Methods for predicting the in vivo pharmacokinetics of molecules

Abstract

The present invention provides: a method for measuring the in vitro pharmacokinetics of a molecule; and others. The method comprises: (a) a step for bringing the molecule into contact with a cell capable of expressing an FcRn in a water-based medium to cause the cell to incorporate the molecule in such a manner that the amount of incorporation becomes higher than 0.068 pmol / 2×105cells, in which the step has at least one characteristic selected from the items (i) to (iii) mentioned below; and (b) a step for measuring the in vitro pharmacokinetics of the molecule. The items (i) to (iii) are as follows: (i) the time of the contact of the molecule with the cell is 5 hours or longer; (ii) the cell that has been contacted with the molecule is not washed under an acidic condition; and (iii) the cell expresses a target for the molecule on the surface thereof. In the method, the molecule contains an FcRn binding domain.

Description

[Technical Field]

[0001] The present invention relates to a method for measuring the in vitro pharmacokinetics of a molecule, a method for predicting the in vivo pharmacokinetics of a molecule, a method for screening a molecule, and the like. [Background technology]

[0002] Laboratory animals such as monkeys and mice are used to evaluate the pharmacokinetics (PK) of therapeutic antibodies and other pharmaceuticals. However, it is difficult to evaluate a large number of samples using laboratory animals, so in vivo pharmacokinetics is evaluated after narrowing down the number of samples in advance. Furthermore, from an ethical standpoint, it is necessary to reduce the number of laboratory animals used. For these reasons, methods for predicting in vivo pharmacokinetics based on the results of in vitro assays are becoming increasingly important.

[0003] Conventionally, methods that utilize the results of in vitro assays using cells have been known as methods for predicting in vivo pharmacokinetics (Non-Patent Documents 1 to 3). Grevys et al. have disclosed a method for predicting the half-life of IgG antibodies in transgenic mice by measuring the amount of IgG antibodies secreted extracellularly via FcRn in vitro using a method they call HERA assay (human endothelial cell-based recycling assay) using cells (HMEC1-hFcRn) in which human fetal Fc receptors (FcRn) have been expressed in a human microvascular endothelial cell line (HMEC1) (Non-Patent Document 1). Jaramillo et al. disclose that they used Madin-Darby canine kidney (MDCK) cells expressing human FcRn or rat FcRn to measure the activity of antibodies to permeate the cells via FcRn, i.e., transcytosis activity, and thereby ranked the in vivo clearance of antibodies (Non-Patent Document 2). Similarly to the method of Jaramillo et al., Chung et al. also measured transcytosis activity using MDCK cells expressing human FcRn, and found a correlation between the measurement results and in vivo clearance in humans (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Grevys et al, Nat Commun. 2018. Vol. 9: 621 [Non-patent document 2] Jaramillo et al, MAbs. 2017. Vol. 9: 781 [Non-patent document 3] Chung et al. J Immunol Methods. 2018. Vol. 462: 101 Summary of the Invention [Problem to be solved by the invention]

[0005] Previous methods for predicting in vivo pharmacokinetics based on the results of in vitro cell-based assays have shown insufficient accuracy. For example, in the method of Grevys et al., the HERA score ((R X / R WT ) / (RA X / RA WT ): R represents the amount of protein taken up by cells and released extracellularly within a given time period, RA represents the remaining amount, X represents the target protein (mutant), and WT represents the parent protein used to normalize the results. Correlation between ) and in vivo pharmacokinetics was observed only when the pharmacokinetics of the antibodies being compared differed significantly. Furthermore, it has not been shown to predict the in vivo pharmacokinetics of antibodies with long blood half-lives, such as those exceeding 11 days. Furthermore, in the method of Jaramillo et al., a correlation was observed between the reciprocal of in vitro transcytosis activity (flux) and in vivo clearance in the case of Fc-modified antibodies, but when comparing antibodies with small differences in in vivo clearance, the accuracy of predicting in vivo clearance from in vitro measurement results was not achieved (see Figure 6A in Non-Patent Document 2). Furthermore, in the results of analyzing antibodies with different antigens, no correlation was observed between in vitro data and in vivo data (see Figure 6B in Non-Patent Document 2). Even with the method of Chung et al., when comparing antibodies with small differences in in vivo clearance, the accuracy of predicting in vivo clearance from in vitro measurement results was not achieved. Therefore, an object of the present invention is to provide a method for predicting the in vivo pharmacokinetics of a molecule with higher sensitivity and accuracy than conventional methods, based on the results of in vitro pharmacokinetic measurements. [Means for solving the problem]

[0006] The present inventors have intensively investigated the reason why conventional methods have insufficient accuracy in predicting in vivo pharmacokinetics based on in vitro pharmacokinetics. As a result, they have found that conventional methods have low prediction accuracy due to insufficient cellular uptake of molecules, and that increasing the amount of cellular uptake of molecules improves prediction accuracy. Based on these findings, the present inventors have conducted further research and have completed the present invention.

[0007] That is, the present invention provides the following inventions. [1] A method for measuring the in vitro pharmacokinetics of a molecule, comprising the steps of: (a) When the molecule was contacted with cells expressing FcRn in an aqueous medium, the uptake was 0.068 pmol / 2 × 10 5A step of incorporating the molecule into cells so that the molecule is elevated above the cells, the step having at least one feature selected from the following (i) to (iii): (i) the contact time between the molecule and the cell is 5 hours or more; (ii) the cells are not washed under acidic conditions after contacting the molecule; and (iii) the cell expresses a target of the molecule on the cell surface; and (b) measuring the in vitro pharmacokinetics of the molecule; Including, The method, wherein the molecule comprises an FcRn-binding domain. [2] The method according to [1], wherein the molecule is an antibody comprising an FcRn-binding domain and a target-binding domain. [3] The method according to [1] or [2], wherein the cell is a cell transformed to express FcRn. [4] The method according to any one of [1] to [3], wherein the cell is a cell transformed so as to express the target of the molecule on the cell surface. [5] The method according to [3] or [4], wherein the cells are CHO cells, HEK293 cells, COS-1 cells, COS-7 cells, MDCK cells, HMEC1 cells, HELA cells, HepG2 cells, or BaF cells. [6] The method according to [1] or [2], wherein the cells are liver parenchymal cells, liver non-parenchymal cells, liver sinusoidal endothelial cells, Kupffer cells, human umbilical vein endothelial cells, peripheral blood mononuclear cells (PBMCs), macrophages, monocytes, B cells, T cells, platelets, NK cells, neutrophils, eosinophils, basophils, granulocytes, or dendritic cells. [7] The uptake was 0.10 pmol / 2×10 5 The method according to any one of [1] to [6], wherein the molecule is taken up by the cells so that the molecule is elevated above the cells. [8] The method according to any one of [1] to [7], wherein the in vitro pharmacokinetics is the excretion rate from cells into the culture medium, the excretion rate from cells into the culture medium, the internalization rate, the transcytosis amount, the Kp value, the intracellular molecule reduction rate, the binding rate to FcRn or a target, or the dissociation rate from FcRn or a target. [9] The method according to any one of [1] to [8], wherein the FcRn is human FcRn, monkey FcRn, miniature pig FcRn, rat FcRn, mouse FcRn, rabbit FcRn, dog FcRn, or guinea pig FcRn.

[10] The following steps: (c) calculating in vitro evaluation parameters from the measurement results obtained in step (b); The method according to any one of [1] to [9], further comprising:

[11] The method according to

[10] , wherein the in vitro evaluation parameter is a clearance index or a HERA score.

[12] The method according to any one of [1] to

[11] , which is used for ensuring the quality or predicting the efficacy of a pharmaceutical containing the molecule.

[13] The method according to any one of [1] to

[12] , wherein the target of the molecule is a membrane protein.

[14] The method according to

[13] , wherein the target of the molecule is human IL6 receptor.

[15] A method for predicting the in vivo pharmacokinetics of a molecule, comprising: (a') measuring in vitro pharmacokinetics by the method according to any one of [1] to

[14] ; and (b') predicting the in vivo pharmacokinetics of the molecule when administered to a living body from the measured values ​​or in vitro evaluation parameters obtained in step (a'); The method comprising:

[16] The method according to

[15] , wherein the in vivo pharmacokinetics is bioavailability, distribution volume, unbound fraction in blood, clearance, urinary excretion rate, blood concentration half-life, or mean residence time.

[17] The method according to

[15] or

[16] , wherein the living organism is a human, monkey, miniature pig, rat, mouse, rabbit, dog, or guinea pig.

[18] The method according to any one of

[15] to

[17] , which is used as an alternative to pharmacokinetic testing using animals.

