide according to any one of claims 1 to 4.

13. In the presence of transglutaminase, a polypeptide containing glutamine residues and a polypeptide represented by the following formula (VI): NH 2 -X-Y-SH (VI) (In the formula, X is a linear alkylene group, Y is a polyethylene glycol chain, and the molecular weight of the polyethylene glycol chain is 1100 or more.) attaching said linker to the carboxamide side chain of the glutamine residue by contacting said linker with obtaining a modified polypeptide formed by coupling the carboxamide side chain to the linker; Including, The modified polypeptide further comprises a side chain of the glutamine residue having the formula (II): 【Chemistry 3】 (In the formula, (C) is the α carbon of the glutamine residue, X is a linear alkylene group, and Y is a polyethylene glycol chain, the molecular weight of the polyethylene glycol chain being 1100 or more.) Represented by Methods for producing modified polypeptides.

14. In the presence of transglutaminase, a polypeptide containing glutamine residues and a polypeptide represented by the following formula (VI): NH 2 -X-Y-SH (VI) (In the formula, X is a linear alkylene group, Y is a polyethylene glycol chain, and the molecular weight of the polyethylene glycol chain is 1100 or more.) attaching said linker to the carboxamide side chain of the glutamine residue by contacting said linker with obtaining a modified polypeptide formed by coupling the carboxamide side chain to the linker; contacting the modified polypeptide with a label having a maleimide group, thereby binding the label to the linker bound to the modified polypeptide; Obtaining the labeled polypeptide produced by binding the modified polypeptide to a label; wherein the side chain of the glutamine residue in the labeled polypeptide is represented by the following formula (I): 【Chemistry 4】 (In the formula, (C) is the α carbon of a glutamine residue, X is a linear alkylene group, Y is a polyethylene glycol chain, Z is a label, L is a spacer or a bond, and the molecular weight of the polyethylene glycol chain is 1100 or more.) Represented by A method for producing a labeled polypeptide.

15. The method according to claim 13 or 14, wherein the polypeptide containing a glutamine residue is a fusion polypeptide of an antibody and a peptide tag containing a glutamine residue.

16. The method according to claim 13 or 14, wherein the weight average molecular weight of the polyethylene glycol chain is 1,300 or more.

17. The method according to claim 13 or 14, wherein the weight average molecular weight of the polyethylene glycol chain is 1700 or more.

18. The polyethylene glycol chain has the following formula (III): -(OCH 2 CH 2 ) n - or -(CH 2 CH 2 O) n -(III) (In the formula, n is an integer of 25 or more.) The method according to claim 13 or 14, wherein the formula is

19. The method according to claim 18, wherein n is an integer of 39 or more.

20. The method according to claim 13 or 14, wherein the linear alkylene group has 2 or more and 10 or less carbon atoms.

21. The method according to claim 14, wherein the label is at least one selected from the group consisting of biotins, enzymes, fluorescent dyes, fluorescent proteins and haptens.

22. The method of claim 15, wherein the antibody is at least one selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, dAb, rIgG, light chain, heavy chain antibody, variable region of a heavy chain antibody, diabody, and triabody.

23. forming an immune complex between the labeled polypeptide according to claim 2 and a target substance; detecting a signal generated by the label contained in the immune complex; A method for measuring a target substance, comprising:

24. The method according to claim 23, wherein in the forming step, the immune complex is formed on a solid phase.

25. 25. The method according to claim 23 or 24, further comprising a step of removing unreacted free components between the forming step and the detecting step.

26. The method according to claim 23 or 24, wherein the target substance is at least one selected from the group consisting of proteins, oligopeptides, nucleic acids, lipids, sugar chains and haptens.

27. The method according to claim 23 or 24, wherein the label is at least one selected from the group consisting of an enzyme, a fluorescent dye, and a fluorescent protein.

28. The measurement method according to claim 23, wherein the label of the labeled polypeptide is a fluorescent dye or a fluorescent protein, and in the detection step, the immune complex is introduced into a flow cell of a flow cytometer, and the signal is detected by the flow cytometer.

29. The measurement method according to claim 28, wherein the target substance is a formed component having at least one type selected from the group consisting of proteins, oligopeptides, nucleic acids, lipids, sugar chains and haptens on its surface.

30. The measurement method according to claim 29, wherein the formed component is at least one selected from the group consisting of cells, extracellular vesicles, microorganisms, viruses, and fragments thereof.

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