Methods for measuring molecular uptake into cells

Abstract

There is a need for an in vitro cell-based assay that mimics the immune complex clearance that occurs in vivo and that can quantify FcγRIIB expression and antibody or antigen binding and internalization. [Solution] A method for measuring the amount of molecule taken up into cells is provided, comprising: (i) adding the molecule to a cell population derived from an organ and incubating the mixture; (ii) selecting the organ-derived cell population based on the expression levels of CD31 and CD45; and (iii) measuring the amount of molecule taken up into the cell population selected in (ii) after steps (i) and (ii), wherein the molecule is taken up into the cells via a receptor present on the cell surface.

Description

[Technical field]

[0001] The present invention relates to a method for measuring in vitro cellular uptake of molecules, such as antigen-antibody complexes (immunocomplexes), antibodies, and nucleic acids, which are taken up into cells via receptors present on the cell surface, and a composition for assaying the uptake of such molecules. [Background technology]

[0002] A complex consisting of an antibody and an antigen is called an immune complex, which is formed by binding an antibody to a foreign substance present in the body. Immune complexes are excreted from the body by the reticuloendothelial system, which removes foreign substances from the body (Non-Patent Document 1). An antibody has a Fab region and an Fc region, and the Fc region is recognized by an Fc receptor present on the cell surface (Non-Patent Document 15). Among the Fc receptors, the Fc gamma receptor (FcγR) is a receptor that recognizes the Fc region of an IgG antibody. There are four subtypes of FcγR, FcγRI, II, III, and IV, and it has been reported that FcγRIIB ("γ" is sometimes written as "g", "II" is sometimes written as "2", and "B" is sometimes written as "b"), which is one of the FcγRII, is a receptor that acts suppressively on the immune system (Non-Patent Document 2, Non-Patent Document 3). FcγRIIB is also known as a receptor that is deeply involved in the disappearance of immune complexes from the blood (Non-Patent Documents 4-7). In recent years, research and development of antibodies as pharmaceuticals has been actively carried out. For example, attempts have been made to produce antibodies with modified amino acid sequences from the viewpoints of ease of immune complex formation, ease of binding to FcγR, etc., and to remove soluble proteins in plasma by administering these to animals (Non-Patent Document 8, Non-Patent Document 9, Patent Documents 1 to 4).

[0003] It has been reported that FcγRIIB is mainly expressed in liver sinusoidal endotherial cells (LSECs). In mice, it has been reported that 3 / 4 of FcγRIIB is strongly expressed in the liver, and 90% of it is expressed in LSECs (Non-Patent Document 10). In addition, as a result of evaluating the migration rate to the liver, lungs, spleen, kidneys, and blood after administration of an immune complex in mice, it has been reported that the migration rate to the liver is higher when an immune complex formed a multimer is administered compared to a monomer or dimer (Non-Patent Document 5). Furthermore, it has been revealed that the disappearance rate of the immune complex is greatly reduced compared to wild-type mice when an immune complex is administered to mice in which FcγRIIB is knocked out (Non-Patent Document 10). Furthermore, it has been reported that the uptake of an immune complex consisting of IgE and an antibody against it was evaluated using non-parenchymal liver cells collected from transgenic mice expressing human FcγRIIB, and the uptake into CD146+CD45low LSECs was confirmed (Patent Document 3). These results suggest that immune complexes are internalized and cleared by LSECs via FcγRIIB in mice.

[0004] It has also been reported that immune complexes are eliminated via FcγRIIB in LSECs in rats (Non-Patent Documents 7 and 11).

[0005] In monkeys, when the distribution of immune complexes in the body 24 hours after administration of radiolabeled antibodies was evaluated by gamma ray imaging, it was reported that, in the case of administration of a control antibody that does not form immune complexes, signals were detected in areas with a large blood flow such as the heart, whereas in the case of administration of an antibody that forms immune complexes, high concentrations of signals were observed in the liver (Non-Patent Document 12). Furthermore, immunostaining has been suggested to cause accumulation of immune complexes in vascular endothelium and Kupffer cells (Non-Patent Document 12). It has also been reported that antibodies that can further reduce the concentration of soluble antigens in the blood can be obtained by enhancing the affinity of the antibody for FcγRIIB (Patent Documents 4 and 5).

[0006] On the other hand, the site of FcγRIIB expression in monkeys and the cells that primarily affect the disappearance of immune complexes have not been identified, and the mechanism and rate of uptake of immune complexes are also unknown.

[0007] In humans, markers expressed in LSECs have been identified, and FcγRIIB has been shown to be expressed in LSECs (Non-Patent Documents 13 and 14), but the mechanism and kinetics of immune complex elimination have not been verified in vitro or in humans.

[0008] Regarding immune complex uptake, a method using cultured cells in which antibody receptors are forcibly expressed and a method using primary cells collected from organs are known as cell-based evaluation systems. Regarding the former method, for example, evaluation of antibody uptake in cells such as macrophage cell line J774 (Non-Patent Document 21) and evaluation of immune complex uptake using MDCK cells have been reported (Patent Document 5). In addition, evaluation of IC uptake using non-parenchymal liver cells including LSEC collected from transgenic mice expressing human FcγRIIB has been reported (Patent Document 3). However, in these evaluation methods, a specific protein is overexpressed, which is significantly different from the in vivo environment and may not correlate with in vivo immune complex disappearance. On the other hand, regarding the latter method using primary cells, it has been reported that the activity of the cells changes over time (Non-Patent Document 22), and it is considered that the method does not correctly reflect in vivo immune complex uptake.

[0009] As described above, while the pharmacokinetics of immune complexes has been evaluated in vivo in mice, rats, and monkeys, an in vitro evaluation system using cells involved in the elimination of immune complexes, such as LSECs, has not been established, and there have been no reports of quantitative evaluation of FcγRIIB expression or cellular uptake of immune complexes using monkey or human cells.

[0010] It has been suggested that nucleic acid drugs, like immune complexes, are eliminated primarily from non-parenchymal cells in the liver. 3 It has been reported that when H-labeled nucleic acid was administered intravenously, 40.5% accumulated in the liver, of which 60.4% accumulated in non-parenchymal cells (Non-Patent Document 26). Another report confirmed that nucleic acid accumulated twice as much in non-parenchymal cells as in parenchymal cells in mice and rats (Non-Patent Document 27). Furthermore, it has been reported that FITC-labeled nucleic acid was taken up by mouse LSECs in vitro (Non-Patent Document 25), and in recent years, it has been revealed that nucleic acid is taken up by receptors called stabilin-1 and stabilin-2 (Non-Patent Document 24). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO 2012 / 132067 A1 [Patent Document 2] WO 2013 / 081143 A1 [Patent Document 3] WO 2014 / 113510 A1 [Patent Document 4] WO 2016 / 117346 A1 [Patent Document 5] WO 2016 / 098357 A1 [Non-patent literature]

[0012] [Non-Patent Document 1] Benacerraf, B., M. Sebestyen, and NS Cooper, The clearance of antigen antibody complexes from the blood by the reticuloendothelial system. J Immunol, 1959. 82(2): p. 131-7. [Non-Patent Document 2] Takai, T., Roles of Fc receptors in autoimmunity. Nat Rev Immunol, 2002. 2(8): p. 580-92. [Non-Patent Document 3] Schwab, I. and F. Nimmerjahn, Intravenous immunoglobulin therapy: how does IgG modulate the immune system? Nat Rev Immunol, 2013. 13(3): p. 176-89. [Non-Patent Document 4] Arend, WP and JC Sturge, Composition and biological properties of soluble IgG-anti-IgG immune complexes: effects of variations in the specificity of rabbit antibodies to different structural components of human IgG. J Immunol, 1979. 123(1): p. 447-54. [Non-Patent Document 5] Finbloom, DS and PH Plotz, Studies of reticuloendothelial function in the mouse with model immune complexes. I. Serum clearance and tissue uptake in normal C3H mice. J Immunol, 1979. 123(4): p. 1594-9. [Non-Patent Document 6] Kurlander, R.J., D.M. Ellison, and J. Hall, The blockade of Fc receptor-mediated clearance of immune complexes in vivo by a monoclonal antibody (2.4G2) directed against Fc receptors on murine leukocytes. J Immunol, 1984. 133(2): p. 855-62.

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[0013] As mentioned above, there are no quantitative in vitro cell evaluation systems that mimic the biological environment. However, unlike animal experiments, such evaluation systems have the advantage that more detailed analysis of mechanisms, evaluation of kinetics such as cellular uptake, and screening of large numbers of candidate substances can be performed. Thus, they are likely to be useful tools not only for analysis of cellular uptake of immune complexes, but also for life science research and drug discovery research.

[0014] Regarding FcγRIIB, the affinity between the recombinant protein and antibody Fc is measured using Biacore (GE Healthcare) or the like (Non-Patent Document 15). This is because the binding and uptake of immune complexes into cells are mediated by FcγRIIB, and therefore affinity for FcγRIIB is considered to be important for the disappearance of immune complexes from plasma. However, it is difficult to measure affinity using Biacore or the like as described above for antibodies with non-specific adsorptivity or large charges. In addition, measurements in a buffer using recombinant proteins may not accurately reflect the affinity in plasma in vivo. It has also been suggested that the uptake of immune complexes involves not only binding to FcγRIIB but also binding to other receptors. For example, it has been reported that neonatal Fc receptor (FcRn) is also involved in the disappearance of immune complexes (Non-Patent Documents 16 and 17).

[0015] Pharmacokinetic analysis, which describes the movement of drugs in vivo using a mathematical model, is useful from the viewpoint of reducing the number of experimental animals and improving the efficiency of clinical trials. By using a mathematical model to empirically scale parameters obtained in non-human animals to humans, it is possible to predict clinical drug concentration trends from non-clinical drug concentration trends (e.g., Non-Patent Document 18). As a mathematical model based on the mechanism that describes the disappearance of drugs via receptors, there is, for example, a method using a target-mediated drug disposition model (e.g., Non-Patent Documents 19 and 20). These models describe the binding of receptors to drugs and the uptake of receptors into cells. Species differences in the amino acid sequence of FcγRIIB are known, and as mentioned above, it has been suggested that the uptake of immune complexes involves binding to receptors other than FcγRIIB. Therefore, when quantitatively predicting the pharmacokinetics of drugs that form immune complexes in humans, it is not sufficient to simply scale the drug concentration trends obtained from non-human animals to humans using empirical rules, and it is necessary to calculate appropriate parameters for each animal species. For this purpose, quantitative kinetics and receptor expression level evaluation using cell systems are necessary.

[0016] Furthermore, screening a large number of drug candidates using animal experiments alone is laborious and requires many experimental animals such as monkeys, and it is impossible to quantify the expression of FcγRIIB and the binding and uptake of immune complexes in cells such as LSECs.

[0017] As mentioned above, the methods using cultured cells overexpressing antibody receptors and the methods using primary cells extracted from organs, which are known as cell-based evaluation systems, are thought to not accurately reflect in vivo immune complex uptake.

[0018] For these reasons, there is a need for an in vitro cell-based assay that mimics the in vivo clearance of immune complexes and allows for quantification of FcγRIIB expression and antibody or antigen binding and uptake. [Means for solving the problem]

[0019] Using nonparenchymal liver cells from transgenic mice expressing human FcγRIIB, + CD45 low There is a report that investigated the uptake of immune complexes into LSECs (Patent Document 3). However, as mentioned above, FcγRIIB is not the only molecule involved in the uptake of immune complexes, so it is extremely difficult to construct an in vitro evaluation system that reflects biological phenomena in humans or monkeys using mouse cells. In addition, even if the uptake of immune complexes can be observed in an evaluation system in which FcγRIIB is forcibly expressed, it is not necessarily possible to evaluate the uptake of immune complexes in a similar manner in non-transgenic cells.

[0020] As a result of intensive research, the present inventors have identified human and monkey non-parenchymal liver cells as multiple CD31-specific CD45 ... + CD45 + We found that the LSECs could be separated into two cell populations, one of which expressed FcγRIIB. CD31 and CD45 are known as markers for LSECs in humans (Non-Patent Documents 28 and 29). + CD45 +It is not known at all that FcγRIIB is specifically expressed in a part of the cell population. In addition, CD31 and CD45 are not known as markers for LSEC in monkeys. The present inventors have succeeded in constructing an evaluation system for measuring the amount of immune complex uptake in the cell population expressing this FcγRIIB. Furthermore, they have also succeeded in constructing an evaluation system for measuring the amount of nucleic acid uptake in the cell population, an evaluation system for measuring the amount of antibody uptake in the cell population via FcγRIIB, and an evaluation system for measuring the amount of antibody uptake that binds to a membrane receptor expressed in the cell population. The present inventors have completed the present invention through further research.