[19] A method for screening a molecule, comprising: (a'') providing two or more different molecules that bind to the same target; (b'') measuring the in vitro pharmacokinetics of each of the two or more molecules prepared in step (a'') by a method described in any one of [1] to

[14] ; and (c'') comparing the measured values ​​or in vitro evaluation parameters for each of the two or more molecules obtained in step (b'') with each other and selecting the molecules that show desirable values; The method comprising: [Effects of the Invention]

[0008] According to the present invention, the in vivo pharmacokinetics of molecules can be predicted more accurately and with higher sensitivity than conventional methods based on the results of in vitro pharmacokinetic measurements, making it possible to predict the in vivo pharmacokinetics of a large number of candidate substances easily and with high accuracy in the early stages of pharmaceutical development. Furthermore, the present invention can contribute to reducing the number of experimental animals used by reducing the number of in vivo pharmacokinetic tests. Furthermore, the present invention can contribute to the development of pharmaceuticals with higher pharmacological effects by providing a method for efficiently screening drugs with desired pharmacokinetics. [Brief explanation of the drawings]

[0009] [Figure 1]The results of pharmacokinetic evaluation of antibodies with different Fc regions (H237-G1d, H237-F1847m, H237-F1886m, H237-F1927m, and H237-F890) in mouse plasma are shown. Each antibody was administered at 1 mg / kg and Sanglobol at 1,000 mg / kg to human FcRn transgenic mice (Tg32). Blood samples were collected up to day 28, and plasma antibody concentrations were measured by ECL assay. The solid black circle represents H237-G1d, the short-dashed black triangle represents H237-F1847m, the solid black circle represents H237-F1886m, the long-dashed black square represents H237-F1927m, and the solid black triangle represents H237-F890. [Figure 2] Figure 1 shows the results of measuring the amount of antibody uptake into cells in vitro for antibodies with different Fc regions. Each antibody was taken up into hFcRn-hIL6R-CHO cells or hFcRn-CHO cells at 37°C for 24 hours, and the amount of uptake into cells is shown after washing with cold 2% FBS-containing PBS. [Figure 3-1] The graph shows the time course of cellular uptake of antibodies with different Fc regions. (a) Measurement results when cells were washed with FBS-PBS. After the antibody was taken up by the cells, the cells were washed with FBS-PBS. Both the antibody bound to the cell surface and the internalized antibody were detected. (b) Measurement results when cells were washed with acidic medium. After the antibody was taken up by the cells, the cells were washed with acidic medium. The antibody bound to the cell surface was removed, and only the internalized antibody was detected. The black solid line and black circle represent H237-G1d, the black short-dashed line and black triangle represent H237-F1847m, the black solid line and white circle represent H237-F1886m, the black long-dashed line and black square represent H237-F1927m, and the black solid line and white triangle represent H237-F890. [Figure 3-2]The graph shows the time course of cellular uptake of antibodies with different Fc regions. (c) shows the results of an integration plot analysis using the measurement results in (a) and (b). The black circle with a solid black line indicates H237-G1d, the black triangle with a short-dashed black line indicates H237-F1847m, the white circle with a solid black line indicates H237-F1886m, the black square with a long-dashed black line indicates H237-F1927m, and the white triangle with a solid black line indicates H237-F890. [Figure 4] The graph shows the time course of intracellular retention and release into the medium of antibodies with different Fc regions. Each antibody was taken up by cells at 37°C for 24 hours, then the medium was replaced with fresh medium and incubated for an additional 4 hours. The amount of antibody remaining in the cells (a) and the amount of antibody released into the medium (b) were measured over time. The black solid circle indicates H237-G1d, the black short-dashed triangle indicates H237-F1847m, the black solid white circle indicates H237-F1886m, the black long-dashed square indicates H237-F1927m, and the black solid white triangle indicates H237-F890. [Figure 5] 1 shows the correlation between the clearance index calculated in Example 4 and the plasma half-life (a) or clearance (b) in mice. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to these, and can be practiced in various modified forms within the described scope. Unless otherwise specified in this specification, "A to B" representing a numerical range means "A or more (including A and greater than A) and B or less (including B and less than B)." Furthermore, unless otherwise specified in this specification, "A and / or B" means "A, B, or both." Furthermore, all prior art documents cited in this specification are incorporated herein by reference.

[0011] I. Methods for measuring the in vitro pharmacokinetics of molecules A first aspect of the present invention relates to a method for measuring the in vitro pharmacokinetics of a molecule (hereinafter also referred to as the measuring method of the present invention).

[0012] As used herein, the term "in vivo pharmacokinetics" refers to the change in the concentration (amount) of a drug (i.e., a molecule of the present invention) in a living body after administration, following a series of processes including absorption, distribution, metabolism, and excretion. After a drug is administered, the processes of absorption, distribution, metabolism, and excretion occur in parallel in the body. The following basic pharmacokinetic (PK) parameters have been established to describe these processes: (1) bioavailability (F), (2) volume of distribution (Vd or V), (3) fraction unbound in blood (fuB), (4) clearance (CL), and (5) cumulative amount of drug excreted in urine (Ae) (Biometrics Vol. 36, Special Issue, S 3-S 18 (2015)). Known bioavailability indices include the area under the blood concentration-time curve (AUC), maximum blood concentration (Cmax), and time to maximum blood concentration (Tmax). As an index of distribution volume, the steady-state distribution volume (V ss ) are known. Other PK parameters include the blood concentration half-life (t 1 / 2 ), mean residence time (MRT), area under the first moment time curve (AUMC), elimination rate constant (kel), and zero concentration after administration (C0) are known.

[0013] As used herein, "in vitro pharmacokinetics" refers to the behavior of a molecule of interest measured by contacting the molecule with cells under artificially constructed conditions outside of a living body. "In vitro pharmacokinetics" can be expressed, for example, by the amount of excretion from inside the cell to outside the cell, the excretion rate from inside the cell to outside the cell, the internalization rate, the amount of transcytosis, the Kp value, the rate of intracellular molecule reduction, the binding rate to FcRn or a target, or the dissociation rate from FcRn or a target, but is not limited to these. The amount of target molecule released from inside the cell to outside the cell (Efflux amount) is determined by contacting the target molecule with the cell for a predetermined time, then replacing the aqueous medium (e.g., culture medium, buffer solution, etc.) with one that does not contain the target molecule, and then detecting the target molecule in the aqueous medium to measure the amount of target molecule released from the cell to outside the cell. The efflux rate from inside to outside the cell (Efflux rate) is determined by measuring the amount of Efflux of the target molecule per unit time. The internalization rate is determined by contacting a target molecule with cells for a predetermined time and measuring the amount of the target molecule taken up by the cells from outside the cells (e.g., from the medium, buffer, etc.) per unit time. In a preferred embodiment, the cells that have been contacted with the target molecule for a predetermined time are washed with an acidic aqueous medium (less than pH 6.0, e.g., pH 5.5 or less, pH 5.0 or less, pH 4.5 or less, pH 4.0 or less, pH 3.5 or less, or pH 3.0 or less) before measuring the amount of the target molecule, thereby removing the target molecule bound to the cell surface. This allows for a more accurate measurement of the amount of the target molecule taken up (internalized) into the cells. The amount of transcytosis can be determined by measuring the amount of permeation from one side of the cell sheet to the other, for example, using a Transwell® system (Corning) (see Non-Patent Documents 2 and 3). The Kp value is known as the tissue-plasma drug concentration ratio (i.e., the ratio of the concentrations of a target molecule between tissue and plasma) in in vivo pharmacokinetics, but herein, in terms of in vitro pharmacokinetics, it refers to the ratio of the concentrations of a target molecule between cells and an aqueous medium (e.g., culture medium). The Kp value is determined by measuring the amount in cells and the amount in aqueous medium. Herein, the Kp value for in vitro pharmacokinetics is a value calculated by (amount in cells) / (amount in aqueous medium). The rate of intracellular molecule depletion is determined by contacting the target molecule with cells for a predetermined time, then replacing the aqueous medium (e.g., culture medium, buffer solution, etc.) with one that does not contain the target molecule, and then detecting the target molecule within the cells to measure the amount of the target molecule depleted from the cells per unit time. The binding rate to FcRn or a target is determined by contacting the target molecule with cells for a predetermined short period of time (e.g., several seconds to several minutes) and measuring the amount of the target molecule bound to FcRn or the target per unit time. The dissociation rate from FcRn or a target is determined by contacting the target molecule with cells for a predetermined time (e.g., a time sufficient to reach equilibrium), then exchanging the aqueous medium (e.g., culture medium, buffer, etc.) for one not containing the target molecule, and detecting the target molecule in the aqueous medium to measure the amount of the target molecule excreted into the aqueous medium per unit time. In a preferred embodiment, the contact of the target molecule with cells and the release of the target molecule into the aqueous medium can be carried out at a temperature that inhibits internalization of the target molecule into cells (e.g., 4°C or lower).

[0014] As used herein, a "molecule" (also referred to as a "molecule of the present invention") has the property of being taken up by the cells used in the measurement method of the present invention and, within the cells, being secreted to the outside of the cells via fetal Fc receptor (FcRn) molecules on endosomes, which are intracellular organelles. This property is due to the molecule containing an FcRn-binding domain.

[0015] FcRn is a receptor that recognizes the Fc region of IgG antibodies. It is expressed in the fetal placenta and mediates the transcytosis of IgG from the mother to the fetus. In adults, FcRn is also expressed in vascular endothelium, intestinal epithelial cells, and blood cells, where it mediates the exocytosis and transcytosis of IgG and albumin from within the cells (Nature Reviews Immunology Vol. 7, pp. 715-725 (2007)). Human FcRn is a dimeric protein consisting of a light chain called the β2m subunit and a heavy chain called the α subunit with a transmembrane domain, and its structure resembles that of major histocompatibility complex (MHC) class I molecules. This FcRn dimer further dimerizes and binds to a single IgG molecule (Annual Review of Cell and Developmental Biology Vol. 12, pp. 181-220 (1996)). Unlike other IgG antibody Fc receptors, FcRn is known to exhibit pH-dependent binding via electrostatic interactions between anionic residues on its own α2 domain and the CH2-CH3 hinge region of IgG (Nature Reviews Immunology Vol. 7, pp. 715-725 (2007)). In endosomes, where the pH is below 6.5, IgG taken up into cells by pinocytosis binds with high affinity to FcRn, escaping degradation in lysosomes. It then dissociates upon migration to the cell surface under neutral conditions (pH 7.4). This pH-dependent binding mode enables IgG transcytosis and exocytosis, contributing to the transport of IgG from the mother to the fetus and the prolonged blood half-life of IgG in vivo (approximately 20 days) (Protein Cell Vol. 9(1), p. 15-32 (2018)).

[0016] Examples of FcRn-binding domains include antibody heavy chain constant regions (Fc regions) and fragments thereof. Another example of an FcRn-binding domain is albumin and fragments thereof. It is known that albumin binds to FcRn (J. Exp. Med. (2003) 197(3), 315-322). The FcRn-binding domain may contain a mutation, as long as it can bind to FcRn in the intraendosomal pH environment of less than pH 6.5. Examples of mutated Fc-binding domains include, but are not limited to, mutated Fc regions of antibodies described in WO 2012 / 133782 A1, WO 2013 / 046704 A2, and WO 2017 / 046994 A1.