[0021] That is, the present invention provides the following inventions. [1] A method for measuring the amount of a molecule taken up into a cell, comprising the steps of: (i) adding the molecule to a population of cells derived from an organ and incubating the mixture; (ii) selecting a population of cells derived from an organ based on the expression levels of CD31 and CD45; and (iii) measuring the uptake of said molecule into the cell population following steps (i) and (ii); Including, The method, wherein the molecule is taken up into the cell via a receptor present on the cell surface. [2] The method according to [1], which comprises step (ii) following step (i), in which a cell population derived from an organ incubated with the molecule is selected based on the expression levels of CD31 and CD45. [3] The method according to [1], further comprising step (ii) prior to step (i), in which the molecule is added to a cell population derived from an organ selected based on the expression levels of CD31 and CD45, and then incubated. [4] The method according to any one of [1] to [3], wherein the molecule is an immune complex or an antibody, and the receptor is an Fc receptor. [5] The method according to any one of [1] to [3], wherein the molecule is an anti-IL-6R antibody and the receptor is IL-6R. [6] The method according to any one of [1] to [3], wherein the molecule is a nucleic acid and the receptor is stabilin. [7] The method according to any of [1] to [6], wherein the organ-derived cell population is a non-parenchymal liver cell population. [8] The non-parenchymal liver cell population is a human non-parenchymal liver cell population, and in step (ii), high CD45 low A method for selecting a cell population, comprising the steps of: [9] CD31 high CD45 low The cell populations were classified into two CD31 subtypes that were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X-axis and Y-axis, respectively. + CD45 + The method according to [8], which is a cell population having a higher expression level of CD31 when compared at the point where the cell density of each cell population is the highest.

[10] CD31 high CD45 low The method according to [8], wherein the cell population has a CD31 fluorescence intensity of 400 to 7000 and a CD45 fluorescence intensity of 100 to 4000 when expression of CD31 and CD45 is detected by flow cytometry.

[11] The non-parenchymal liver cell population is a monkey non-parenchymal liver cell population, and in step (ii), intermediate CD45 intermediate A method for selecting a cell population, comprising the steps of:

[12] CD31 intermediate CD45 intermediate The cell populations were classified into three CD31 subtypes, which were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X and Y axes, respectively. + CD45 + The method according to

[11] , wherein the cell population has the second highest CD31 expression level when compared at the point where the cell density of each cell population is the highest.

[13] CD31 intermediateCD45 intermediate The method according to

[11] , wherein the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000 when expression of CD31 and CD45 is detected by flow cytometry.

[14] A composition for use in a molecule uptake assay, comprising an isolated, organ-derived cell population expressing CD31 and CD45, wherein the molecule is taken up into the cells via a receptor present on the cell surface.

[15] The composition according to

[14] , wherein the assay is performed according to the method described in any one of [1] to

[13] .

[16] The composition described in

[14] or

[15] , wherein the cell population is a crudely purified cell population.

[17] A method for producing the composition according to any one of

[14] to

[16] , (i) preparing organ-derived cells from an organ removed from a living body; and (ii) selecting a population of organ-derived cells based on the expression levels of CD31 and CD45; The method comprising:

[18] A method for predicting in vivo clearance of a molecule, comprising: (i) measuring the amount of the molecule taken up into cells using the method according to any one of [1] to

[13] ; and (ii) predicting the in vivo clearance of the molecule when the molecule is administered to a living body from the amount of uptake measured in (i) above; Including, The method, wherein the molecule is taken up into the cell via a receptor present on the cell surface.

[19] A method for screening a molecule, comprising: (i) providing two or more different molecules that bind to the same receptor; (ii) measuring the amount of each of the molecules prepared in (i) above taken up into cells by using a method described in any one of [1] to

[13] ; and (iii) comparing the amounts of the molecules taken up into cells measured in (ii) above with each other and selecting the molecule having the highest amount of uptake; Including, The method, wherein the molecule is taken up into the cell via a receptor present on the cell surface.

[20] The method according to any of [1] to [7], wherein the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), a cell population shown as P2 in Figure 8 is selected.

[21] The method according to any of [1] to [7], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), a cell population shown as P2 in Figure 2 is selected.

[22] The composition according to any one of

[14] to

[16] , wherein the organ-derived cell population is a human liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 8.

[23] The composition according to any one of

[14] to

[16] , wherein the organ-derived cell population is a monkey liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 2.

[0022] The present invention also includes the following aspects. [a1] A method for measuring the amount of immune complex taken up into cells, comprising the steps of: (i) adding and incubating an immune complex with a population of cells derived from an organ; (ii) selecting a population of cells derived from an organ based on the expression levels of CD31 and CD45; and (iii) measuring the amount of immune complex uptake into the cell population following steps (i) and (ii); The method comprising: [a2] The method according to [a1], which comprises step (ii) following step (i), in which a cell population derived from an organ incubated with an immune complex is selected based on the expression levels of CD31 and CD45. [a3] The method according to [a1], which comprises step (ii) prior to step (i), in which an immune complex is added to a cell population derived from an organ selected based on the expression levels of CD31 and CD45, and then incubated. [a4] The method according to any of [a1] to [a3], wherein the immune complex is taken up into the cell via FcγRIIB. [a5] The method according to any of [a1] to [a4], wherein the organ-derived cell population is a liver non-parenchymal cell population. [a6] The non-parenchymal liver cell population is a human non-parenchymal liver cell population, and in step (ii), high CD45 low A method according to [a5] for selecting a cell population. [a7]CD31 high CD45 low The cell populations were classified into two CD31 subtypes that were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X-axis and Y-axis, respectively. + CD45 + The method according to [a6], which is a cell population having a higher expression level of CD31 when compared at the point where the cell density of each cell population is the highest. [a8]CD31 high CD45 low The method according to [a6], wherein the cell population has a CD31 fluorescence intensity of 400 to 7000 and a CD45 fluorescence intensity of 100 to 4000 when expression of CD31 and CD45 is detected by flow cytometry. [a9] The non-parenchymal liver cell population is a monkey non-parenchymal liver cell population, and in step (ii), intermediate CD45 intermediate A method according to [a5] for selecting a cell population. [a10]CD31 intermediate CD45 intermediate The cell populations were classified into three CD31 subtypes, which were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X and Y axes, respectively. + CD45 + The method according to [a9], wherein the cell population has the second highest CD31 expression level when compared at the point where the cell density of each cell population is the highest. [a11]CD31 intermediate CD45 intermediate The method according to [a9], wherein the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000 when expression of CD31 and CD45 is detected by flow cytometry. [a12] A composition for use in an immune complex uptake assay, comprising an isolated, organ-derived cell population that expresses CD31 and CD45. [a13] The composition described in [a12], wherein the assay is performed according to a method described in any one of [a1] to [a11]. [a14] The composition described in [a12] or [a13], wherein the cell population is a crudely purified cell population. [a15] A method for producing the composition according to any one of [a12] to [a14], (i) preparing organ-derived cells from an organ removed from a living body; and (ii) selecting a population of organ-derived cells based on the expression levels of CD31 and CD45; The method comprising: [a16] A method for predicting an in vivo blood antigen reduction rate due to administration of an antibody, comprising: (i) forming an immune complex from an antigen and an antibody; (ii) measuring the amount of the immune complex (i) taken up into cells by using any one of the methods described in [a1] to [a11]; and (iii) predicting, from the amount of uptake measured in (ii) above, the in vivo blood reduction rate of the antigen when the antibody is administered to a living body; The method comprising: [a17] A method for screening for an antibody having an antigen-removing effect, comprising the steps of: (i) providing two or more different antibodies that bind to the same antigen; (ii) preparing an immune complex between each of the two or more antibodies prepared in (i) above and the antigen; (iii) measuring the amount of each of the immune complexes prepared in (ii) above taken up into cells by using any of the methods described in [a1] to [a11]; and (iv) comparing the amounts of the immune complexes taken up into cells measured in (iii) above with each other and selecting the immune complex having the largest amount taken up; The method comprising: [a18] The method according to any of [a1] to [a5], wherein the organ-derived cell population is a human liver non-parenchymal cell population, and in the step (ii), a cell population shown as P2 in Figure 8 is selected. [a19] The method according to any of [a1] to [a5], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), a cell population shown as P2 in Figure 2 is selected. [a20] The composition described in [a12], wherein the organ-derived cell population is a human liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 8. [a21] The composition described in [a12], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 2.

[0023] The present invention also includes the following aspects. [b1] A method for measuring the amount of antibody uptake into cells, comprising the steps of: (i) adding the antibody to a population of cells derived from an organ and incubating the same; (ii) selecting a population of cells derived from an organ based on the expression levels of CD31 and CD45; and (iii) measuring the amount of uptake of the antibody into the cell population following steps (i) and (ii); Including, The method, wherein the antibody binds to a soluble antigen. [b2] The method according to [b1], which comprises step (ii) following step (i), in which a cell population derived from an organ incubated with the antibody is selected based on the expression levels of CD31 and CD45. [b3] The method according to [b1], which comprises step (ii) prior to step (i), in which the antibody is added to a cell population derived from an organ selected based on the expression levels of CD31 and CD45, and then incubated. [b4] The method according to any one of [b1] to [b3], wherein the antibody is taken up into cells via an Fc receptor. [b5] The method according to any one of [b1] to [b4], wherein the organ-derived cell population is a non-parenchymal liver cell population. [b6] The non-parenchymal liver cell population is a human non-parenchymal liver cell population, and in step (ii), high CD45 low A method according to [b5] for selecting a cell population. [b7]CD31 high CD45 low The cell populations were classified into two CD31 subtypes that were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X-axis and Y-axis, respectively. + CD45 + The method according to [b6], which is a cell population having a higher expression level of CD31 when compared at the point where the cell density of each cell population is the highest. [b8]CD31 high CD45 low The method according to [b6], wherein the cell population has a CD31 fluorescence intensity of 400 to 7000 and a CD45 fluorescence intensity of 100 to 4000 when expression of CD31 and CD45 is detected by flow cytometry. [b9] The non-parenchymal liver cell population is a monkey non-parenchymal liver cell population, and in step (ii), intermediate CD45 intermediate A method according to [b5] for selecting a cell population. [b10]CD31 intermediate CD45 intermediate The cell populations were classified into three CD31 subtypes, which were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X and Y axes, respectively. + CD45+ The method according to [b9], which is the cell population having the second highest CD31 expression level when compared at the point where the cell density of each cell population is the highest. [b11]CD31 intermediate CD45 intermediate The method according to [b9], wherein the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000 when expression of CD31 and CD45 is detected by flow cytometry. [b12] A composition for use in an antibody uptake assay, comprising an isolated, organ-derived cell population expressing CD31 and CD45, wherein the antibody binds to a soluble antigen. [b13] The composition described in [b12], wherein the assay is performed according to a method described in any one of [b1] to [b11]. [b14] The composition described in [b12] or [b13], wherein the cell population is a crudely purified cell population. [b15] A method for producing the composition according to any one of [b12] to [b14], (i) preparing organ-derived cells from an organ removed from a living body; and (ii) selecting a population of organ-derived cells based on the expression levels of CD31 and CD45; The method comprising: [b16] A method for predicting antibody clearance in vivo, comprising: (i) measuring the amount of a target antibody taken up into cells by using a method according to any one of [b1] to [b11]; and (ii) predicting the in vivo clearance of the antibody when the antibody is administered to a living body from the amount of uptake measured in (i) above; Including, The method, wherein the antibody binds to a soluble antigen. [b17] A method for screening an antibody, comprising the steps of: (i) providing two or more different antibodies that bind to the same soluble antigen; (ii) measuring the amount of each of the antibodies prepared in (i) above taken up into cells by using a method described in any one of [b1] to [b11]; and (iii) comparing the amounts of uptake into cells of the antibodies measured in (ii) above with each other and selecting the antibody with the highest amount of uptake; The method comprising: [b18] The method according to any one of [b1] to [b5], wherein the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), a cell population shown as P2 in Figure 8 is selected. [b19] The method according to any one of [b1] to [b5], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population, and in the step (ii), a cell population shown as P2 in Figure 2 is selected. [b20] The composition described in [b12], wherein the organ-derived cell population is a human liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 8. [b21] The composition described in [b12], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 2.