[0017] The molecules of the present invention may further have the property of binding to a target (i.e., target binding activity) or the property of catalyzing a reaction in the target (i.e., enzymatic or catalytic activity). Molecules with target binding activity may function as agonists or antagonists. When possessing these properties, the molecule has a target binding domain or a catalytic domain. In a preferred embodiment, the molecules of the present invention contain a target binding domain, which can increase the amount of uptake into cells expressing the target on their cell surface.

[0018] As used herein, the term "target" refers to another molecule or structure that binds to a molecule of the present invention, or another molecule or structure that is catalytically acted upon by a molecule of the present invention. "Target" includes proteins, nucleic acids, sugar chains, etc. "Target" may also be referred to as an antigen, receptor, substrate, etc. in relation to the molecule of the present invention.

[0019] The structure of the target-binding domain is not particularly limited, and any structure can be used as long as it is capable of binding to a target. Examples of target-binding domains include the antigen-binding domain of an antibody, Avimers (International Publication Nos. WO2004 / 044011 and WO2005 / 040229) containing modules of approximately 35 amino acids (A domains) contained in various cell membrane proteins in vivo, Adnectins (International Publication No. WO2002 / 032925) containing the 10Fn3 domain of fibronectin, a glycoprotein expressed on cell membranes, Affibodies (International Publication No. WO1995 / 001937) using an IgG-binding domain consisting of 58 amino acids from Protein A as a scaffold, DARPins (Designed Ankyrin Repeat proteins) (International Publication No. WO2002 / 020565) containing ankyrin repeats (AR), a repeating sequence of 33 amino acids, as a backbone, and neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin). Examples include Anticalin (International Publication WO2003 / 029462), which contains lipocalins such as lipocalin (NGAL) as its skeleton, and variable lymphocyte receptor (VLR), a protein that functions in the adaptive immune system of jawless fish such as lampreys and hagfish and contains a leucine-rich-repeat (LRR) module (International Publication WO2008 / 016854). As used herein, an antigen-binding domain can be provided by one or more antibody variable domains. Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Suitable examples of such antigen-binding domains include "scFv (single chain Fv)," "single chain antibody," "Fv," "scFv2 (single chain Fv 2)," "Fab," and "F(ab')2." In certain embodiments, the target binding domain comprises the variable region of an antibody heavy and / or light chain. In preferred embodiments, the target binding domain comprises or consists of the variable region of an antibody heavy and light chain.

[0020] Catalytic domains include those in enzymes.

[0021] In a preferred embodiment, the molecule of the present invention comprises an FcRn-binding domain and a target-binding domain, more preferably an Fc region and heavy and light chain variable regions of an antibody.

[0022] Molecules of the present invention include pharmaceuticals and drug candidates, including, but not limited to, proteins such as antibodies, which are molecules described in the Examples, as well as peptide compounds, nucleic acids, toxins, viruses, and DDS preparations such as nanoparticles and microparticles, as long as they can bind to FcRn. Molecules of the present invention can be prepared by standard methods using techniques known in the art, depending on the type of molecule. According to the measurement methods of the present invention, the in vitro pharmacokinetics of these molecules can be measured in the same manner as in the Examples, and their in vivo pharmacokinetics can be predicted.

[0023] As used herein, the term "protein" refers to a polymer of amino acids linked via peptide bonds, and may include peptide compounds. Proteins may be naturally occurring or non-naturally occurring, such as recombinant proteins. Examples of proteins include cytokines, bioactive peptides, biological enzymes, antibodies, and variants thereof.

[0024] As used herein, the term "antibody" refers to a natural or partially or completely synthetically produced immunoglobulin. Antibodies can be isolated from natural sources such as plasma or serum, or from the culture supernatant of antibody-producing hybridoma cells, or can be partially or completely synthesized using techniques such as genetic recombination. Preferred examples of antibodies include immunoglobulin isotypes (i.e., IgG, IgA, IgD, IgE, and IgM) and their isotype subclasses. Nine subclasses of human immunoglobulins are known: IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. In a preferred embodiment, the antibody used in the measurement method of the present invention is IgG.

[0025] The antibody may be either a polyclonal antibody or a monoclonal antibody. Furthermore, in the present invention, recombinant antibodies that have been artificially modified for purposes such as reducing heterologous antigenicity, such as chimeric antibodies, humanized antibodies, and human antibodies, can be used. The antibody may also be a bispecific antibody. The antibody may also be an antibody fragment, so long as it contains an "FcRn-binding domain." Examples of such antibody fragments include Fc fragments and scFv-CH1-Fc. The "FcRn-binding domain" of an antibody may be any domain capable of binding to FcRn, and examples thereof include the heavy chain constant region (Fc region) of an antibody. The antibody molecule of the present invention preferably comprises an "antigen-binding domain," more preferably comprising the variable regions of the heavy and light chains of the antibody. This can increase the amount of uptake of the molecule of the present invention into cells that express the antigen on their cell surface. Methods for producing these antibodies are known to those skilled in the art (for example, WO 2013 / 081143, etc.).

[0026] As used herein, the structure of an "antigen" is not particularly limited as long as it contains an epitope to which an antigen-binding domain binds. The antigen may be an inorganic or organic substance. In some embodiments, antigens include 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 Antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6, Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, c AMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL2 8, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD 4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD3 0L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / E phB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractal Kine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), G DF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high-molecular-weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 , IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor receptor 1 (IGFR), insulin-like growth factor receptor 2 (IGFR), insulin-like growth factor receptor 3 (IGFR), insulin-like growth factor receptor 4 (IGFR), insulin-like growth factor receptor 5 (IGFR), insulin-like growth factor receptor 6 (IGFR), insulin-like growth factor receptor 7 (IGFR), insulin-like growth factor receptor 8 (IGFR), insulin-like growth factor receptor 9 (IGFR), insulin-like growth factor receptor 1 ... Factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES , MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B(OPG OCIF, TR1), TNFRSF12(TWEAK R FN14), TNFRSF13B(TACI), TNFRSF13C(BAFF R), TNFRSF14(HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17(BCMA), TNFRSF18(GITR AITR), TNFRSF19(TROY TAJ, TRADE), TNFRSF19L(RELT), TNFRSF1A(TNF RI CD120a, p55-60), TNFRSF1B(TNF RIICD120b, p75-80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3) Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α Connectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand) gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL-R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor)receptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP High-pressure washer CA125, high-pressure washers and wheelbarrows TW EAK, TXB2, Ung, uPAR, uPAR-1, VC AM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGF R, VEGFR-3(flt-4), VEGI, VIM, VLA, VLA-1, VLA-4, and VNR Liquid classes WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WN T7A、WNT7B、WNT8A、WNT8B、WNT9A、WNT9A、WNT9B、WNT10A、WNT10B、WNT11、WNT16、X CL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1. DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, low-density LDL, PCSK9, prekallikrein RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B tau VAP1 IL-31 IL-31R Nav1.1 Nav 1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1 Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9 factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, antioxidant factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, and factor IX、factor IXa、factor X、factor Xa、factor XI、factor Xia、factor XII、factorXIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, etc. In some embodiments, antigens include receptors for hormones and growth factors, etc.

[0027] When an antibody binds to multiple epitopes in an antigen molecule, such as a bispecific antibody, the antigen capable of forming a complex with the antibody may be any of the antigens exemplified above or a combination thereof, in other words, a monomer or a heteromultimer. Non-limiting examples of heteromultimers include heterodimers such as IL-12 comprising IL-12p40 and IL-12p35, IL-23 comprising IL-12p40 and IL-23p19 (also known as IL-30B), IL-23 comprising EBI-3 and IL27p28, or IL-35 comprising IL-12p35 and EBI-3.

[0028] The above examples of antigens also include receptors, which may exist in soluble form in biological fluids such as plasma. Such soluble receptors are also included in the antigens of the present invention. A non-limiting example of a soluble receptor is the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968) (e.g., a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1 in WO 2013 / 081143).

[0029] Although the above examples of antigens include soluble antigens, the solution in which the antigen exists is not limited, and the soluble antigen can exist in biological fluids, i.e., all fluids filling the vessels or tissues and cells in the body. In a non-limiting embodiment, the antigen bound by the antibody can be present in extracellular fluid. In vertebrates, extracellular fluid refers to components in bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, spinal fluid, aspirate, or synovial fluid, as well as transcellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activity of cells, and fluids in the digestive tract and other body cavities) such as alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor).

[0030] The above-mentioned examples of antigens include soluble antigens and membrane antigens, and it is well known to those skilled in the art which antigen each corresponds to. For example, classification can be achieved by searching for individual antigens on websites such as UniProtKB (https: / / www.uniprot.org / ) and Human Protein Atlas (https: / / www.proteinatlas.org / ).

[0031] When the molecule of the present invention is an antibody, the antibody may preferably be an IgG. In a specific embodiment, the molecule of the present invention may be an anti-IL-6R antibody, more particularly a humanized anti-IL-6R antibody.

[0032] In the present invention, "nucleic acid" refers to DNA, RNA, and their analogs, and may be natural or synthetic. Analogs include artificial nucleic acids such as PNA and LNA. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may also be modified. Modifications include those chemically modified in the internucleoside linkage, base, and / or sugar, and those with modified groups at the 5' and / or 3' ends. Modifications of internucleoside linkages include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, methylphosphonate linkages, phosphoramidate linkages, non-phosphate linkages, and methylphosphonothioate linkages, or combinations thereof. Base modifications include those to 5-propynyluracil, 2-aminoadenine, etc. Sugar modifications include those to 2'-fluororibose, 2'-O-methylribose, etc. Nucleic acids may be referred to as siRNA, antisense RNA, miRNA, shRNA, ribozymes, or aptamers depending on their function or use. The nucleic acids used in the present invention also include CpG oligonucleotides that act on Toll-like receptor 9 (TLR9) to activate innate immunity. The base length of the nucleic acid may be any length that allows it to be taken up into cells via stabilin, and is, for example, in the range of 4 to 100 bases, 10 to 50 bases, 10 to 40 bases, or 10 to 30 bases.