[0024] The present invention also includes the following aspects. [c1] A method for measuring the amount of antibody uptake into cells, comprising the steps of: (i) adding the antibody to a population of cells derived from an organ and incubating the same; (ii) selecting a population of cells derived from an organ based on the expression levels of CD31 and CD45; and (iii) measuring the amount of uptake of the antibody into the cell population following steps (i) and (ii); Including, The method, wherein the antibody binds to a membrane-type receptor. [c2] The method according to [c1], which comprises step (ii) following step (i), in which a cell population derived from an organ incubated with the antibody is selected based on the expression levels of CD31 and CD45. [c3] The method according to [c1], which comprises step (ii) prior to step (i), in which the antibody is added to a cell population derived from an organ selected based on the expression levels of CD31 and CD45, and then incubated. [c4] The method according to any one of [c1] to [c3], wherein the antibody is taken up into the cell via the membrane receptor. [c5] The method according to any one of [c1] to [c4], wherein the antibody is an anti-IL-6R antibody and the membrane receptor is IL-6R. [c6] The method according to any one of [c1] to [c5], wherein the organ-derived cell population is a non-parenchymal liver cell population. [c7] The non-parenchymal liver cell population is a human non-parenchymal liver cell population, and in step (ii), high CD45 low A method according to [c6] for selecting a cell population. [c8]CD31 high CD45 low The cell populations were classified into two CD31 subtypes that were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X-axis and Y-axis, respectively. + CD45 + The method according to [c7], which is a cell population having a higher expression level of CD31 when compared at the point where the cell density of each cell population is the highest. [c9]CD31 high CD45 low The method according to [c7], wherein the cell population has a CD31 fluorescence intensity of 400 to 7000 and a CD45 fluorescence intensity of 100 to 4000 when expression of CD31 and CD45 is detected by flow cytometry. [c10] The non-parenchymal liver cell population is a monkey non-parenchymal liver cell population, and in step (ii), intermediate CD45 intermediate A method according to [c6] for selecting a cell population. [c11]CD31 intermediate CD45 intermediateThe cell populations were classified into three CD31 subtypes, which were distinguished from each other based on cell density in a development diagram in which the expression of CD31 and CD45 was detected by flow cytometry and the expression levels of CD31 and CD45 were plotted on the X and Y axes, respectively. + CD45 + The method according to [c10], wherein the cell population has the second highest CD31 expression level when compared at the point where the cell density of each cell population is the highest. [c12]CD31 intermediate CD45 intermediate The method according to [c10], wherein the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000 when expression of CD31 and CD45 is detected by flow cytometry. [c13] A composition for use in an antibody uptake assay, comprising an isolated, organ-derived cell population expressing CD31 and CD45, wherein the antibody binds to a membrane receptor. [c14] The composition described in [c13], wherein the assay is performed according to a method described in any one of [c1] to [c12]. [c15] The composition described in [c13] or [c14], wherein the cell population is a crudely purified cell population. [c16] A method for producing the composition according to any one of [c13] to [c15], (i) preparing organ-derived cells from an organ removed from a living body; and (ii) selecting a population of organ-derived cells based on the expression levels of CD31 and CD45; The method comprising: [c17] A method for predicting antibody clearance in vivo, comprising: (i) measuring the amount of the antibody taken up into cells by using the method according to any one of [c1] to [c12]; and (ii) predicting the in vivo clearance of the antibody when the antibody is administered to a living body from the amount of uptake measured in (i) above; Including, The method, wherein the antibody binds to a membrane-type receptor. [c18] A method for screening an antibody, comprising: (i) providing two or more different antibodies that bind to the same antigen; (ii) measuring the amount of each of the antibodies prepared in (i) above taken up into cells by using a method described in any one of [c1] to [c12]; and (iii) comparing the amounts of uptake into cells of the antibodies measured in (ii) above with each other and selecting the antibody with the highest amount of uptake; Including, The method, wherein the antibody binds to a membrane-type receptor. [c19] The method according to any one of [c1] to [c6], wherein the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), a cell population shown in P2 of Figure 8 is selected. [c20] The method according to any one of [c1] to [c6], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), a cell population shown in P2 of Figure 2 is selected. [c21] The composition described in [c13], wherein the organ-derived cell population is a human liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 8. [c22] The composition described in [c13], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 2.

[0025] The present invention also encompasses an embodiment of an in vitro evaluation system for nucleic acid uptake. As in vitro evaluation systems for nucleic acid uptake into cells, a method using cultured cells in which receptors are forcibly expressed and a method using primary cells collected from organs are known, similar to the immune complex uptake evaluation system. As the former, for example, a nucleic acid uptake evaluation system using HEK-293 cells in which Stabilin-1 and Stabilin-2, known as nucleic acid receptors, are forcibly expressed is known (Non-Patent Document 24). However, such a method is significantly different from the in vivo environment because a specific protein is overexpressed, and may not correlate with in vivo nucleic acid uptake. As the latter method using primary cells collected from organs, a method of isolating and evaluating LSECs from rats (Non-Patent Document 25) and mice (Non-Patent Document 24) is known, but there is a problem that the activity of the cells changes over time.

[0026] Moreover, it is unclear whether the systems using rat and mouse cells reflect the uptake of nucleic acids in monkeys, humans, etc.

[0027] To address these problems, the inventors conducted intensive research and succeeded in constructing an evaluation system capable of evaluating in vivo nucleic acid uptake by constructing an evaluation system using organ-derived cells, similar to the evaluation system for quantitatively measuring the uptake of immune complexes described above.

[0028] That is, the present invention includes the following aspects. [A1] A method for measuring an amount of nucleic acid taken up into a cell, comprising the steps of: (i) adding nucleic acid to a population of cells derived from an organ and incubating the cell population; (ii) selecting a cell population derived from an organ that expresses stabilin (a nucleic acid uptake receptor); and (iii) measuring the amount of nucleic acid uptake into the cell population following steps (i) and (ii); The method comprising: [A2] The method according to [A1], which comprises step (ii) following step (i), and comprises selecting a cell population expressing Stabilin from a cell population derived from an organ to which nucleic acid has been added and incubated. [A3] The method according to [A1], which comprises step (ii) prior to step (i), in which nucleic acid is added to a selected cell population derived from an organ expressing Stabilin, and the cell population is incubated. [A4] The method according to any one of [A1] to [A3], wherein the cell population selected in (ii) expresses CD31 and CD45. [A5] The method according to any one of [A1] to [A4], wherein the organ-derived cell population is a liver non-parenchymal cell population. [A6] A composition for use in a nucleic acid uptake assay, comprising an isolated, organ-derived cell population that expresses Stabilin. [A7] The composition according to [A6], wherein the assay is carried out according to a method according to any one of [A1] to [A5]. [A8] The composition according to [A6] or [A7], wherein the cell population is a crudely purified cell population. [A9] A method for producing the composition according to any one of [A6] to [A8], (i) preparing organ-derived cells from an organ removed from a living body; and (ii) selecting a cell population derived from an organ that expresses Stabilin; The method comprising: [A10] A method for screening a nucleic acid, comprising: (i) providing two or more nucleic acids having the same nucleotide sequence but different chemical modifications; (ii) measuring the amount of each of the two or more nucleic acids prepared in (i) above taken up into cells by using a method described in any one of [A1] to [A5]; and (iii) comparing the amounts of nucleic acid uptake into cells measured in (ii) above with each other and selecting a nucleic acid that shows a desired amount of uptake; The method comprising: [A11] The method according to any of [A1] to [A5], wherein the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), a cell population shown as P1 or P2 in Figure 8 is selected. [A12] The method according to any one of [A1] to [A5], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), a cell population shown as any one of P1, P2, and P3 in Figure 2 is selected. [A13] The composition according to [A6], wherein the organ-derived cell population is a human liver non-parenchymal cell population and consists of the cell population shown in P2 of Figure 8. [A14] The composition according to [A6], wherein the organ-derived cell population is a monkey liver non-parenchymal cell population and consists of a cell population shown as any one of P1, P2, and P3 in Figure 2. Effect of the Invention

[0029] The method of the present invention for measuring the amount of a molecule taken up into a cell via a receptor present on the cell surface more accurately reflects the uptake of the molecule into a cell in vivo compared to conventional measurement methods, and enables more accurate prediction of the dynamics of the molecule in vivo.

[0030] Furthermore, the method of measuring the amount of immune complex uptake into cells of the present invention reflects the uptake of immune complexes into cells in vivo more accurately than conventional measurement methods, and the results obtained by the present invention show a high correlation with the reduction rate of antigens in plasma in vivo. Therefore, according to the present invention, it is possible to efficiently select antibodies capable of efficiently eliminating immune complexes in vivo, and it can also contribute to reducing animal experiments using monkeys and the like. Furthermore, the data obtained by the measurement method of the present invention contributes to the construction of a pharmacokinetic model that can predict the concentration transition of antibodies and antigens in vivo.

[0031] Furthermore, according to the method of the present invention for measuring the amount of nucleic acid uptake into cells, nucleic acid pharmaceuticals with improved uptake into cells can be efficiently screened during research and development of nucleic acid pharmaceuticals. [Brief description of the drawings]

[0032] [Figure 1] Figure 1 is a scatter plot showing the distribution of cells based on forward scattered light (FSC) and side scattered light (SSC) detected when monkey liver non-parenchymal cells are passed through a flow cytometer as shown in Example 3. When measuring with FACS Canto II, data on 20,000 cells excluding debris was acquired and plotted. [Diagram 2] After staining monkey liver non-parenchymal cells with Pacific Blue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody, the respective fluorescence intensities were measured by FACS. All cells shown in the scatter plot in Figure 1 were expanded according to Pacific Blue fluorescence intensity and APC expression intensity. Signals from three cell populations (referred to as P1, P2, and P3, respectively) are confirmed: CD31Low CD45High, CD31Intermediate CD45Intermediate, and CD31High CD45Low. In other areas, cells not expressing these cell markers and signals thought to be noise or autofluorescence can be confirmed, and these are also confirmed in unstained cells. [Diagram 3] Monkey liver non-parenchymal cells were stained with Pacific Blue-labeled anti-CD31 antibody, APC-labeled anti-CD45 antibody, and Alexa488-labeled anti-monkey FcγRIIB antibody, and then their respective fluorescence intensities were measured by FACS. The histograms show the Alexa488 fluorescence intensities for each of the cell populations P1 to P3 in Figure 2. The black dashed line shows the fluorescent signal when stained with Alexa488-labeled negative control antibody, and the black solid line shows the fluorescent signal when stained with Alexa488-labeled anti-monkey FcγRIIB antibody. [Figure 4]The antibody and Alexa488-labeled salmiostatin shown in Example 4 were mixed to form an antibody-antigen complex, which was then added to the cell solution and taken up, and the cells were stained with Pacific Blue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody. The cell population P2 was identified from the development diagrams shown in Figures 1 and 2, and the fluorescence intensity of Alexa488-labeled salmiostatin in P2 is shown in a histogram. The black shading indicates the condition where no antibody was added (only Alexa488-labeled salmiostatin was added), the black short dashed line indicates SG1, the black long dashed line indicates SG145, the black solid line indicates SG141, and the black thick line indicates the histogram when SG143 was mixed with Alexa488-labeled latent salmiostatin and added. Each antibody was added at a concentration of 0.5 (A), 2.5 (B), 10 (C), and 40 (D) μg / mL, respectively. [Diagram 5] The amount of cellular uptake obtained for each added amount of each antibody from the histogram shown in Figure 4. Black circles indicate SG1, black triangles indicate SG141, white circles indicate SG143, and black squares indicate SG145. [Figure 6] For each sample, the reduction rate of myostatin in in vivo monkeys and the amount of myostatin cellular uptake in monkey liver nonparenchymal cells were plotted, and the correlation between the two was shown. The reduction rate of myostatin in monkey plasma was measured 14 days after administration of SG1 (black circle), SG141 (black square), SG143 (black triangle), and SG145 (white circle) to monkeys, and the reduction rate of myostatin in plasma at the time of administration of SG1 was set to 1, and the reduction rate of myostatin in each sample was shown. In addition, the cellular uptake clearance in monkey liver nonparenchymal cells was calculated using the amount of cellular uptake calculated from the fluorescence intensity of Alexa488-labeled myostatin when the antibody was added at 10 μg / mL. [Figure 7] Figure 7 is a scatter plot of the distribution of cells based on forward scattered light (FSC) and side scattered light (SSC) detected when human liver non-parenchymal cells were run through a flow cytometer. The area enclosed by the square was the subject of evaluation. [Figure 8]Human liver non-parenchymal cells were stained with FITC-labeled anti-CD31 antibody and VioBlue-labeled anti-CD45 antibody, and their respective fluorescence intensities were measured by FACS. The cells in the area enclosed by the square in the scatter plot shown in Figure 7 were expanded according to the expression intensity of FITC and VioBlue. Two cell populations, CD31Low CD45High and CD31High CD45Low (referred to as P1 and P2, respectively), were confirmed. In other areas, cells not expressing these cell markers and signals thought to be noise or autofluorescence were confirmed, and these were also confirmed in unstained cells. [Figure 9] Human liver non-parenchymal cells were stained with FITC-labeled anti-CD31 antibody, VioBlue-labeled anti-CD45 antibody, anti-human FcγRIIB human IgG antibody, and Alexa647-labeled anti-human antibody, and then the fluorescence intensity of each was measured by FACS. The histograms show the Alexa647 fluorescence intensity for cell populations P1 and P2 in Figure 8. The black dashed line shows the fluorescence signal when stained with the negative control antibody, and the black solid line shows the fluorescence signal when stained with the anti-human FcγRIIB antibody. (A) P1, (B) P2. [Figure 10] The TT91 antibody and Alexa488-labeled human myostatin shown in Example 9 were mixed to form an antibody-antigen complex, which was then added to a cell solution and taken up, and the cells were stained with VioBlue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody. Cell population P2 was identified from the developments shown in Figures 7 and 8, and the fluorescence intensity of Alexa488-labeled human myostatin in P2 is shown in a histogram. The black shading indicates the condition where no antibody was added (only Alexa488-labeled human myostatin was added), the long dashed black line indicates the antibody at 0.1 μg / mL, the solid black line indicates the antibody at 1 μg / mL, the short solid black line indicates the antibody at 10 μg / mL, and the thick black line indicates the antibody at 100 μg / mL. [Figure 11] The amount of cellular uptake obtained from the histogram shown in FIG. 10 is shown for each amount of antibody added. [Figure 12]Monkey liver non-parenchymal cells were stained with Pacific Blue-labeled anti-CD31 antibody, APC-labeled anti-CD45 antibody, and Alexa488-labeled anti-Stabilin1 antibody or anti-Stabilin2 antibody, and then their fluorescence intensities were measured by FACS. The histograms show the Alexa488 fluorescence intensities for each of the cell populations P1 to P3 in Figure 2. The black dashed line shows the fluorescence signal when stained with Alexa488-labeled negative control antibody, the thin black solid line shows the fluorescence signal when stained with Alexa488-labeled Stabilin1 antibody, and the thick black solid line shows the fluorescence signal when stained with Alexa488-labeled Stabilin2 antibody. [Figure 13] FITC-labeled nucleic acid was added to the cell solution and allowed to be taken up, and then the cells were stained with Pacific Blue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody. Cell populations P1, P2, and P3 were identified from the developments shown in Figures 1 and 2, and the fluorescence intensity of FITC-labeled nucleic acid in each cell population is shown in a histogram for each cell population P1 to P3. The black shading indicates the condition without nucleic acid addition, the black short dashed line indicates the histogram when 0.5 μg / mL nucleic acid was added, the black solid line indicates the histogram when 2.5 μg / mL nucleic acid was added, the black thick line indicates the histogram when 10 μg / mL nucleic acid was added, and the black long wavy line indicates the histogram when 40 μg / mL nucleic acid was added. [Figure 14] The mean fluorescence intensity of cellular uptake obtained from the histogram shown in FIG. 13 is shown for each amount of cell population antibody added. [Figure 15] The Alexa488-labeled antibody shown in Example XXX was added to the cell solution and allowed to be taken up, and then the cells were stained with Pacific Blue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody. The cell population P2 was identified from the developments shown in Figures 1 and 2, and the fluorescence intensity of the Alexa488-labeled antibody in P2 was shown in a histogram. The short black dashed line is the histogram of Alexa488 obtained when SG1 was added, the long black dashed line is the histogram of SG1081, the solid black line is the histogram of SG141, and the thick black line is the histogram of Alexa488 obtained when SG143 was added. Each antibody was added at a concentration of 10 (A), 30 (B), 100 (C), and 200 (D) μg / mL, respectively. [Figure 16]The amount of cellular uptake obtained from the histogram shown in Figure 15 is shown for each amount of antibody added. Black circles indicate SG1, black triangles indicate SG141, white circles indicate SG143, and black squares indicate SG1081. [Figure 17] The clearance of antibodies from plasma in vivo in monkeys and the amount of cellular uptake of Alexa488-labeled antibodies in vitro were plotted to show the correlation between the two. The clearance of antibodies in monkey plasma (plasma clearance) was calculated by measuring the plasma antibody concentrations up to 56 days after administration of SG1 (black circle), SG141 (black square), SG143 (black triangle), and SG1081 (white circle) to monkeys, and using non-compartment model analysis. For the amount of cellular uptake in monkey liver non-parenchymal cells, the amount of cellular uptake was calculated from the fluorescence intensity of Alexa488-labeled antibodies when the antibody was added at 200 μg / mL and used for the plot. [Figure 18] Monkey liver non-parenchymal cells were stained with Pacific Blue-labeled anti-CD31 antibody, APC-labeled anti-CD45 antibody, and Alexa488-labeled tocilizumab, and then their respective fluorescence intensities were measured by FACS. Histograms of Alexa488 fluorescence intensity for cell populations located at P1 to P3, identified from the same development as in Figure 2, are shown in (A) to (C), respectively. The black dashed line shows the results of reaction with an Alexa488-labeled negative control antibody, the black solid line shows Alexa488-labeled tocilizumab, and the gray shadow shows the results of reaction with a solution of Alexa488-labeled tocilizumab with an excess amount of unlabeled tocilizumab. [Figure 19](A) Alexa488-labeled tocilizumab was added to the cell solution at 3 μg / mL and allowed to take up for 2, 5, 10, 15, or 30 minutes at 37°C. After staining with Pacific Blue-labeled anti-CD31 antibody and APC-labeled anti-CD45 antibody, cell population P2 was identified from the same development as in Figure 2, and the fluorescence intensity of Alexa488-labeled tocilizumab in P2 was shown in a histogram. (B) An excess of 1 mg / mL unlabeled tocilizumab was added to 3 μg / mL Alexa488-labeled tocilizumab, and the results were treated in the same manner as in (A). Histograms with different intensities, from light gray to black, show the results of uptake over 2 to 30 minutes. (C) The uptake amount obtained from the histograms shown in (A) and (B) is plotted on the Y axis and the uptake time on the X axis, with the values ​​at each time point. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] I. Cellular uptake of molecules that are taken up into cells via receptors present on the cell surface I-1. A method for measuring the amount of molecules taken up into cells via receptors present on the cell surface A first aspect of the present invention relates to a method for measuring the amount of a molecule taken up into a cell via a receptor present on the cell surface (hereinafter also referred to as measurement method I of the present invention).

[0034] In the present invention, the "molecule that is taken into the cell via a receptor present on the cell surface" refers to a molecule that has a receptor present on the surface of a cell contained in a cell population derived from an organ described below, and that is taken into the cell by binding to the receptor. The molecule that is taken into the cell may be a single molecule or a complex consisting of two or more molecules. The "molecule that is taken into the cell via a receptor present on the cell surface" may be a structure or substance consisting of a large number of molecules. Examples of the molecule include, but are not limited to, the antibody-antigen complex (immune complex), nucleic acid, an antibody that binds to a soluble antigen, and an antibody that binds to a membrane-type receptor, which are molecules described in the Examples, as well as peptide compounds, toxins, viruses, and DDS preparations such as nanoparticles and microparticles.

[0035] According to the measurement method of the present invention, the amount of these molecules taken up into cells can be evaluated in the same manner as in the Examples of the present application, and their dynamics in vivo can be predicted.

[0036] In the present invention, an "immune complex" refers to a complex containing an antibody and an antigen, which is formed by the binding of at least one antibody to at least one antigen. In one embodiment, the immune complex is composed of an antibody and an antigen, and in this case, it can be referred to as an antigen-antibody complex.

[0037] In the present specification, the term "antibody" refers to an immunoglobulin that is natural or partially or completely synthetically produced. Antibodies can be isolated from natural sources such as plasma or serum in which they are naturally present, or from culture supernatants of hybridoma cells that produce antibodies, or can be partially or completely synthesized by using techniques such as genetic recombination. 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 that constitutes the immune complex in the measurement method I of the present invention is IgG.

[0038] The antibody may be either a polyclonal antibody or a monoclonal antibody. In the present invention, a genetically engineered antibody that has been artificially modified for the purpose of reducing heterologous antigenicity, such as a chimeric antibody or a humanized antibody, may be used. The antibody may be a bispecific antibody.

[0039] The antibody may be an antibody fragment so long as it contains an "antigen-binding domain" and an "Fc receptor-binding domain". The "antigen-binding domain" of an antibody may be any domain that binds to an antigen of interest, such as the variable region of the heavy or light chain of an antibody. The "Fc receptor-binding domain" of an antibody may be any domain that binds to an Fc receptor, such as the constant (Fc) region of an antibody. Examples of Fc receptors include FcγR and FcRn. FcγR is preferably FcγRII, more preferably FcγRIIB.

[0040] Methods for producing these antibodies are known to those skilled in the art (e.g., WO 2013 / 081143, etc.).

[0041] As used herein, the term "antigen" is not limited to a specific structure as long as it contains an epitope to which an antigen-binding domain binds. In another sense, an antigen can be inorganic or organic. Antigens include the following molecules: 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, and 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, A RC, 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-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-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, cAMP, 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, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (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,UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, 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, サイトケラチン tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factorfactor), 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, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalka In, 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), GD F-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-23, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, insulin Tegrin 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, NCADHERIN, 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, 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-a 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, TRAILR, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressed Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Virus rus antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, 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, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,Examples include factor XI, factor XIa, factor XII, factor XIIa, 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 and receptors for hormones and growth factors.

[0042] 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, which includes IL-12p40 and IL-12p35, IL-23, which includes IL-12p40 and IL-23p19 (also called IL-30B), or IL-23, which includes EBI-3 and IL27p28, or IL-35, which includes IL-12p35 and EBI-3.

[0043] The above-mentioned examples of antigens also include receptors, and when these receptors are present in a soluble form in a biological fluid such as plasma, they can form a complex with an antibody, and therefore, as long as the above-mentioned receptors are present in a soluble form in a biological fluid such as plasma, they can be used as antigens to which an antibody can bind to form an immune complex. A non-limiting example of such a soluble receptor is the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968) (for example, a protein consisting of amino acids 1 to 357 of the IL-6R polypeptide sequence represented by SEQ ID NO: 1 described in WO 2013 / 081143).

[0044] The above-mentioned examples of antigens include soluble antigens, but the solution in which the antigen exists is not limited, and the soluble antigen may exist in biological fluids, i.e., all fluids that fill blood vessels or tissues and cells in a living body. In a non-limiting embodiment, the antigen that the antibody binds to may exist in extracellular fluid. In vertebrates, the term "extracellular fluid" refers to a collective term for plasma, interstitial fluid, lymphatic fluid, dense connective tissue, cerebrospinal fluid, spinal fluid, aspirate, or synovial fluid, and other components in bones and cartilage, alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor), and other intercellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activities of cells, and fluids in the digestive tract and other body cavities).

[0045] When the molecule that is taken up into cells via the receptor present on the cell surface is an immune complex or an antibody, the antibody is preferably IgG, and the receptor can be Fc receptor.The Fc receptor is preferably FcγR or FcRn.The FcγR is more preferably FcγRII, and more preferably FcγRIIB.

[0046] In the present invention, the term "nucleic acid" refers to DNA, RNA, and analogs thereof, 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 bond, base, and / or sugar, and those having a modification group at the 5' end and / or 3' end. Modifications of internucleoside bonds include changes to any of phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, methylphosphonate bonds, phosphoramidate bonds, non-phosphate bonds, and methylphosphonothioate bonds, or combinations thereof. Modifications of bases include changes to 5-propynyluracil, 2-aminoadenine, and the like. Modifications of sugars include changes to 2'-fluororibose, 2'-O-methylribose, and the like.

[0047] Depending on its function or use, the nucleic acid may be called siRNA, antisense RNA, miRNA, shRNA, ribozyme, or aptamer. The nucleic acid used in the present invention also includes CpG oligonucleotides that act on Toll-like receptor 9 (TLR9) to activate natural immunity.

[0048] 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.

[0049] When the molecule that is taken up into a cell via a receptor present on the cell surface is a nucleic acid, the receptor is preferably stabilin.

[0050] In the present invention, stabilin refers to a protein belonging to a family of transmembrane proteins known as nucleic acid receptors. In mammals, two types of homologues, stabilin-1 and stabilin-2, are known, and the stabilin of the present invention may be either of them. In humans, stabilin-1 (NCBI accession number: NP#055951.2) and stabilin-2 (NCBI accession number: NP#060034.9) are known, and they have been reported to be expressed in LSECs, spleen, adrenal cortex, lymph nodes, and sinusoidal macrophages (Patent Document 24).

[0051] When the molecule that is taken up into cells via the receptor present on the cell surface is an antibody that binds to a soluble antigen, the antibody is preferably IgG, and the receptor can be an Fc receptor.The Fc receptor is preferably FcγR or FcRn.The FcγR is more preferably FcγRII, and more preferably FcγRIIB.

[0052] When the molecule that is taken up into the cell via a receptor present on the cell surface is an antibody that binds to a membrane-type receptor, the receptor may be IL-6R, IL-4R, IL-5R, IL-17R, EGFR, HER2, RANKL, PD-1, PD-L1, etc., and is preferably IL-6R. RANKL and PD-L1 are also called membrane-type ligands, and it is known that antibodies against them also have the same pharmacokinetics as antibodies that bind to membrane-type receptors. Therefore, molecules classified as membrane-type ligands are also included in the "membrane-type receptor" in this specification. In a preferred embodiment, the molecule that is taken up into the cell via a receptor present on the cell surface is an anti-IL-6R antibody, and the receptor is IL-6R. The anti-IL-6R antibody is more preferably a humanized anti-IL-6R antibody, and even more preferably tocilizumab.

[0053] A peptide compound is a compound formed by amide bonds or ester bonds between amino acids or amino acid analogues. The molecular form of a peptide compound may be linear, cyclic, or cyclic with a linear portion.