[0033] In one embodiment, when the molecule of the present invention is a nucleic acid, its target (or receptor) may be stabilin. As used herein, stabilin refers to a protein belonging to a family of transmembrane proteins known as nucleic acid receptors. Two homologs, stabilin-1 and stabilin-2, are known in mammals, and either of these may be used as stabilin in the present invention. In humans, stabilin-1 (NCBI accession number: NP_055951.2) and stabilin-2 (NCBI accession number: NP_060034.9) are known and have been reported to be expressed in LSECs, spleen, adrenal cortex, lymph nodes, and sinusoidal macrophages.

[0034] In the present invention, a "peptide compound" refers to a compound formed by amide bonds or ester bonds between amino acids or amino acid analogs. The molecular form of a peptide compound may be linear, cyclic, or cyclic with a linear portion. The number of amide bonds or ester bonds (number and length of amino acids or amino acid analogs) is not particularly limited, but when a linear portion is present, the total number of residues in the cyclic portion and linear portion is preferably 30 or less. The total number of amino acids in the cyclized portion and linear portion is more preferably 13 or less. To achieve high metabolic stability, the total number of amino acids is more preferably 9 or more. In addition to the above, the number of amino acids and amino acid analogs constituting the cyclic portion is preferably 5 to 12. Furthermore, in addition to the above, the number of amino acids and amino acid analogs constituting the cyclic portion is more preferably 5 to 11, and even more preferably 7 to 11 residues. 9 to 11 residues are particularly preferred. The number of amino acids and amino acid analogs (number of units) in the linear portion is preferably 0 to 8, and even more preferably 0 to 3. In this application, unless otherwise specified, amino acids may also include amino acid analogs.

[0035] In this specification, the "amino acids" and "amino acid analogs" that constitute the peptide compounds may be referred to as "amino acid residues" and "amino acid analog residues," respectively. The amino acids are α-, β-, and γ-amino acids, and are not limited to naturally occurring amino acids (in this application, naturally occurring amino acids refer to the 20 types of amino acids contained in proteins, specifically Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, and Pro), but may also be unnatural amino acids. In the case of α-amino acids, they may be L-amino acids or D-amino acids, or α,α-dialkylamino acids. There are no particular restrictions on the selection of amino acid side chains, and in addition to hydrogen atoms, they may be freely selected from, for example, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups, aralkyl groups, and cycloalkyl groups. Each of these may have a substituent, and the substituent is also freely selected from any functional group containing, for example, an N atom, an O atom, an S atom, a B atom, a Si atom, or a P atom (i.e., an optionally substituted alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group, cycloalkyl group, etc.). The "amino acids" and "amino acid analogs" that constitute the peptide compounds include all corresponding isotopes. An isotope of an "amino acid" or "amino acid analog" is one in which at least one atom has been replaced with an atom that has the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons). Examples of isotopes contained in the "amino acids" and "amino acid analogs" that constitute the peptide compounds of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, and each of these isotopes is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Includes Cl etc.

[0036] When using the Alexa Fluor® 488 Protein Labeling Kit (Invitrogen) to detect peptide compounds, it is desirable to have an amino acid with an amino group. Examples of such amino acids include Lys (lysine). Other amino acids that have a thiol group can also be labeled with thiol-reactive fluorescent dyes. Examples of such amino acids include Cys (cysteine).

[0037] When the molecule of the present invention is a peptide compound, preferably its target (or receptor) is PEPT1 or PEPT2.

[0038] Nanoparticles and microparticles are known to be used in formulations for drug delivery (DDS). Examples include, but are not limited to, liposomes, micelles, dendrimers, nanoemulsions, iron nanoparticles, gold nanoparticles, and PLGA particles (Organ Biology VOL.24 NO.1 2017, 54-60). In a preferred embodiment, the molecules of the present invention include nanoparticles or microparticles to which a molecule that specifically binds to a specific cell is attached. For example, an antigen-binding molecule for a surface antigen of the cell can be attached to these particles. Alternatively, for example, a molecule comprising an FcRn-binding domain can be attached to these particles. In one embodiment, the molecules of the present invention can be nanoparticles or microparticles to which an antibody comprising an FcRn-binding domain and / or a target-binding domain is attached.

[0039] In the present invention, the term "toxin" is not particularly limited as long as it can specifically deliver a cytotoxic agent, toxin, or radioisotope to a specific cell and injure it. For example, a molecule can be prepared by binding a cytotoxic agent, toxin, or radioisotope to a molecule that specifically binds to the cell (e.g., an antigen-binding molecule for a cell surface antigen of the cell). Examples of "molecules that specifically bind to cells" include the above-mentioned antibodies, nucleic acids, and peptide compounds. Using such molecules, a cytotoxic agent, toxin, or radioisotope can be efficiently delivered to the cell. As a result, the cell can be specifically injured. Examples of cytotoxic agents include maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al. al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065. Examples of toxins include enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. Examples of radioisotopes include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 These include radioactive isotopes of Pb and Lu.

[0040] Viruses can also be used as molecules in the present invention. According to the measurement method of the present invention, for example, the in vitro dynamics of the following viruses or viral proteins or portions thereof can be measured. Examples of viruses include viruses used in gene therapy, such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, lentiviruses, poxviruses, and Epstein-Barr viruses (Adv Biomed Res. (2012) 1: 27. doi:10.4103 / 2277-9175.98152), and viruses used for drug delivery, such as Red clover necrotic mosaic virus (RCNMV) (Methods Mol Biol. 2011;726:207-221). Examples of viral proteins or portions thereof include partial peptides of HIV-1 tat protein, L2 peptides of human papillomavirus, and envelope L protein of HBV.

[0041] In one embodiment, the measurement method of the present invention comprises the following steps: (a) When the molecule was contacted with cells expressing FcRn in an aqueous medium, the uptake was 0.068 pmol / 2 × 10 5 allowing the molecule to enter the cells so that it is elevated above the cells; and (b) measuring the in vitro pharmacokinetics of the molecule; Includes.

[0042] In the measurement method of the present invention, step (b) does not have to be initiated after step (a) is completed. That is, step (b) may be initiated after the uptake of the molecule of the present invention into cells is completed in step (a), or when the molecule is placed in a state in which it can be taken up by cells in step (a).

[0043] The cells used in the measurement methods of the present invention are not particularly limited as long as they can be contacted with a target molecule in vitro and express FcRn, and can be, for example, cells collected from a living organism, primary cultured cells, or established cell lines. FcRn expression can be confirmed by staining the cells with a fluorescently labeled anti-FcRn antibody, measuring the fluorescence by FACS, and observing a histogram shift toward higher fluorescence intensity than that of a control antibody (e.g., an isotype control antibody). For more quantitative evaluation, the cells may be analyzed by liquid chromatography-mass spectrometry (LC-MS) to measure the amount of FcRn-derived peptides present.

[0044] In a preferred embodiment, the FcRn can be that of a biological species for which in vivo pharmacokinetics is desired to be predicted, such as human FcRn, monkey FcRn, minipig FcRn, rat FcRn, mouse FcRn, rabbit FcRn, dog FcRn, guinea pig FcRn, hamster FcRn, chimpanzee FcRn, marmoset FcRn, ferret FcRn, or cat FcRn.

[0045] In one embodiment, the cells may be cells transformed to express FcRn. Such transformation can be achieved, for example, by introducing a polynucleotide encoding FcRn into the cells. The promoter used can be a promoter commonly used for expression in animal cells, such as CMV, PGK, RSV, CAG, EF-1 alpha, SV40, TRE, Oct3 / 4, and Nanog (PLoS One. 2010; 5(5): e10611). Use of such promoters allows for the expression of a sufficient amount of FcRn.

[0046] In one embodiment, the cell may be a cell transformed to express the target of the molecule of the present invention on the cell surface. When the target is a protein, such transformation can be achieved by introducing a polynucleotide encoding the protein into the cell. The greater the expression level of the target, the greater the amount of the molecule of the present invention taken up by the cell. The same promoter as used to express FcRn can be used, thereby allowing for the expression of a sufficient amount of the target.

[0047] The cells used to prepare the transformed cells are not particularly limited as long as they are amenable to transformation techniques for introducing foreign genes into cells, such as transfection and transduction. Examples of such cells include CHO cells, HEK293 cells, COS-1 cells, COS-7 cells, MDCK cells, HMEC1 cells, HELA cells, HepG2 cells, and BaF cells, and in certain embodiments, may be CHO cells.

[0048] In one embodiment, the cells may be cells that express endogenous FcRn, i.e., cells that express FcRn without being subjected to manipulation to force expression of exogenous FcRn. Examples of such cells include liver parenchymal cells, non-parenchymal liver cells, liver sinusoidal endothelial cells, Kupffer cells, human umbilical vein endothelial cells, peripheral blood mononuclear cells (PBMCs), macrophages, monocytes, B cells, T cells, platelets, NK cells, neutrophils, eosinophils, basophils, granulocytes, and dendritic cells.

[0049] In one embodiment, the cells may be cells that express an endogenous protein that is the target of the molecule of the present invention on their cell surface, i.e., cells that express the target protein on their cell surface without being subjected to a manipulation that forces the expression of an exogenous target protein. Such cells may be selected appropriately depending on the target protein.

[0050] In one embodiment, cells or cell lines with high endocytic activity may be used as cells. This may increase the amount of molecules of the present invention taken up by the cells. Examples of such cells or cell lines include phagocytes such as macrophages, neutrophils, eosinophils, and monocytes, or established cell lines thereof. Phagocytes have strong phagocytic activity and high uptake capacity. Examples of macrophages include Kupffer cells in the liver, alveolar macrophages, and microglia in the brain.

[0051] In a preferred embodiment, the cell is a cell transformed to express FcRn, and more preferably, the cell may be a cell transformed to express FcRn and to express the target molecule of the present invention on the cell surface.

[0052] As used herein, "aqueous medium" refers to a liquid containing water as an essential component. There are no particular limitations on the aqueous medium, so long as the cells used in the measurement method of the present invention do not lose cellular functions such as endocytosis and the molecules of the present invention can be present stably in the aqueous medium. Examples of aqueous media include buffer solutions such as phosphate-buffered saline (PBS) and liquid media such as Dulbecco's Modified Eagle's medium (DMEM). In a preferred embodiment, the aqueous medium may be a liquid medium, from the viewpoint of reducing the load on the cells.

[0053] In one embodiment of step (a), the uptake of the molecule of the present invention into cells can be carried out by contacting the molecule with the cells in an aqueous medium under conditions that do not cause the cells to lose cellular functions such as endocytosis. Such conditions can be appropriately set depending on the cells used. For example, when mammalian cells such as CHO cells are used, incubation can be carried out in a liquid medium at 30 to 40°C, preferably 36 to 38°C. In a specific embodiment of step (a), the uptake of a molecule of the present invention into cells can be carried out by contacting the molecule with cells in an aqueous medium at a temperature at which the internalization of the molecule into cells is suppressed (e.g., 4°C or lower). This allows the measurement of the molecule that is bound to the cell surface but not internalized, and allows, for example, more accurate measurement of the dissociation rate from FcRn or a target.

[0054] The uptake of the molecule into cells in the present invention was 0.068 pmol / 2×10 5 The uptake is measured so that the concentration is higher than that of the cells. The uptake is measured by contacting the molecule of the present invention with cells for a predetermined time depending on the in vitro pharmacokinetics to be measured, removing the aqueous medium containing the molecule not taken up by the cells, and measuring the amount of the molecule internalized in the cells and / or bound to the cell surface. Measurement of the uptake can be performed using a measurement method appropriate for the molecule, including measurement methods using labels such as fluorescent dyes, measurement methods using antibodies against the molecule such as ELISA (Enzyme-Linked Immunosorbent Assay), and measurement methods that quantify the molecule or its fragments using liquid chromatography mass spectrometry (LC-MS). When using ELISA or LC-MS, the cells are solubilized and the concentration of the molecule contained in the cell lysate is quantified, which can be performed by conventional methods using techniques commonly used in the field.

[0055] In one embodiment, the amount of uptake can be measured using a label attached to the molecule of the present invention. For example, when the molecule of the present invention is a protein, a label such as a fluorescent dye can be attached to the protein, and the amount of the protein present can be measured using the label. The method for labeling the protein is not limited to a specific method and can be performed by a conventional method using techniques commonly used in the field. Methods for labeling proteins include, for example, fluorescent labeling, biotin labeling, labeling with peptide tags (His tag, FLAG tag, HA tag, etc.), labeling with gold colloids, labeling with magnetic beads, RI (radioisotope) labeling, and enzyme labeling (HRP (horse radish peroxydase), AP (alkaline phosphatase), etc.). Commonly used fluorescent labels include, for example, Rhodamin, VioBlue, DyLight 405, DY-405, Alexa Fluor 405, AMCA, AMCA-X, Pacific Blue, DY-415, Royal Blue, ATTO 425, Cy2, ATTO 465, DY-475XL, NorthernLights 493, DY-490, DyLight 488, Alexa Fluor 488, 5-FITC, 5-FAM, DY-495-X5, DY-495, Fluorescein, FITC, ATTO 488, HiLyte Flour 488, MFP488, ATTO 495, and Oyster 500.

[0056] The uptake was 0.068 pmol / 2×10 5 In a preferred embodiment, the uptake of a molecule of the present invention into cells is at an amount of 0.070 pmol / 2×10 cells or higher, which can improve the accuracy of predicting in vivo pharmacokinetics from in vitro pharmacokinetics. 5 0.080 pmol / 2×10 cells 5 cells, 0.090 pmol / 2×10 5cells, or 0.10 pmol / 2 x 10 5 The upper limit of the amount of uptake is not particularly limited, but for example, 0.42 pmol / 2×10 5 Less than 0.40 pmol / 2×10 cells 5 Less than 0.30 pmol / 2×10 cells 5 Less than 0.20 pmol / 2×10 cells 5 cells, or 0.16 pmol / 2×10 5 The uptake of the molecule of the present invention into cells can be, for example, 0.068 pmol / 2×10 5 cells, preferably 0.070 pmol / 2×10 5 cells, 0.080 pmol / 2×10 5 cells, 0.090 pmol / 2×10 5 cells, or 0.10 pmol / 2×10 5 cells and 0.42 pmol / 2×10 5 cells, preferably less than 0.40 pmol / 2×10 5 Less than 0.30 pmol / 2×10 cells 5 Less than 0.20 pmol / 2×10 cells 5 cells, or 0.16 pmol / 2×10 5 It can be performed so that the number of cells is less than 1.

[0057] In one embodiment, the molecule of the present invention is a protein, and in step (a), the amount of uptake measured using a fluorescent dye attached to the molecule is 0.068 pmol / 2×10 5 It can be performed so that the height is higher than the cells.

[0058] In one embodiment, step (a) has at least one feature selected from the following (i) to (iii): (i) the contact time between the molecule and the cell is 5 hours or more; (ii) the cells are not washed under acidic conditions after contacting the molecule; and (iii) the cell expresses the target of the molecule on the cell surface.

[0059] Regarding (i), the contact time can be 5 hours or more, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more. The upper limit of the contact time is not particularly limited, as long as the molecule of the present invention is stable and cellular functions such as endocytosis are not lost. The contact time can be, for example, 72 hours or less, 48 ​​hours or less, or 36 hours or less. Thus, the contact time can be 5 hours or more (for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more) and 72 hours or less (for example, 48 hours or less or 36 hours or less).

[0060] Regarding (ii), cells may be washed before measuring the in vitro pharmacokinetics in step (b) (e.g., when measuring the amount of excretion from intracellular to extracellular, the rate of excretion from intracellular to extracellular, the rate of intracellular molecule reduction, or the rate of dissociation from FcRn or a target as in vitro pharmacokinetics). In this case, washing under acidic conditions can remove molecules bound to the cell surface. Therefore, not washing under acidic conditions can increase the amount of molecules of the present invention taken up into cells. Here, acidic conditions refer to pH below 6.0, for example, pH 5.5 or lower, pH 5.0 or lower, pH 4.5 or lower, pH 4.0 or lower, pH 3.5 or lower, or pH 3.0 or lower. Furthermore, in this embodiment, the contact time between the molecule of the present invention and the cells is not particularly limited, as long as the molecule of the present invention is present stably and cellular functions such as endocytosis are not lost, but may be, for example, 5 hours or more, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or more, or 72 hours or less, 48 ​​hours or less, or 36 hours or less.

[0061] Regarding (iii), the cells may be either cells transformed to express the target of the molecule of the present invention on the cell surface, or cells that have not been transformed in this way and express an endogenous protein that is the target of the molecule of the present invention on the cell surface. By binding the molecule of the present invention to the target present on the cell surface, the amount of the molecule taken up by the cell can be increased. The higher the expression level of the target, the more preferable it is. For example, a promoter commonly used for expression in animal cells, such as CMV, PGK, RSV, CAG, EF-1 alpha, SV40, TRE, Oct3 / 4, or Nanog, can be used as a promoter to express a sufficient amount of the target.

[0062] In one embodiment, the amount of the molecule of the present invention taken up into cells can be increased by including at least one step selected from the following steps (iv) to (vii) in step (a): (iv) adjusting the pH of the aqueous medium to 5.0 to 6.0; (v) when the molecule is an antibody, forming an immune complex (IC) between the antibody and its antigen; (vi) adding an anti-Fc antibody to the aqueous medium when the molecule contains an Fc region; and (vii) adding an uptake enhancer to the aqueous medium.

[0063] Regarding (iv), in an aqueous medium adjusted to a pH of 5.0 to 6.0, the molecules of the present invention are positively charged, which facilitates their entry into cells. Furthermore, when the molecules of the present invention contain an Fc region, the binding strength of the Fc region to FcRn increases in the intraendosomal pH environment of less than pH 6.5, which facilitates their entry into cells. Therefore, adjusting the pH of the aqueous medium to 5.0 to 6.0 can increase the amount of the molecules of the present invention taken up into cells. Therefore, in a preferred embodiment, the molecules of the present invention contain an Fc region, and step (a) can include step (iv).

[0064] Regarding (v), it is known that a complex between an antibody and an antigen, i.e., an immune complex, is easily taken up by cells (J Immunol. 1962 Oct;89:471-82 and Int Arch Allergy Appl Immunol. 1974;46(2):230-48). When the molecule of the present invention is an antibody, the amount of the molecule taken up by cells can be increased by contacting an immune complex containing the molecule with cells.

[0065] Regarding (vi), it is known that binding of an anti-Fc antibody to the Fc region of a molecule of the present invention causes multiple molecules to associate, thereby facilitating cellular uptake (J Immunol. 2013 Jun 15;190(12):6694-706). When a molecule of the present invention contains an Fc region, the amount of the molecule taken up by cells can be increased by adding an anti-Fc antibody to bring the associated molecules into contact with cells.