[0054] The number of amide bonds or ester bonds (number and length of amino acids or amino acid analogs) is not particularly limited, but in the case of a linear portion, the total number of residues in the cyclic portion and the linear portion is preferably 30 or less. The total number of amino acids in the cyclized portion and the linear portion is more preferably 13 or less. In order to obtain 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 further preferably 7 to 11 residues. In particular, 9 to 11 residues are preferred. The number of amino acids and amino acid analogs in the linear portion (number of units) is preferably 0 to 8. More preferably, 0 to 3. In addition, in the present application, unless otherwise specified, amino acids may also include amino acid analogs.

[0055] In this specification, the "amino acids" and "amino acid analogs" constituting a peptide compound may be referred to as "amino acid residues" and "amino acid analog residues", respectively.

[0056] The amino acids are α, β and γ amino acids, and are not limited to natural amino acids (in the present application, natural 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), and may 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 the amino acid side chain, 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.).

[0057] The "amino acids" and "amino acid analogs" constituting the peptide compound include all of their 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" constituting the peptide compound of the present invention include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, and chlorine atoms, each of which is represented by the following: 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 31 P, 32 P, 35 S,18 F, 36 Cl etc. are included.

[0058] When using the fluorescent labeling kit Alexa FluorR 488 Protein Labeling Kit (Invitrogen) to detect peptide compounds, it is desirable to have an amino acid having an amino group. Such an amino acid includes Lys (lysine). In addition, amino acids having a thiol group can also be labeled with a thiol-reactive fluorescent dye. Such an amino acid includes Cys (cysteine).

[0059] When the molecule that is taken up into cells via a receptor present on the cell surface is a peptide compound, the receptor is preferably PEPT1 or PEPT2.

[0060] Nanoparticles and microparticles are known to be used in formulations for drug delivery (Drug Delivery System, commonly known as 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).

[0061] In a preferred embodiment, the molecules that are taken up into cells via receptors present on the cell surface in the measurement method I of the present invention include nanoparticles / microparticles to which a molecule that specifically binds to a specific cell population is bound. For example, an antigen-binding molecule for a surface antigen of the cell population can be bound to these particles. In addition, for example, an antigen-binding molecule that binds to an Fc receptor can be bound to these particles. In one embodiment, the molecules that are taken up into cells via receptors present on the cell surface in the measurement method I of the present invention are nanoparticles / microparticles to which an antibody that binds to the receptor is bound, and the receptor can be an Fc receptor or the above-mentioned membrane-type receptor.

[0062] In addition, hyaluronic acid receptors (CD44, LYVE) are expressed in LSECs, and it is known that hyaluronic acid administered to rats accumulates in LSECs (Cell Tissue Res. 1985;242(3):505-10;J Hepatol. 2017 Jan;66(1):212-227;J Biomater Sci Polym Ed. 2009;20(1):83-97). It has also been shown in experiments using mice that exogenous genes can be delivered to LSECs by binding hyaluronic acid to liposomes (J Pharm Sci. 2013 Sep;102(9):3119-27). Therefore, in one embodiment, the molecule that is taken up into the cell via the receptor present on the cell surface in the measurement method I of the present invention is a nanoparticle or microparticle bound to hyaluronic acid, and the receptor can be a hyaluronic acid receptor.

[0063] Furthermore, it is known that LSECs express mannose receptors and that lysosomal enzymes are recruited to LSECs (Hepatology. 2008 Dec;48(6):2007-15). Therefore, in one embodiment, in measurement method I of the present invention, the molecule that is taken up into a cell via a receptor present on the cell surface is a nanoparticle or microparticle coupled with a molecule that binds to the mannose receptor (e.g. a glycoprotein such as a lysosomal enzyme), and the receptor can be the mannose receptor.

[0064] In the present invention, the "toxin" is not particularly limited as long as it can specifically deliver a cytotoxic agent, a toxin, or a radioisotope to a specific cell population and injure the cell population. For example, a molecule can be prepared by binding a cytotoxic agent, a toxin, or a radioisotope to a molecule that specifically binds to the cell population (for example, an antigen-binding molecule for a cell surface antigen of the cell population). Examples of the "molecule that specifically binds to a cell population" include the above-mentioned antibodies, nucleic acids, and peptide compounds. By using such a molecule, a cytotoxic agent, a toxin, or a radioisotope can be efficiently delivered to the cell population. As a result, the cell population can be specifically injured.

[0065] Exemplary 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 monomethylauristatin 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.

[0066] 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 proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0067] 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.

[0068] Viruses can also be used to damage specific cell populations. According to the measurement method I of the present invention, for example, the uptake of the following viruses or viral proteins or parts thereof into cells can be measured.

[0069] Gene therapy exerts a therapeutic effect by introducing a foreign gene into a host cell. Viruses used in gene therapy include, for example, 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). By producing a recombinant virus by incorporating a gene having cytotoxic activity into these viruses and delivering the recombinant virus to the above-mentioned cell population, the above-mentioned cell population can be damaged.

[0070] There have also been reports of drugs being directly encapsulated in viruses for use in drug delivery (Methods Mol Biol. 2011;726:207-221). In this report, nucleic acids (RNA and DNA) were encapsulated in Red clover necrotic mosaic virus (RCNMV) to create a virus for drug delivery.

[0071] In addition, parts of the proteins that make up the virus can also be used for drug delivery. Here, an example is where a part of the protein that the virus uses to enter the cell is bound to another molecule (Nanotheranostics. 2017; 1(4): 415-429.).

[0072] For example, a partial peptide of the HIV-1 tat protein is used as a cell membrane-permeable peptide. The L2 peptide of human papillomavirus has the activity of destabilizing cell membranes under acidic conditions. The envelope L protein of HBV interacts with the HBV receptor with high affinity. When a partial peptide of this L protein is bound to a synthetic nanocarrier, the nanocarrier is delivered specifically to human liver cells.

[0073] In addition, it has been shown in experimental animals that adenovirus and hepatitis B virus are cleared by LSECs (PLoS Pathog 2011; 7(9): e1002281, Hepatology. 2001;34(4 Pt 1):803-8.).

[0074] The measurement method I of the present invention comprises the following steps (i) to (iii). (i) adding a molecule that is taken up into cells via a receptor present on the cell surface to a population of cells derived from an organ, and incubating the mixture; (ii) selecting a population of cells derived from an organ based on the expression levels of CD31 and CD45; and (iii) measuring the uptake of said molecule into the cell population following steps (i) and (ii).

[0075] As described below, either step (i) or (ii) may be carried out first, and step (iii) is carried out after steps (i) and (ii).

[0076] Measurement method I of the present invention targets mammals. In one embodiment, the mammal is a primate, such as a human, a monkey (such as a cynomolgus monkey, a marmoset, or a rhesus monkey), or a chimpanzee, and is preferably a human or a monkey.

[0077] The "organ" in step (i) includes an organ in a living body that contains a cell population, including blood and bone marrow. In one embodiment, the organ is the liver, mesenteric lymph, blood, bone marrow, stomach, lung, or spleen, and preferably the liver.

[0078] The organ-derived cell population can be prepared by a method commonly used in the art. Alternatively, a commercially available product can be used as the organ-derived cell population. For example, human liver non-parenchymal cells can be obtained from Sekisui Xenotech, Inc., and monkey liver non-parenchymal cells can be obtained from Ina Research, Inc. The cell population can be suspended in an appropriate medium (e.g., OptiThaw Kupffer Cell Thaw / Culture Media (Sekisui XenoTech), HCM (LONZA), etc.), buffer solution, etc.

[0079] In one embodiment, the cell population subjected to the measurement method I of the present invention is a crudely purified cell population. Here, the crudely purified cell population refers to a cell population consisting of cells released from tissue collected from an organ, but which has not been subjected to a purification process to obtain a cell population having the desired characteristics. By not performing excessive purification, the cells can be maintained in a fresh state, and changes in the properties of the cells can be avoided. For example, in the case of a solid organ, a crudely purified cell population can be obtained by enzymatically treating tissue collected from the organ to release the cells, removing impurities with gauze, etc., and removing unnecessary cell populations by a method such as centrifugation, if necessary. An example of a crudely purified cell population is a non-parenchymal cell population prepared from the liver. A liver non-parenchymal cell population can be prepared by resecting tissue from the liver, enzymatically treating it with collagen to release the cells, filtering out impurities with gauze, centrifuging the obtained cell population, removing the precipitated cell population as liver parenchymal cells, and recovering the cells contained in the supernatant (Organ Biology Vol.16 No.3 2009, 361-370).

[0080] In step (i), when the molecule that is taken up into the cells via a receptor present on the cell surface is an immune complex, the antibody and the antigen may be mixed in advance to prepare an immune complex, and this may be added to the cell population, or the antibody and the antigen may be added separately to the cell population and allowed to form an immune complex during incubation.

[0081] The organ-derived cell population to which a molecule that is taken up into cells via a receptor present on the cell surface has been added is incubated for 10 seconds to 24 hours, for example, 1 to 60 minutes, at the physiological temperature of the cell population. The incubation time can be appropriately set by a person skilled in the art within a range in which the amount of uptake increases linearly with the increase in the molecule and the species of organism. The physiological temperature may vary depending on the species of organism, but is, for example, 35 to 38°C, for example, 36 to 37°C for humans, and 35 to 38°C, for example, 36 to 37°C for monkeys. In a preferred embodiment, the cells are incubated at 37°C for 60 minutes for humans, and at 37°C for 15 minutes for monkeys.

[0082] In step (ii), the organ-derived cell population is selected based on the expression levels of CD31 and CD45.

[0083] In step (ii), selecting the organ-derived cell population based on the expression levels of CD31 and CD45 means dividing the organ-derived cell population expressing CD31 and CD45 into smaller cell populations and selecting a cell population having a predetermined range of expression levels of CD31 and CD45. The selected cell population may or may not be isolated from the original cell population.

[0084] When isolating a cell population, the method to be used is not particularly limited, but examples include a method of separating cells using a flow cytometer. In addition, the "panning method," which is known as a method of separating cells by placing a cell population on a plate coated with an antibody that binds to a cell surface marker, and the "immunomagnetic method" using "immunomagnetic beads" in which an antibody against a cell surface marker is immobilized on beads, can also be used (Non-Patent Document 15).

[0085] In order to measure the uptake of a molecule that is taken up into a cell via a receptor present on the cell surface, a cell population expressing the receptor is used. If such a cell population is selected using the expression level of the receptor as an index, the uptake of the molecule may not be measured accurately. For example, if cells are stained with a labeled antibody that binds to the receptor and the molecule is added as is, the antibody may compete with the molecule, which may affect the uptake of the molecule. In contrast, in the present invention, a cell population derived from an organ is selected based on the expression levels of CD31 and CD45, and a cell population expressing the receptor can be identified, so that the measurement can be performed without affecting the uptake of the molecule.

[0086] In one embodiment, measurement method I of the present invention comprises step (ii) following step (i), in which a cell population derived from an organ is incubated with a molecule that is taken up into cells via a receptor present on the cell surface, and the cell population is selected based on the expression levels of CD31 and CD45.

[0087] In another embodiment, the measurement method I of the present invention includes a step (ii) before the step (i), in which a molecule that is incorporated into cells via a receptor present on the cell surface is added to a cell population derived from an organ selected based on the expression levels of CD31 and CD45, and incubated. In this case, the cell population selected in the step (ii) is usually isolated from the original cell population.

[0088] As used herein, "CD31" refers to a molecule identified as the 31st molecule in the CD (cluster of differentiation, cluster of designation, or classification determinant) classification, a method for classifying leukocytes by surface antigens (Histopathology. 1988 May;12(5):461-80.). CD31, also known as Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1), is a single-chain membrane glycoprotein with a molecular weight of 140 kDa that belongs to the immunoglobulin superfamily (Woodfin A., et al. PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol, 2007. 27 (12): 2514-23). ​​The amino acid sequence of human CD31 can be, for example, the sequence described in NCBI Reference Sequence: NP#000433.4. An example of the amino acid sequence of monkey CD31 is the sequence described in GenBank: AFH32784.1. Expression is observed in hematopoietic progenitor cells of the bone marrow system, platelets, and endothelial cell junctions. CD31 is known as an endothelial cell marker, but it has been reported that in normal LSECs, it is expressed in the cytoplasm rather than on the cell surface (Deleve, LD, J Clin Invest. 2013 123(5):1861-6.).

[0089] In this specification, "CD45" refers to a molecule identified as the 45th molecule in the CD classification (Penninger JM., Et al. CD45: new jobs for an old acquaintance. Nat Immunol, 2001. 2 (5): 389-96.). The CD45 molecule is a single-chain transmembrane protein with at least five isoforms ranging in molecular weight from 180 to 235 kDa. These isoforms are formed by combinations of three exons (A, B, and C) on the gene sequence by alternative splicing. The amino acid sequence of the extracellular domain near the membrane, which is common to all CD45 isoform structures, is the leucocyte common antigen (LCA), which is a non-limiting CD45 antigen. All monoclonal antibodies belonging to the CD45 cluster react to this common portion and can recognize all CD45 isoforms. CD45 is known as a hematopoietic cell marker, but it has also been reported that it is expressed in LSECs. It has been reported that CD45 is highly expressed in periportal LSECs, less expressed in midlobular LSECs, and not expressed in centrilobular LSECs (Deleve, LD, J Clin Invest. 2013 123(5):1861-6.). Examples of the amino acid sequence of human CD45 include the sequence described in NCBI Reference Sequence: NP#002829.3. Examples of the amino acid sequence of monkey CD45 include the sequences described in NCBI Reference Sequence: XP#021532837.1, XP#021532838.1, or XP#021532839.1.