[0066] Regarding (vii), examples of the uptake enhancer include those for proteins, such as BioPORTER® Protein Delivery Reagent (Genlantis Inc.), PULSin® Kit (Polyplus-transfection® SA), Pro-DeliverIN (OZ Biosciences), and L17E Cytosolic Delivery Peptide (Peptide Institute, Inc.). The uptake enhancer also includes an endocytosis enhancer, such as okadaic acid (Drug Delivery System, 2016, Vol. 31, No. 1, pp. 83-84). The uptake enhancer also includes substances that inhibit the excretion of the molecules of the present invention from cells. Examples of such substances include inhibitors of ABC transporters (ATP-binding cassette transporters). As the ABC transporter inhibitor, those commonly used in the art can be used, such as MK571 and Ceefourin, which are known as MRP2 inhibitors. TM 1 (Abcam), Ceefourin TM 2 (manufactured by Abcam), erythromycin, thienylbutyl isothiocyanate, etc.

[0067] In step (a), a pharmaceutically active ingredient may be further added to the aqueous medium. This allows the in vitro pharmacokinetics of the molecule of the present invention to be evaluated in the presence of the ingredient. The pharmaceutically active ingredient is not particularly limited as long as it is a drug that can be used in combination with the molecule in vivo, and examples include therapeutic agents for diseases such as cancer, autoimmune diseases, infectious diseases, neurological diseases, osteoporosis, knee osteoarthritis / shoulder periarthritis, and hemophilia A.

[0068] In the measurement of in vitro pharmacokinetics in step (b), appropriate values ​​are measured depending on the type of in vitro pharmacokinetics. The measurements may be performed at a specific time point or multiple times over time.

[0069] In one embodiment, when the in vitro pharmacokinetics is represented by the amount of efflux from inside the cells to outside the cells (efflux amount), the efflux amount is determined by replacing the aqueous medium such as the culture medium with one that does not contain the molecule of the present invention after step (a), and then detecting the molecule in the aqueous medium to measure the amount of the molecule effluxed from the cells to outside the cells. In one embodiment, when the in vitro pharmacokinetics is the efflux rate from inside the cell to outside the cell (efflux rate), the efflux rate is determined by replacing the aqueous medium, such as the culture medium, with one that does not contain the molecule of the present invention after step (a), and then detecting the molecule in the aqueous medium to measure the amount of excretion of the molecule per unit time. In one embodiment, when the in vitro pharmacokinetics is represented by the rate of decrease of the intracellular molecule, the rate of decrease of the intracellular molecule is determined by replacing the aqueous medium, such as the culture medium, with one that does not contain the molecule of the present invention after step (a), and then detecting the molecule within the cells, thereby measuring the amount of the molecule decreased from the cells per unit time. In one embodiment, when the in vitro pharmacokinetics is represented by the dissociation rate from FcRn or a target, the dissociation rate from FcRn or a target is determined by, after step (a), replacing the aqueous medium such as a culture medium with one that does not contain the molecule of the present invention, and then detecting the molecule in the aqueous medium, thereby measuring the amount of the molecule excreted into the aqueous medium per unit time.

[0070] Step (b) may be a step of measuring in vitro pharmacokinetics during cellular uptake of the molecule of the present invention in step (a). Examples of in vitro pharmacokinetics measured in this manner include internalization rate, transcytosis amount, Kp value, and binding rate to FcRn or a target. In one embodiment, when the in vitro pharmacokinetics is expressed by the internalization rate, the internalization rate is determined by measuring the amount of the molecule of the present invention taken up into the cells from the outside per unit time after the molecule of the present invention is placed in a state where it can be taken up by the cells in step (a). In a preferred embodiment, the cells contacted with the molecule of the present invention for a predetermined time are washed with an acidic aqueous medium (pH less than 6.0, e.g., pH 5.5 or less, pH 5.0 or less, pH 4.5 or less, pH 4.0 or less, pH 3.5 or less, or pH 3.0 or less) to remove the molecule bound to the cell surface before measuring the amount of the molecule. Alternatively, for example, the amount of the molecule taken up into the cells over time after the start of uptake can be measured, and an integration plot analysis can be performed using the obtained measurements to calculate the internalization rate from the initial slope. In one embodiment, when in vitro pharmacokinetics is expressed by the amount of transcytosis, the amount of transcytosis can be determined by measuring the amount of the molecule that penetrates the cells after the molecule of the present invention is placed in a state where it can be taken up by the cells in step (a). For this purpose, cells cultured in a sheet form can be used. Commercially available products that can be used to measure the amount of transcytosis (e.g., Transwell® Permeable Support (Corning)) can also be used. In one embodiment, when the in vitro pharmacokinetics is expressed by the Kp value, the Kp value is determined by placing the molecule of the present invention in a state in which it can be taken up by cells in step (a), and measuring the amount of the molecule in the cells and in the aqueous medium after a predetermined time has passed. The Kp value is calculated by (amount in the cells) / (amount in the aqueous medium). In one embodiment, when the in vitro pharmacokinetics is represented by the binding rate to FcRn or a target, the binding rate to FcRn or a target is determined by measuring the amount of the molecule of the present invention bound to FcRn or a target per unit time after placing the molecule in a state where it can be taken up by cells in step (a).

[0071] In one embodiment, the measurement method of the present invention comprises the following steps: (c) calculating in vitro evaluation parameters from the measurement results obtained in step (b); It may further include:

[0072] As used herein, the term "in vitro evaluation parameter" refers to an index calculated from values ​​measured as in vitro pharmacokinetics. Calculating the in vitro evaluation parameter facilitates evaluation of in vitro pharmacokinetics and prediction of in vivo pharmacokinetics.

[0073] In vitro evaluation parameters include, for example, indicators such as "clearance index" and "HERA index."

[0074] The "clearance index" is calculated based on the amount of drug released from inside the cell to outside the cell (Efflux amount) using one of the following three methods. Method 1: The amount of intracellular molecules at 0 minutes after the start of excretion and the amount of extracellular molecules at 240 minutes after the start of excretion are measured, and the clearance index is calculated as (amount of extracellular molecules at 240 minutes) / (amount of intracellular molecules at 0 minutes). Method 2: The amount of intracellular molecules at 0 minutes after the start of excretion and the amount of extracellular molecules at 120 and 240 minutes after the start of excretion are measured, and the clearance index is calculated by dividing the average amount of extracellular molecules at 120 and 240 minutes by the amount of intracellular molecules at 0 minutes. Method 3: The amount of intracellular molecules at 0 minutes after the start of excretion and the amount of extracellular molecules at 60, 120, and 240 minutes after the start of excretion are measured, and the clearance index is calculated by dividing the average amount of extracellular molecules at 60, 120, and 240 minutes by the amount of intracellular molecules at 0 minutes.

[0075] In a preferred embodiment, the clearance index is calculated by Method 3 as an in vitro evaluation parameter.

[0076] The "HERA index" is calculated based on the amount of efflux from inside the cell to outside the cell using the following method. The molecule of the present invention is taken up into the cells by incubating the molecule with cells in a buffer solution at pH 6.0 for 4 hours. The cells are then washed, and a buffer solution at pH 7.4 is added to expel the molecule from the cells. The amount of the molecule expelled into the buffer solution (Rx) and the amount of the molecule remaining in the cells (RAx) are measured. The amount of excreted (Rwt) and the amount remaining (RAwt) of a reference molecule (e.g., a wild-type protein when the molecule of the present invention is a mutant protein) are also measured in the same way. The value calculated by (Rx / Rwt) / (RAx / RAwt) is the HERA score (Non-Patent Document 1).

[0077] In a preferred embodiment, the measurement method of the present invention is a method for measuring the in vitro pharmacokinetics of an antibody, comprising the following steps: (a) When the antibody was contacted with cells expressing FcRn in an aqueous medium, the uptake was 0.068 pmol / 2 × 10 5 a step of incorporating the antibody into the cells so that the antibody is elevated above the cells, the step having the following characteristics (i) to (iii): (i) the contact time between the antibody and the cells is 24 hours or longer; (ii) the cells are not washed under acidic conditions after contacting with the antibody; and (iii) the cell expresses the target of the antibody on the cell surface; (b) measuring the in vitro pharmacokinetics of the antibody; and (c) calculating in vitro evaluation parameters from the measurement results obtained in step (b); Including, the antibody comprises an FcRn-binding domain and a target-binding domain; In vitro pharmacokinetics is the efflux from inside the cell to outside the cell. The in vitro evaluation parameter may be the clearance index.

[0078] The measurement method of the present invention can be used to ensure the quality or predict the efficacy of pharmaceuticals containing the molecules of the present invention.

[0079] In one embodiment, to ensure the quality of a pharmaceutical product, for example, the method of the present invention can be incorporated into a part of the pharmaceutical manufacturing process as a pharmaceutical specification test. By defining a range that should contain in vitro pharmacokinetic measurements or in vitro evaluation parameters as a specification and manufacturing a product that meets the specification, the quality of the pharmaceutical product can be maintained at a constant level.

[0080] In one embodiment, the efficacy of a drug can be predicted by measuring in vitro pharmacokinetics using the assay method of the present invention. For example, it is known that in some autoimmune diseases, autoantibodies against autoantigens increase and attack the periphery, resulting in an autoimmune reaction. The use of immunoglobulin preparations (e.g., Hizentra® (CSF Behring)) that intravenously inject large amounts of human plasma-derived IgG has been reported as an attempt to reduce autoantibodies by inhibiting their extracellular secretion via FcRn. It has also been reported that FcRn inhibitors may be developed as therapeutic agents for autoimmune diseases (Folia Pharmacol. Jpn., 136, 280-284 (2010)). Therefore, the efficacy of a drug may be predicted by measuring the in vitro pharmacokinetics of a target molecule using the assay method of the present invention and evaluating the FcRn binding affinity of the molecule based on the results. Furthermore, by measuring in vitro pharmacokinetics using the measurement method of the present invention and predicting in vivo pharmacokinetics based on the results, it is possible to predict drug efficacy (see Section II below). In a specific embodiment, the prediction of drug efficacy can be a prediction of drug-drug interactions. For example, in step (a), the molecule of the present invention and another pharmaceutically active ingredient are contacted with cells, and the in vitro pharmacokinetics in the presence of the ingredient is measured, thereby predicting how the ingredient will affect the drug efficacy of the molecule.