[0090] To detect cell surface markers expressed in a cell population, (i) mRNA of the gene encoding the cell surface marker protein ( M essenger R ibo n ucleic aThese methods can be broadly divided into (i) methods that detect the specific antigen (cid) and (ii) methods that detect cell surface marker proteins.

[0091] Examples of methods for detecting mRNA include the Northern hybridization method, the RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) method, DNA chip analysis, etc. Details of these methods are described in textbooks (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989, or Current Protocols in Molecular Biology, John Wiley & Sons Inc., 2003).

[0092] Methods for detecting proteins include, for example, Western blotting, immunohistochemical staining, flow cytometry, ELISA (Enzyme-Linked ImmunoSorbent Assay), surface plasmon resonance analysis, and protein arrays.

[0093] In step (ii), the expression levels of CD31 and CD45 may be measured using any of the above methods. In a preferred embodiment, the cell population expressing the cell surface markers CD31 and CD45 is analyzed by flow cytometry, but other methods may be used. As an example of a method for isolating and analyzing a cell population expressing a specific cell surface marker, a "panning method" is known, which is a method for separating cells by placing a cell population on a plate coated with an antibody that binds to the cell surface marker. In addition, a method of cell separation using "immunomagnetic beads" (immunomagnet method) in which an antibody against a cell surface marker is immobilized on beads is also known. These methods can also be used to separate and analyze a cell population expressing a specific cell surface marker (Non-Patent Document 15).

[0094] When using antibodies to measure the expression levels of CD31 and CD45, labeled antibodies can be used. The method of labeling the antibodies is not limited to a specific method, and any method commonly used in the art can be used, as long as the two types of antibodies used to select the cell population can be distinguished from each other. Methods for labeling antibodies include fluorescent labeling, biotin labeling of antibodies, labeling with peptide tags (His tag, FLAG tag, HA tag, etc.), labeling with gold colloids, labeling with magnetic beads, RI (Radio Isotope) labeling, and enzyme labeling (HRP (Horse Radish Peroxydase) and AP (Alkaline Phosphatase)). Commonly used fluorescent labels include 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, Oyster 500, etc. Any combination can be used as long as the excited labels have different fluorescence spectra.

[0095] Although it is common in the art to refer to cells as "positive" or "negative" for a particular marker, the actual expression level is a quantitative trait. The number of molecules on the cell surface can vary by several orders of magnitude and still be characterized as "positive". It is also well known in the art that negative cells, i.e., cells with no detectable difference from the control, can still express small amounts of the marker. Analysis of expression levels allows fine selection between cell populations.

[0096] In a preferred embodiment, the expression levels of CD31 and CD45 are measured using fluorescently labeled antibodies.

[0097] When fluorescent reagents are used, the staining intensity of cells (i.e., expression of CD31 and CD45) can be monitored by flow cytometry, where a laser detects the quantitative level of fluorochrome (proportional to the amount of cell surface marker bound by a particular reagent, e.g., an antibody). Flow cytometry, or FACS, can also be used to separate cell populations based on the strength of binding to a particular reagent, as well as other parameters such as cell size and light scatter. Although absolute levels of staining may vary with particular fluorochromes and reagent preparations, data can be normalized to controls.

[0098] To normalize the distribution to a control, each cell is recorded as a data point with a particular intensity of staining. These data points can be displayed using a logarithmic scale with arbitrary units of measurement for staining intensity. In one example, the most brightly staining cells in a sample are three orders of magnitude more intense than the negative cells. When displayed in this way, it is clear that the cells at the highest orders of magnitude of staining intensity are bright and the cells at the lowest intensity are negative. A "low" positive staining cell has a staining level that exceeds the brightness of the isotype-same control, but does not stain as intensely as the most brightly staining cells typically found in the cell population. Low positive cells may have unique properties that are different from the negative and brightly staining positive cells in a sample.

[0099] In this specification, a difference of "single digit" represents a difference of roughly the same to 10 times, a difference of "double digit" represents a difference of roughly 10 to 100 times, a difference of "triple digit" represents a difference of roughly 100 to 1000 times, and a difference of "four digits" represents a difference of roughly 1000 to 10,000 times.

[0100] In a preferred embodiment of the present invention, the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), high CD45 low The cell population is sorted.

[0101] In this embodiment, CD31high CD45 low The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "high" and the expression level of CD45 is "low." Here, for CD31, "high" staining intensity means that, when the staining intensity of the brightest stained cell is three orders of magnitude stronger than that of the negative cells, the staining intensity is two to three orders of magnitude stronger than that of the negative cells. "Low" staining intensity means that the staining intensity is one order of magnitude or more, but less than two orders of magnitude stronger than that of the negative cells.

[0102] Regarding CD45, "high" staining intensity means that the staining intensity is two to three orders of magnitude stronger than that of negative cells, when the staining intensity of the brightest stained cells is three orders of magnitude stronger than that of negative cells, and "low" staining intensity means that the staining intensity is one or more orders of magnitude stronger than that of negative cells, but less than two orders of magnitude stronger.

[0103] Here, negative cells are the cell population that exhibits the lowest fluorescence intensity, for example, the cell population that appears on the lower left side in FIG.

[0104] When the expression of CD31 and CD45 is detected by flow cytometry in a human liver non-parenchymal cell population and the expression levels of CD31 and CD45 are plotted on the X-axis and Y-axis, respectively, two distinct cell populations (areas) with high cell density appear in addition to signals thought to be cells not expressing these molecules, autofluorescence, and nonspecific fluorescence due to debris. These two areas can be shown as areas surrounded by contour lines that are not connected to other areas in a contour display based on cell density. high CD45 low The cell population is one of them, that is, a cell population having a higher expression level of CD31 and a cell population having a lower expression level of CD45 when compared at the point where the cell density of each cell population is the highest.

[0105] CD31 above high CD45 lowWhen expression of CD31 and CD45 is detected by flow cytometry, the cell population has a CD31 fluorescence intensity of 400 to 7000 and a CD45 fluorescence intensity of 100 to 4000, where the average CD31 fluorescence intensity of negative cells is approximately 7, the average CD45 fluorescence intensity is approximately 70, and the CD31 staining intensity of the most brightly stained cells is approximately 7000 and the CD45 fluorescence intensity is approximately 70,000.

[0106] In a preferred embodiment of the present invention, the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), the cell population shown as P2 in FIG. 8 is selected.

[0107] In a preferred embodiment of the present invention, the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), intermediate CD45 intermediate The cell population is sorted.

[0108] In this embodiment, CD31 intermediate CD45 intermediate The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "intermediate" and the expression level of CD45 is "intermediate." Here, "intermediate" staining intensity for CD31 means that, when the staining intensity of the most brightly stained cell is three orders of magnitude stronger than that of the negative cell, the staining intensity is about the same as or one order of magnitude stronger than that of the negative cell. Also, "intermediate" staining intensity for CD45 means that, when the staining intensity of the most brightly stained cell is two orders of magnitude stronger than that of the negative cell, the staining intensity is one to two orders of magnitude stronger than that of the negative cell.

[0109] Here, negative cells are the cell population that exhibits the lowest fluorescence intensity, for example, the cell population that appears on the lower left side in FIG.

[0110] When expression of CD31 and CD45 is detected by flow cytometry in monkey liver non-parenchymal cell populations and the expression levels of CD31 and CD45 are plotted on the X-axis and Y-axis, respectively, three distinct cell populations (areas) with high cell density appear in addition to signals thought to be cells not expressing these molecules, autofluorescence, and nonspecific fluorescence due to debris. These three areas can be shown as areas surrounded by contour lines that are not connected to other areas in a contour display based on cell density. intermediate CD45 intermediate The cell population is one of them, i.e., the cell population with the second highest expression of CD31 and the cell population with the second highest expression of CD45 when compared at the highest cell density point of each cell population.

[0111] CD31 above intermediate CD45 intermediate When the expression of CD31 and CD45 is detected by flow cytometry, the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000, where the average fluorescence intensity of CD31 for negative cells is approximately 200, the average fluorescence intensity of CD45 is approximately 200, and the staining intensity of CD31 for the most brightly stained cells is approximately 150,000 and the staining intensity of CD45 is approximately 70,000.

[0112] If the average fluorescence intensity of the reference negative cells changes, intermediate CD45 intermediate The range of fluorescence intensities of cell populations may also vary. intermediate CD45 intermediate The cell population may be a cell population in which the CD31 fluorescence intensity of negative cells is approximately 100, the CD45 fluorescence intensity of negative cells is approximately 20, the CD31 staining intensity of the brightest stained cells is approximately 150,000, and the CD45 staining intensity of negative cells is approximately 70,000, the CD31 fluorescence intensity is 200 to 900, and the CD45 fluorescence intensity is 1,500 to 8,000.

[0113] In a preferred embodiment of the present invention, the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), the cell population shown as P2 in FIG. 2 is selected.

[0114] In step (iii), the amount of the molecule taken up into the cells via a receptor present on the cell surface is measured for the cell population that has been subjected to steps (i) and (ii).

[0115] The amount of a molecule taken up into a cell via a receptor present on the cell surface can be quantified by labeling the molecule and detecting the signal intensity of the label. Labels for proteins include those used for labeling the above-mentioned antibodies. Labeling methods can be used that are common in the art.

[0116] A labeling method for each molecule can be appropriately selected by those skilled in the art, but a method for labeling compounds in general is a method using stable isotopes. This method is generally known as a labeling method used for the pharmacokinetic analysis of drug candidate compounds (Pharmacokinetics vol.8, No.3,1993, 99-109).

[0117] Labeling may also be performed using a commercially available kit. For example, a commercially available kit such as Alexa Fluor (registered trademark) 488 Protein Labeling Kit can be used to fluorescently label a protein.

[0118] The label of the molecule that is internalized into the cell via a receptor present on the cell surface is one that can be distinguished from the label used to label CD31 and CD45.

[0119] In a preferred embodiment, the amount of a molecule taken up into a cell via a receptor present on the cell surface is measured by labeling the molecule with a fluorescent label and using a flow cytometer.

[0120] I-2. Composition for assaying the uptake of molecules taken into cells via receptors present on the cell surface A second aspect of the present invention relates to a composition for an uptake assay of a molecule that is taken up into a cell via a receptor present on the cell surface (hereinafter, also referred to as composition I of the present invention). An uptake assay of a molecule that is taken up into a cell via a receptor present on the cell surface is a test for evaluating the uptake of the molecule into a cell, and in a preferred embodiment, the assay is carried out by measurement method I of the present invention.

[0121] Composition I of the present invention comprises an isolated cell population derived from an organ, which expresses CD31 and CD45. In the present invention, "isolated" refers to a state of being separated from an organ. In one embodiment, the cell population is a crudely purified cell population.

[0122] In the present invention, the term "organ" refers to an organ in a living body that contains a cell population, including blood and bone marrow. In one embodiment, the organ is the liver, mesenteric lymph, blood, bone marrow, stomach, lung, or spleen, and preferably the liver.

[0123] The expression of CD31 and CD45 in the cell population may be detected by a method commonly used in the art (eg, the method for detecting cell surface markers described in I-1 above).

[0124] In a preferred embodiment, the organ-derived cell population contained in Composition I of the present invention is a human liver non-parenchymal cell population, and is CD31 high CD45 low In this embodiment, the composition I of the present invention is a CD31 low CD45 high Does not contain cell populations.

[0125] In a more preferred embodiment, the organ-derived cell population contained in composition I of the present invention is a human liver non-parenchymal cell population, and consists of the cell population shown in P2 of Figure 8. In this embodiment, composition I of the present invention does not include the cell population shown in P1 of Figure 8.

[0126] In yet another preferred embodiment, the organ-derived cell population is a monkey liver non-parenchymal cell population and is CD31 intermediate CD45 intermediate In this embodiment, the composition I of the present invention is a CD31 low CD45 high Cell population and / or CD31 high CD45 low Does not contain cell populations.

[0127] In a more preferred embodiment, the organ-derived cell population is a monkey liver non-parenchymal cell population, consisting of the cell population shown as P2 in Figure 2. In this embodiment, composition I of the present invention does not include the cell populations shown as P1 and P3 in Figure 2.

[0128] These cell populations express FcγRIIB and are suitable for assessing the uptake of immune complexes or antibodies.

[0129] Furthermore, these cell populations express IL-6R and are suitable for evaluating the uptake of anti-IL-6R antibodies.