[0081] II. Methods for predicting the in vivo pharmacokinetics of molecules A second aspect of the present invention relates to a method for predicting the in vivo pharmacokinetics of a molecule (hereinafter referred to as the prediction method of the present invention).

[0082] The prediction method of the present invention comprises the following steps: (a') measuring in vitro pharmacokinetics by the measurement method of the present invention; and (b') predicting the in vivo pharmacokinetics of the molecule when administered to a living body from the measured values ​​or in vitro evaluation parameters obtained in step (a'); Includes.

[0083] Step (a') is carried out as described in I above.

[0084] In step (b'), in vivo pharmacokinetics is predicted based on the correlation between the in vitro pharmacokinetic measurements or in vitro evaluation parameters previously calculated from the measurements or in vitro evaluation parameters obtained in step (a') and the in vivo pharmacokinetics. The correlation is determined depending on each molecule, biological species, and the type of in vitro pharmacokinetics and in vivo pharmacokinetics, as in the specific example using mice shown below.

[0085] Here, we present an example in which the intracellular to extracellular efflux of a reference molecule is measured as an in vitro pharmacokinetic parameter, a clearance index (Method 3) is calculated as an in vitro evaluation parameter, and the plasma half-life or clearance is predicted as an in vivo pharmacokinetic parameter. The reference molecule is selected from molecules of the same type as the molecule of the present invention (e.g., proteins, peptide compounds, nucleic acids, toxins, viruses, DDS formulations such as nanoparticles and microparticles, etc.) and molecules with the same target. For example, if the molecule of the present invention and the reference molecule are antibodies, they bind to the same antigen (preferably the same epitope). For example, if the molecule of the present invention is an artificial molecule (e.g., a mutant protein, a mutant peptide compound, a mutant nucleic acid, etc.), the reference molecule can be the molecule used as a reference in its creation (e.g., a wild-type protein, a wild-type peptide compound, a wild-type nucleic acid, etc.) and / or another molecule created in a similar manner to the artificial molecule. The number of reference molecules used to determine the correlation is one or more, preferably two or more (e.g., three or more, four or more, five or more, ten or more).

[0086] The in vitro pharmacokinetics of the reference molecule is measured by the measurement method of the present invention, similar to that of the molecule of the present invention. If necessary, in vitro evaluation parameters are calculated from the in vitro pharmacokinetic measurement results.

[0087] For in vivo pharmacokinetics, a reference molecule is administered via the tail vein to FcRn-expressing mice, plasma antibody concentrations are measured over time up to 28 days after administration, and plasma half-life or clearance is calculated using non-compartmental model analysis.

[0088] For the reference molecule, the measured in vitro pharmacokinetics or in vitro evaluation parameter values ​​are plotted against the plasma half-life or clearance value. A regression line may be constructed based on the obtained data. In this way, the correlation between the measured in vitro pharmacokinetics or in vitro evaluation parameter values ​​and in vivo pharmacokinetics can be calculated.

[0089] The in vivo pharmacokinetics in the prediction method of the present invention is not particularly limited and may be, for example, bioavailability, distribution volume, unbound fraction in blood, clearance, urinary excretion rate, blood concentration half-life, or mean residence time. In a preferred embodiment, the in vivo pharmacokinetics is clearance or blood concentration half-life, and the in vitro evaluation parameter is a clearance index.

[0090] In one embodiment, the living organism may be a human, monkey, miniature pig, rat, mouse, rabbit, dog, guinea pig, hamster, chimpanzee, marmoset, ferret, or cat. When the prediction method of the present invention is used for the purpose of reducing the number of experimental animals used, the living organism may be a non-human animal, for example, a monkey, miniature pig, rat, mouse, rabbit, dog, or guinea pig.

[0091] According to the prediction method of the present invention, in vivo pharmacokinetics such as plasma half-life and clearance can be predicted by in vitro testing. Therefore, the prediction method of the present invention can be used as an alternative to in vivo pharmacokinetic testing using animals. As a result, the number of in vivo pharmacokinetic tests and the number of experimental animals used can be reduced, and the present invention is also useful from the viewpoint of animal ethics.

[0092] III. Molecular Screening Methods A third aspect of the present invention relates to a method for screening molecules (hereinafter referred to as the screening method of the present invention).

[0093] The screening method of the present invention comprises the following steps: (a'') providing two or more different molecules that bind to the same target; (b'') measuring the in vitro pharmacokinetics of each of the two or more molecules prepared in step (a'') by the measurement method of the present invention; and (c'') comparing the measured values ​​or in vitro evaluation parameters for each of the two or more molecules obtained in step (b'') with each other and selecting the molecules that show desirable values; Includes.

[0094] The two or more molecules in step (a'') are each a molecule of the present invention described in I above. The two or more molecules are of the same type, have the same target, and are different from each other. For example, when the two or more molecules are antibodies, they may be different variants derived from the same parent antibody.

[0095] Step (b'') is carried out for each of the two or more molecules as described in I above.

[0096] In step (c''), molecules that exhibit desirable in vitro pharmacokinetic measurements or values ​​of in vitro evaluation parameters are selected. The desirable value may vary depending on the type of in vitro pharmacokinetics, but may be, for example, a value that indicates higher binding activity to FcRn or a target. When the in vitro pharmacokinetics is the amount of efflux from intracellular to extracellular space, the rate of efflux from intracellular to extracellular space, the amount of transcytosis, or the rate of intracellular molecule decrease, a higher value indicates higher binding activity to FcRn. When the in vitro pharmacokinetics is the internalization rate and the cells express the target, a higher value indicates higher binding activity to the target. Each selected molecule can be used for purposes (such as pharmaceuticals) according to its characteristics, or may be subjected to further testing.

[0097] According to the screening method of the present invention, molecules having desired characteristics can be selected without performing in vivo pharmacokinetic tests, which reduces the number of in vivo pharmacokinetic tests and the number of experimental animals used, making the present invention useful from the viewpoint of animal ethics.

[0098] All prior art documents cited in this specification are hereby incorporated by reference.

[0099] The present invention will now be described in more detail with reference to examples. [Example]

[0100] Example 1: Evaluation of pharmacokinetics in mouse plasma of each Fc variant (1-1) Characteristics of the Fc region of the antibody used for uptake evaluation The anti-IL-6R antibodies H237-G1d, H237-F1847m, H237-F1886m, H237-F1927m, and H237-F890, which have Fc domains described in WO 2012 / 133782 A1, WO 2013 / 046704 A2, WO 2017 / 046994 A1, and WO 2009 / 125825 A1, were used. The heavy chain sequence of H237-G1d is the amino acid sequence of SEQ ID NO: 79 of WO 2012 / 133782 A1. The heavy chain sequence of H237-F1847m is the amino acid sequence of SEQ ID NO: 50 of WO 2017 / 046994 A1. The heavy chain sequence of H237-F1886m is the amino acid sequence of SEQ ID NO: 52 of WO 2017 / 046994 A1. The heavy chain sequence of H237-F1927m is the amino acid sequence of SEQ ID NO: 54 of WO 2017 / 046994 A1. The heavy chain sequence of H237-F890 is the amino acid sequence of SEQ ID NO: 6 of WO 2013 / 046704 A2. All of these light chain sequences are the amino acid sequences of SEQ ID NO: 27 in WO 2009 / 125825 A1.

[0101] (1-2) Plasma pharmacokinetics in mice Mouse FcRn knockout / human FcRn transgenic mice (Tg#32, male) were administered 1 mg / kg of one of the antibodies H237-G1d, H237-F1847m, H237-F1886m, H237-F1927m, or H237-F890 and 1,000 mg / kg of Sanglopor (dried pH4-treated human immunoglobulin, CSL Behring) via the tail vein. Blood samples were collected from the jugular vein 5 minutes after administration and periodically thereafter up to 28 days after administration. The collected blood was centrifuged (12,000 rpm, 4°C, 5 minutes) to obtain plasma. Plasma antibody concentrations were measured using electrochemiluminescence immunoassay (ECL) with capture and detection antibodies against the administered antibody. Furthermore, non-compartmental model analysis was performed using the obtained PK profiles to calculate half-lives and clearance.

[0102] The results are shown in Figure 1. H237-F1847m, H237-F1886m, and H237-F1927m exhibited a gentler elimination slope during the elimination phase compared with H237-G1d and H237-F890, indicating a tendency for slower elimination from plasma. The calculated PK parameters are shown in Table 1. The terminal half-life of H237-F1886m was the longest, and H237-F1927m and H237-F1847m were also longer than those of H237-G1d. H237-F890 exhibited the shortest half-life. Furthermore, clearance of H237-F1886m was the shortest, and H237-F1927m and H237-F1886m were also shorter than those of H237-G1d.

[0103] [Table 1]

[0104] Example 2 Comparison of cellular uptake of each Fc variant (2-1) Alexa647 labeling of Fc region-modified antibodies H237-G1d, H237-F1847m, H237-F1886m, H237-F1927m, and H237-F890 were labeled with Alexa 647 (AF647) using the Alexa flour 647 labeling kit (Thermo Fisher Scientific) according to the attached protocol. The concentration of each antibody and the labeling efficiency of the fluorescent substance were calculated by measuring the absorbance using a Nanodrop (Thermo Fisher Scientific) and using the formula described in the attached protocol.

[0105] (2-2) Evaluation of Fc region-modified antibody uptake in hFcRn-hIL6R-CHO cells and hFcRn-CHO cells 2 x 10 cells in complete medium (CHO-S-SFM II (Invitrogen)) 5 AF647-labeled antibodies were added to 50 μL of a cell solution containing CHO cells overexpressing human FcRn and human IL-6R (hFcRn-hIL6R-CHO cells (Chiome Bioscience); generated by introducing an expression vector containing a CMV promoter (pcDNA3.1 vector, Invitrogen) into CHO cells) or CHO cells overexpressing human FcRn alone (hFcRn-CHO cells (Chiome Bioscience); generated by introducing an expression vector containing a CMV promoter (pcDNA3.1 vector, Invitrogen) into CHO cells). The AF647-labeled antibodies were added to a final concentration of 50 μg / mL in a 96-well plate (100 μL / well). The mixture was then incubated at 37°C in a CO2 incubator for 24 hours. The plates were then cooled on ice and washed with cold 2% FBS-containing PBS (FBS-PBS). The fluorescence intensity of the cells was measured using a FACS Canto II (Becton, Dickinson and Company). In addition to the cells used in the assay, the fluorescence intensity of fluorescently labeled standard beads was also measured using Quantum MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard, and the amount of antibody uptake was calculated from the geometric mean fluorescence intensity of the samples to which each antibody had been added.

[0106] The results are shown in Figure 2. The amount of uptake of each antibody was increased in the range of 2.2 to 36 times in hFcRn-hIL6R-CHO cells compared to hFcRn-CHO cells.

[0107] Example 3: Evaluation of cellular uptake over time of each Fc variant 2x10 5 AF647-labeled antibody was added to 50 μL of cell solution containing 10 hFcRn-hIL6R-CHO cells at a final concentration of 50 μg / mL, and 100 μL was added per well of a 96-well plate. The plate was then incubated at 37°C with stirring for up to 24 hours. The plate was then cooled on ice, and cold 2% FBS-containing PBS was added. The cells were washed once with FBS-PBS or medium (Acid) adjusted to pH 3.0. The cells were then collected by centrifugation (1000 g, 3 min). The fluorescence intensity of the cells was measured using a FACS Canto II. In addition to the cells used in the assay, the fluorescence intensity of fluorescently labeled standard beads was also measured using Quantum MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard, and the amount of antibody uptake was calculated from the geometric mean fluorescence intensity of the samples to which each antibody had been added.

[0108] The results are shown in Figure 3(a) and (b). A time-dependent increase in uptake was observed for each antibody. H237-F890 and H237-F1886m showed slightly higher uptake than H237-G1d, H237-F1847m, and H237-F1927m, both after FBS-PBS and acid washes. The obtained antibody uptake measurements were used for integration plot analysis, and the internalization rate was calculated from the initial slope. The results of the integration plot analysis are shown in Figure 3(c). The calculated internalization rates are shown in Table 2. H237-F890 showed a slightly higher value than H237-G1d, H237-F1847m, H237-F1886m, and H237-F1927m.

[0109] [Table 2]

[0110] Example 4: Evaluation of the amount of intracellular antibodies and the amount of efflux into the medium of each Fc variant over time (4-1) Time-dependent evaluation of intracellular antibody levels and their release into the medium 2x10 5 AF647-labeled antibody was added to 50 μL of cell suspension containing 10 hFcRn-hIL6R-CHO cells at a final concentration of 50 μg / mL, and 100 μL of antibody was added per well of a 96-well plate. The mixture was then incubated at 37°C for 24 hours. The mixture was then cooled on ice, and cold 2% BSA-containing medium was added and removed. 100 μL of fresh 2% BSA-containing medium was added, and the mixture was incubated at 37°C for up to 4 hours. Samples were collected over time. After centrifugation, the supernatant was collected and the cells were washed with FBS-PBS. The antibody concentration in the supernatant was measured using electrochemiluminescence immunoassay (ECL). The fluorescence intensity of the cells was measured using a FACS Canto II, and the amount of antibody contained in the cells was calculated from the fluorescence intensity of the sample beads.

[0111] The results are shown in Figure 4(a). A time-dependent decrease in the amount of intracellular antibody was confirmed for all antibodies. The amount of intracellular antibody for H237-F890 remained high. Figure 4(b) shows the time course of the amount of antibody excreted into the medium. All antibodies were rapidly excreted until approximately 30 minutes after the start of excretion, after which they reached a plateau. Up until 10 minutes after the start of excretion, the graphs for all antibodies showed similar slopes, but after 60 minutes, differences in the amount of antibody in the medium were observed for each antibody, with H237-F1886m showing the highest amount and H237-G1d showing the lowest.

[0112] (4-2) Calculation of the clearance index The clearance index was calculated using the amount of intracellular antibody at 0 minutes after the start of excretion and the amount of antibody excreted into the medium up to each time point using the three formulas shown below. Method 1: (amount of antibody in the medium at 240 minutes) / (amount of antibody in the cells at 0 minutes) Method 2: (average amount of antibody in the medium at 120 and 240 minutes) / (amount of antibody in the cells at 0 minutes) Method 3: (amount of antibody in the medium at 60, 120, and 240 minutes) / (amount of antibody in the cells at 0 minutes) The clearance index values ​​calculated by each of the above methods are shown in Table 4. For all antibodies, the values ​​were approximately 0.30 to 0.70. The values ​​calculated by the three methods for each antibody were roughly similar. The values ​​between antibodies also showed the same tendency across the three methods.

[0113] [Table 3]

[0114] Example 5: Correlation between clearance index and in vivo pharmacokinetics The correlation between the clearance index calculated in Example 4 and the in vivo half-life or clearance measured in Example 1 was evaluated. The results are shown in Figure 5. A strong correlation was observed between both the in vivo half-life and clearance and the clearance index (R 2 =0.961 and R 2= 0.822). Therefore, it was demonstrated that the clearance index calculated by Methods 1 to 3 can predict the in vivo plasma half-life or clearance. [Industrial Applicability]

[0115] According to the present invention, it is possible to predict the in vivo pharmacokinetics of a large number of drug candidate substances more simply and with higher accuracy than conventional methods. Furthermore, the present invention can contribute to reducing the number of experimental animals used and to the development of drugs with stronger pharmacological effects.

Claims

1. 1. A method for measuring the in vitro pharmacokinetics of a molecule, comprising the steps of: (a) When the molecule was contacted with cells expressing FcRn in an aqueous medium, the uptake was 0.068 pmol / 2×10 5 uptake of the molecule into cells so that the molecule is elevated above cells, the cells expressing a target of the molecule on their cell surface; and (b) measuring the in vitro pharmacokinetics of the molecule; Including, The method, wherein the molecule comprises an FcRn-binding domain.

2. Step (a) (i) the contact time between the molecule and the cell is 5 hours or more; and (ii) the cells are not washed under acidic conditions after contact with the molecule; The method of claim 1 , having at least one feature selected from:

3. The method of claim 1 , wherein the molecule is an antibody comprising an FcRn-binding domain and a target-binding domain.

4. The method according to any one of claims 1 to 3, wherein the cell is a cell transformed to express FcRn.

5. The method according to any one of claims 1 to 3, wherein the cell is a cell transformed to express the target of the molecule on the cell surface.

6. The method of claim 4, wherein the cell is a CHO cell, a HEK293 cell, a COS-1 cell, a COS-7 cell, an MDCK cell, a HMEC1 cell, a HELA cell, a HepG2 cell, or a BaF cell.

7. The method of any one of claims 1 to 3, wherein the cells are liver parenchymal cells, liver non-parenchymal cells, liver sinusoidal endothelial cells, Kupffer cells, human umbilical vein endothelial cells, peripheral blood mononuclear cells (PBMCs), macrophages, monocytes, B cells, T cells, platelets, NK cells, neutrophils, eosinophils, basophils, granulocytes, or dendritic cells.

8. The uptake was 0.10 pmol / 2×10 5 The method of any one of claims 1 to 3, wherein the molecule is taken up by the cells so that it is elevated above the cells.

9. The method according to any one of claims 1 to 3, wherein the in vitro pharmacokinetics is the amount of excretion from cells into a culture medium, the excretion rate from cells into a culture medium, the internalization rate, the amount of transcytosis, the Kp value, the rate of decrease in intracellular molecules, the binding rate to FcRn or a target, or the dissociation rate from FcRn or a target.

10. The method according to any one of claims 1 to 3, wherein the FcRn is human FcRn, monkey FcRn, minipig FcRn, rat FcRn, mouse FcRn, rabbit FcRn, dog FcRn, or guinea pig FcRn.

11. The following process: (c) calculating in vitro evaluation parameters from the measurement results obtained in step (b); The method of any one of claims 1 to 3, further comprising:

12. The method of claim 11, wherein the in vitro evaluation parameter is a clearance index or a HERA score.

13. The method according to any one of claims 1 to 3, which is used for quality assurance or prediction of efficacy of a pharmaceutical containing the molecule.

14. The method of any one of claims 1 to 3, wherein the molecular target is a membrane protein.

15. The method of claim 14, wherein the target of the molecule is the human IL6 receptor.

16. 1. A method for predicting the in vivo pharmacokinetics of a molecule, comprising: (a') measuring in vitro pharmacokinetics by the method according to any one of claims 1 to 3; and (b') predicting the in vivo pharmacokinetics of the molecule when administered to a living body from the measured values ​​or in vitro evaluation parameters obtained in step (a'). Including, The method, wherein the in vivo pharmacokinetics is clearance or blood concentration half-life.

17. 17. The method of claim 16, wherein the living organism is a human, monkey, minipig, rat, mouse, rabbit, dog, or guinea pig.

18. 17. The method of claim 16, which is used as an alternative to pharmacokinetic studies using animals.

19. 1. A method for screening a molecule, comprising: (a'') providing two or more different molecules that bind to the same target; (b'') measuring the in vitro pharmacokinetics of each of the two or more molecules prepared in step (a'') by the method of any one of claims 1 to 3; and (c'') comparing the measured values ​​or in vitro evaluation parameters for each of the two or more molecules obtained in step (b'') with each other and selecting a molecule that exhibits a desirable value; The method comprising:

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