[0130] Composition I of the present invention may contain a culture medium in addition to the cell population. The culture medium can be appropriately selected depending on the type of organ and the animal species. For example, a culture medium for a cell population derived from human liver may be OptiThaw Kupffer Cell Thaw / Culture Media (Sekisui XenoTech). For a cell population derived from monkey liver, a culture medium may be HCM (LONZA).

[0131] Composition I of the present invention can also be produced by a method comprising the steps of (i) preparing organ-derived cells from an organ removed from a living body, and (ii) selecting a cell population based on the expression levels of CD31 and CD45.

[0132] Steps (i) and (ii) can be carried out as described in I-1 above.

[0133] I-3. A method for predicting in vivo clearance of molecules taken up into cells via receptors present on the cell surface A third aspect of the present invention relates to a method for predicting the in vivo clearance of a molecule that is taken up into a cell via a receptor present on the cell surface (hereinafter also referred to as prediction method I of the present invention).

[0134] The prediction method I of the present invention comprises the following steps. (i) measuring the amount of a molecule taken up into a cell via a receptor present on the cell surface by using the measurement method of the present invention; and (ii) predicting the in vivo clearance of the molecule when the molecule is administered to a living body from the amount of uptake measured in (i) above.

[0135] When the molecule that is taken up into cells via a receptor present on the cell surface is an immune complex, a step of forming an immune complex from an antigen and an antibody may be included prior to step (i), and this step can be carried out by mixing the antigen and the antibody. Alternatively, in step (i), the antibody and the antigen may be added to the cell population, and the immune complex may be formed during incubation.

[0136] Step (i) can be carried out as described in I-1 above.

[0137] In step (ii), the in vivo elimination clearance is predicted from the uptake measured in step (i) based on the correlation between the "in vitro uptake of the molecule" and the "in vivo elimination clearance of the molecule" calculated in advance. The correlation is calculated for each molecule and species, similarly to the specific example using monkeys shown below.

[0138] The amount of the test molecule taken up in vitro is measured and calculated by the following method.

[0139] Monkey liver non-parenchymal cells are commercially available from, for example, Ina Research.

[0140] For detection, the test molecule fluorescently labeled with Alexa488 is added to monkey liver non-parenchymal cells and incubated at 37°C for 15 minutes. Antibodies against the cell surface markers CD31 and CD45 are then added, each labeled with a different fluorescent label. CD31 and CD45 positive cell groups are identified by flow cytometry, and the amount of the test molecule taken up by the receptor positive cell group is measured. The fluorescence intensity detected by flow cytometry is converted to mass. Quantum kit for standardizing fluorescence intensity and mass is used. TM MESF (Bangs Laboratories) or the like may also be used.

[0141] The in vivo elimination clearance (plasma clearance) of the test molecule is calculated by administering the test molecule to monkeys (e.g., cynomolgus monkeys), measuring the plasma antibody concentration up to 56 days after administration, and using non-compartment model analysis.

[0142] The "in vitro uptake of the test molecule" and the "in vivo clearance of the test molecule" are plotted for each molecule. In this way, the correlation between the in vivo clearance of the test molecule and the in vitro uptake of the test molecule can be calculated.

[0143] In the binding activity evaluation using Biacore or the like, the affinity of the test molecule to the receptor can be examined, but the in vivo behavior of the test molecule cannot be predicted. In contrast, the prediction method I of the present invention can estimate the in vivo behavior of the test molecule from the in vitro test results. The prediction method I of the present invention can also be applied to humans, and can improve the accuracy of pharmacokinetic prediction in humans compared to conventional prediction methods based on tests using non-human animals.

[0144] I-4. Method for predicting in vivo antigen reduction rate in blood following antibody administration When the molecule that is taken up into a cell via a receptor present on the cell surface is an immune complex, a fourth aspect of the present invention relates to a method for predicting the in vivo blood antigen reduction rate due to administration of an antibody (hereinafter also referred to as prediction method Ia of the present invention).

[0145] The prediction method Ia of the present invention comprises the following steps: (i) forming an immune complex from an antigen and an antibody; (ii) measuring the amount of the immune complex of (i) above taken up into cells using the measurement method I of the present invention; and (iii) predicting, from the amount of uptake measured in (ii) above, the in vivo blood reduction rate of the antigen when the antibody is administered to a living body.

[0146] Step (i) can be carried out by mixing the antigen and the antibody, or in step (ii), the antibody and the antigen can be added to the cell population and immune complexes formed during incubation.

[0147] Step (ii) can be carried out as described in I-1 above.

[0148] In step (iii), the uptake efficiency (clearance) of the immune complex is calculated from the uptake amount measured in step (ii), and the in vivo blood antigen reduction rate is predicted based on the correlation between the previously calculated "in vitro immune complex uptake efficiency (clearance)" and the "in vivo blood antigen reduction rate." The correlation is calculated for each antigen and species, similar to the specific example using monkeys shown below.

[0149] The in vitro immune complex uptake efficiency (clearance) is measured and calculated by the following method.

[0150] Monkey liver non-parenchymal cells are commercially available from, for example, Ina Research.

[0151] For detection, salmiostatin fluorescently labeled with Alexa488, unmodified anti-salmiostatin antibody (SG1) or various modified anti-salmiostatin antibodies are added to monkey liver non-parenchymal cells and reacted at 37°C for 15 minutes. Antibodies against the cell surface markers CD31 and CD45, each labeled with a different fluorescent label, are then added, and CD31 and CD45 positive cell groups are identified by flow cytometry, and the amount of salmiostatin taken up by FcγRIIB positive cell groups is measured. The fluorescence intensity detected by flow cytometry is converted to mass. Quantum kit for standardizing fluorescence intensity and mass is used. TM MESF (Bangs Laboratories) or the like may also be used. From the calculated results, the in vitro uptake clearance is calculated according to the following formula. In vitro uptake clearance (mL / day / 2x10 5 cells) = [uptake (pg / 2x10 5 cells)] / [reaction time (min) × reaction concentration (pg / mL)] × 60 × 24 In the formula, the reaction concentration is the concentration of the antigen, salmyostatin, when reacting with monkey liver non-parenchymal cells.

[0152] The in vivo antigen reduction rate is measured by administering various modified anti-salmyostatin antibodies to monkeys (e.g., cynomolgus monkeys) and measuring the salmyostatin concentration 14 days after administration (the method is described, for example, in Example 21 of WO2016 / 098357).

[0153] The myostatin plasma concentration upon administration of unmodified anti-salmyostatin antibody SG1 is set to 1, and the in vivo salmyostatin reduction rate can be calculated from the myostatin plasma concentration upon administration of each antibody using the following formula. In vivo plasma myostatin reduction rate = (plasma myostatin concentration upon administration of SG1) / (plasma myostatin concentration upon administration of each antibody) In the formula, the plasma concentration is the plasma concentration 14 days after antibody administration.

[0154] The "in vitro uptake clearance" value calculated based on the "amount of salmiostatin uptake in cells in vitro" and the "in vivo reduction rate of salmiostatin from plasma" are plotted for each antibody (Figure 6). In this way, the correlation between the reduction rate of salmiostatin in plasma of each sample relative to SG1 in vivo and the amount of salmiostatin uptake by monkey liver non-parenchymal cells in vitro can be calculated.

[0155] I-5. Screening method for molecules that are taken up into cells via receptors present on the cell surface The fifth aspect of the present invention relates to a screening method for a molecule that is taken up into cells via a receptor present on the cell surface (hereinafter also referred to as screening method I of the present invention).

[0156] Screening method I of the present invention comprises the following steps: (i) preparing a molecule that is taken up into a cell via two or more different cell surface receptors that bind to the same receptor; (ii) measuring the amount of each of the molecules prepared in (i) above taken up into cells using the measurement method I of the present invention; and (iii) A step of comparing the amounts of the molecules taken up into cells measured in (ii) above with each other and selecting the molecule with the highest amount taken up.

[0157] The two or more molecules in step (i) have binding activity to the same receptor and are different from each other. The molecules that are taken up into cells via receptors present on the cell surface are as described above in I-1.

[0158] When the molecule that is taken up into cells via a receptor present on the cell surface is an immune complex, step (i) can be carried out by mixing an antigen and an antibody, and in step (ii), an antibody and an antigen may be added to a cell population and an immune complex may be formed during incubation.

[0159] Step (ii) can be carried out for each of the two or more molecules as described in I-1 above.

[0160] In step (iii), the molecule with the highest cellular uptake is selected.

[0161] Each selected molecule can be used for purposes according to its characteristics. For example, when the selected molecule is an immune complex, an antibody contained in the immune complex can be used as the antibody with the highest antigen removal activity (see I-6 below).

[0162] Furthermore, when the selected molecule is a DDS preparation such as a toxin, a virus, or nanoparticles or microparticles, the molecule can be used to exert a medicinal effect by directly damaging cells in a living body corresponding to the cell population used in the screening method of the present invention. For example, a compound having cytotoxicity can be delivered to a cell population in a living body by a DDS preparation, thereby damaging the cell population.

[0163] I-6. Screening method for antibodies with antigen removal activity When the molecule that is taken up into the cell via a receptor present on the cell surface is an immune complex, a sixth aspect of the present invention relates to a method for screening an antibody having an antigen-removing effect (hereinafter also referred to as screening method Ia of the present invention).

[0164] The screening method Ia of the present invention comprises the following steps: (i) providing two or more different antibodies that bind to the same antigen; (ii) preparing an immune complex between each of the two or more antibodies prepared in (i) above and the antigen; (iii) measuring the amount of each of the immune complexes prepared in (ii) above taken up into cells using the measurement method I of the present invention; and (iv) A step of comparing the amounts of the immune complexes taken up into cells measured in (iii) above, and selecting the immune complex having the largest amount taken up.

[0165] The two or more antibodies in step (i) have binding activity to the same antigen but differ from each other in amino acid sequence. The antibodies and antigens are as described in I-1 above.

[0166] Step (ii) can be carried out by mixing the antigen and the antibody, or in step (iii), the antibody and the antigen can be added to the cell population and immune complexes formed during incubation.

[0167] Steps (ii) and (iii) can be carried out for each of the two or more antibodies as described in I-1 above.

[0168] In step (iv), by selecting the immune complex that is taken up into the cells in the highest amount, the antibody contained in the immune complex can be selected as the antibody having the highest antigen removal activity.

[0169] II. Cellular uptake of nucleic acids II-1. Method for measuring the amount of nucleic acid uptake into cells A first aspect of the present invention relates to a method for measuring the amount of nucleic acid taken up into a cell (hereinafter also referred to as measurement method II of the present invention).

[0170] The term "nucleic acid" is as described in I above.

[0171] Measurement method II of the present invention comprises the following steps (i) to (iii). (i) adding nucleic acid to a population of cells derived from an organ and incubating the cell population; (ii) selecting a cell population derived from an organ that expresses Stabilin; and (iii) measuring the amount of nucleic acid uptake into the cell population following steps (i) and (ii).

[0172] As described below, either step (i) or (ii) may be carried out first, and step (iii) is carried out after steps (i) and (ii).

[0173] The measurement method II of the present invention is directed to a mammal. In one embodiment, the mammal is a primate, such as a human, a monkey (such as a cynomolgus monkey, a marmoset, or a rhesus monkey), or a chimpanzee, and is preferably a human or a monkey.

[0174] The "organ" in step (i) includes an organ in a living body that contains a cell population, including blood and bone marrow. In one embodiment, the organ is the liver, mesenteric lymph, blood, bone marrow, stomach, lung, or spleen, and preferably the liver.

[0175] The organ-derived cell population can be prepared by a method commonly used in the art. Alternatively, a commercially available product can be used as the organ-derived cell population. For example, human liver non-parenchymal cells can be obtained from Sekisui Xenotech, Inc., and monkey liver non-parenchymal cells can be obtained from Ina Research, Inc. The cell population can be suspended in an appropriate medium (e.g., OptiThaw Kupffer Cell Thaw / Culture Media (Sekisui XenoTech), HCM (LONZA), etc.), buffer solution, etc.

[0176] In one embodiment, the cell population subjected to the measurement method II of the present invention is a crudely purified cell population. Here, the crudely purified cell population refers to a cell population consisting of cells released from tissue collected from an organ, but which has not been subjected to a purification process to obtain a cell population having the desired characteristics. By not performing excessive purification, the cells can be maintained in a fresh state, and changes in the properties of the cells can be avoided. For example, in the case of a solid organ, a crudely purified cell population can be obtained by enzymatically treating tissue collected from the organ to release the cells, removing impurities with gauze, etc., and removing unnecessary cell populations by a method such as centrifugation, if necessary. An example of a crudely purified cell population is a non-parenchymal cell population prepared from the liver. A liver non-parenchymal cell population can be prepared by excising tissue from the liver, enzymatically treating it with collagen to release the cells, filtering out impurities with gauze, centrifuging the obtained cell population, removing the precipitated cell population as liver parenchymal cells, and recovering the cells contained in the supernatant (Organ Biology Vol.16 No.3 2009, 361-370).

[0177] The organ-derived cell population to which the nucleic acid has been added in step (i) is incubated for 10 seconds to 24 hours, for example, 1 to 60 minutes, at the physiological temperature of the cell population. The physiological temperature is, for example, 35 to 38° C., for example, 36 to 37° C. for humans, and 35 to 38° C., for example, 36 to 37° C. for monkeys. In a preferred embodiment, the cell population is incubated at 37° C. for 60 minutes for humans, and at 37° C. for 15 minutes for monkeys.

[0178] In step (ii), a cell population derived from an organ that expresses Stabilin is selected.

[0179] Stabilin is as described in I above.

[0180] The cell population sorted in step (ii) may or may not be isolated from the original cell population.

[0181] When isolating a cell population, the method to be used is not particularly limited, but examples include a method of separating cells using a flow cytometer. In addition, the "panning method," which is known as a method of separating cells by placing a cell population on a plate coated with an antibody that binds to a cell surface marker, and the "immunomagnetic method" using "immunomagnetic beads" in which an antibody against a cell surface marker is immobilized on beads, can also be used (Non-Patent Document 15).

[0182] In one embodiment, measurement method II of the present invention comprises step (ii) following step (i), in which a cell population expressing Stabilin is selected from a cell population derived from an organ incubated with the addition of nucleic acid.

[0183] In another embodiment, the measurement method II of the present invention includes a step (ii) prior to the step (i), in which a nucleic acid is added to a selected cell population derived from an organ expressing Stabilin, followed by incubation. In this case, the cell population selected in the step (ii) is usually isolated from the original cell population.

[0184] In step (ii), a cell population derived from an organ expressing Stabilin can be selected using a method generally used in the art. For example, the expression of Stabilin can be detected using a method for detecting the mRNA of a gene encoding Stabilin and a method for detecting Stabilin protein.

[0185] Examples of methods for detecting mRNA include the Northern hybridization method, the RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) method, DNA chip analysis, etc. Details of these methods are described in textbooks (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989, or Current Protocols in Molecular Biology, John Wiley & Sons Inc., 2003), etc.

[0186] Methods for detecting proteins include, for example, Western blotting, immunohistochemical staining, flow cytometry, ELISA (Enzyme-Linked ImmunoSorbent Assay), surface plasmon resonance analysis, and protein arrays.

[0187] In step (ii), the expression level of Stabilin may be measured using any of the above methods. In a preferred embodiment, the cell population expressing Stabilin is analyzed by flow cytometry, but other methods may be used. As an example of a method for isolating and analyzing a cell population expressing a specific protein, a "panning method" is known, which is a method for separating cells by placing a cell population on a plate coated with an antibody that binds to the protein. In addition, a cell separation method (immunomagnetic method) using "immunomagnetic beads" in which an antibody against the protein is immobilized on beads is also known. These methods can also be used to separate and analyze a cell population expressing a specific protein (Non-Patent Document 15).

[0188] In the present invention, when a cell population expressing Stabilin is selected using the expression level of Stabilin as an index, it may not be possible to accurately measure the uptake of nucleic acid. For example, if cells are stained with a labeled anti-Stabilin antibody and nucleic acid is added directly, the antibody may compete with the nucleic acid, affecting the uptake of nucleic acid.

[0189] Therefore, in a preferred embodiment of the present invention, a cell population expressing Stabilin is selected without using the expression level of Stabilin as an index. For example, a cell population expressing Stabilin can be identified by selecting an organ-derived cell population based on the expression levels of CD31 and CD45. In this embodiment, the cell population selected in step (ii) is a cell population expressing CD31 and CD45.

[0190] In addition, in the above-mentioned preferred embodiment, selecting an organ-derived cell population based on the expression levels of CD31 and CD45 in step (ii) means dividing an organ-derived cell population expressing CD31 and CD45 into smaller cell populations and selecting a cell population having expression levels of CD31 and CD45 within a predetermined range.

[0191] The expression levels of CD31 and CD45 were measured as described in I-1 above.

[0192] When antibodies are used to measure the expression levels of stabilin, CD31, and CD45, labeled antibodies can be used. The method of labeling the antibodies is not limited to a specific method, and any method commonly used in the art can be used, as long as it can distinguish the two types of antibodies used to select the cell population. Methods for labeling antibodies include fluorescent labeling, biotin labeling of antibodies, labeling with peptide tags (His tag, FLAG tag, HA tag, etc.), labeling with gold colloids, labeling with magnetic beads, RI (Radio Isotope) labeling, and enzyme labeling (HRP (Horse Radish Peroxydase) and AP (Alkaline Phosphatase)). Commonly used fluorescent labels include 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, Oyster 500, etc. Any combination can be used as long as the excited labels have different fluorescence spectra.

[0193] Although it is common in the art to refer to cells as "positive" or "negative" for a particular marker, the actual expression level is a quantitative trait. The number of molecules on the cell surface can vary by several orders of magnitude and still be characterized as "positive". It is also well known in the art that negative cells, i.e., cells with no detectable difference from the control, can still express small amounts of the marker. Analysis of expression levels allows fine selection between cell populations.

[0194] In a preferred embodiment, the expression levels of Stabilin, as well as CD31 and CD45, are measured using fluorescently labeled antibodies.

[0195] When fluorescent reagents are used, the staining intensity of cells (i.e., expression of Stabilin, CD31, and CD45) can be monitored by flow cytometry, where a laser detects the quantitative level of fluorochrome (proportional to the amount of cell surface marker bound by a particular reagent, e.g., an antibody). Flow cytometry, or FACS, can also be used to separate cell populations based on the strength of binding to a particular reagent, as well as other parameters such as cell size and light scatter. Although absolute levels of staining may vary with particular fluorochromes and reagent preparations, data can be normalized to controls.

[0196] To normalize the distribution to a control, each cell is recorded as a data point with a particular intensity of staining. These data points can be displayed using a logarithmic scale with arbitrary units of measurement for staining intensity. In one example, the most brightly staining cells in a sample are three orders of magnitude more intense than the negative cells. When displayed in this way, it is clear that the cells at the highest orders of magnitude of staining intensity are bright and the cells at the lowest intensity are negative. A "low" positive staining cell has a staining level that exceeds the brightness of the isotype-same control, but does not stain as intensely as the most brightly staining cells typically found in the cell population. Low positive cells may have unique properties that are different from the negative and brightly staining positive cells in a sample.

[0197] In this specification, a difference of "single digit" represents a difference of roughly the same to 10 times, a difference of "double digit" represents a difference of roughly 10 to 100 times, a difference of "triple digit" represents a difference of roughly 100 to 1000 times, and a difference of "four digits" represents a difference of roughly 1000 to 10,000 times.

[0198] In a preferred embodiment of the present invention, the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), high CD45 lowCell population or CD31 low CD45 high Select the group.

[0199] In this embodiment, CD31 high CD45 low The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "high" and the expression level of CD45 is "low." low CD45 high The population refers to a cell population in which the staining intensity (expression level) of CD31 is "low" and the expression level of CD45 is "high." Here, for CD31, "high" staining intensity means that, when the staining intensity of the brightest stained cell is three orders of magnitude stronger than that of the negative cells, the staining intensity is two to three orders of magnitude stronger than that of the negative cells. Furthermore, "low" staining intensity means that the staining intensity is one order of magnitude stronger than that of the negative cells, but less than two orders of magnitude stronger.

[0200] Regarding CD45, "high" staining intensity means that, when the staining intensity of the brightest stained cells is three orders of magnitude stronger than that of negative cells, the staining intensity is two to three orders of magnitude stronger than that of negative cells, whereas "low" staining intensity means that the staining intensity is one or more orders of magnitude stronger than that of negative cells but less than two orders of magnitude stronger.

[0201] Here, negative cells are the cell population that exhibits the lowest fluorescence intensity, for example, the cell population that appears on the lower left side in FIG.

[0202] When the expression of CD31 and CD45 is detected by flow cytometry in a human liver non-parenchymal cell population and the expression levels of CD31 and CD45 are plotted on the X-axis and Y-axis, respectively, two distinct cell populations (areas) with high cell density appear in addition to signals thought to be cells not expressing these molecules, autofluorescence, and nonspecific fluorescence due to debris. These two areas can be shown as areas surrounded by contour lines that are not connected to other areas in a contour display based on cell density. high CD45 lowThe cell population is one of them, that is, a cell population with a higher expression level of CD31 and a lower expression level of CD45 when compared at the highest cell density point of each cell population. low CD45 high The population is a cell population that expresses lower amounts of CD31 and higher amounts of CD45 when compared to the other one, ie, at the point of highest cell density of each cell population.

[0203] CD31 above high CD45 low The cell population is a cell population in which, when the expression of CD31 and CD45 is detected by flow cytometry, the average fluorescence intensity of CD31 of negative cells is about 7, the average fluorescence intensity of CD45 is about 70, the staining intensity of CD31 of the brightest stained cell is about 7000, and the fluorescence intensity of CD45 is about 70000. The cell population is a cell population in which the fluorescence intensity of CD31 is 400 to 7000 and the fluorescence intensity of CD45 is 100 to 4000. low CD45 high When expression of CD31 and CD45 is detected by flow cytometry, the population is a cell population in which the CD31 fluorescence intensity is 30 to 1000 and the CD45 fluorescence intensity is 5000 to 70000, where the average fluorescence intensity of CD31 for negative cells is about 7, the average fluorescence intensity of CD45 is about 70, and the staining intensity of CD31 for the most brightly stained cells is about 7000 and the fluorescence intensity of CD45 is about 70000.

[0204] In a more preferred embodiment of the present invention, the organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), a cell population shown as P1 or P2 in FIG. 8 is selected.

[0205] In a preferred embodiment of the present invention, the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), low CD45 high Cell population, CD31 intermediate CD45 intermediate Cell population, or CD31 high CD45low The cell population is sorted.

[0206] In this embodiment, CD31 low CD45 high The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "low" and the expression level of CD45 is "high." CD31 intermediate CD45 intermediate The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "intermediate" and the expression level of CD45 is "intermediate." CD31 high CD45 low The cell population refers to a cell population in which the staining intensity (expression level) of CD31 is "high" and the expression level of CD45 is "low."

[0207] Here, "intermediate" staining intensity for CD31 means that, when the staining intensity of the brightest stained cells is three orders of magnitude stronger than that of negative cells, the staining intensity is the same as or one order of magnitude stronger than that of negative cells. "High" staining intensity means that the staining intensity is one to three orders of magnitude stronger than that of negative cells. "Low" staining intensity means that the staining intensity is the same as or one order of magnitude stronger than that of negative cells.

[0208] Regarding CD45, "intermediate" staining intensity means that, when the staining intensity of the brightest stained cells is two orders of magnitude stronger than that of negative cells, the staining intensity is one to two orders of magnitude stronger than that of negative cells. "High" staining intensity means that the staining intensity is one to two orders of magnitude stronger than that of negative cells. "Low" staining intensity means that the staining intensity is the same or one order of magnitude stronger than that of negative cells.

[0209] Here, negative cells are the cell population that exhibits the lowest fluorescence intensity, for example, the cell population that appears on the lower left side in FIG.

[0210] In Figure 2, P1 is CD31 low CD45 highP2 is a CD31 cell population. intermediate CD45 intermediate Although they are cell populations, the fluorescence intensity of CD31 in P2 is stronger than that in P1, and the fluorescence intensity of CD45 in P2 is weaker than that in P1, and the two are distinguishable from each other as shown in FIG. 2.

[0211] When expression of CD31 and CD45 is detected by flow cytometry in monkey liver non-parenchymal cell populations and the expression levels of CD31 and CD45 are plotted on the X-axis and Y-axis, respectively, three distinct cell populations (areas) with high cell density appear in addition to signals thought to be cells not expressing these molecules, autofluorescence, and nonspecific fluorescence due to debris. These three areas can be shown as areas surrounded by contour lines that are not connected to other areas in a contour display based on cell density. low CD45 high The cell population is the one that expresses the lowest amount of CD31 and the highest amount of CD45 when compared in one of the three regions, i.e., at the point of highest cell density of each cell population. intermediate CD45 intermediate The cell population is the cell population with the second highest expression of CD31 and the second highest expression of CD45 when compared at one of the three regions, i.e., the highest cell density point of each cell population. high CD45 low The cell population is the one with the highest expression of CD31 and the lowest expression of CD45 when compared in one of three regions, i.e., at the point of highest cell density for each cell population.

[0212] CD31 above low CD45 highWhen expression of CD31 and CD45 is detected by flow cytometry, the cell population has a CD31 fluorescence intensity of 50 to 1,500 and a CD45 fluorescence intensity of 8,000 to 70,000, where the average fluorescence intensity of CD31 for negative cells is approximately 200, the average fluorescence intensity of CD45 is approximately 200, and the staining intensity of CD31 for the most brightly stained cells is approximately 150,000 and the staining intensity of CD45 is approximately 70,000.

[0213] CD31 above intermediate CD45 intermediate When the expression of CD31 and CD45 is detected by flow cytometry, the cell population has a CD31 fluorescence intensity of 500 to 3000 and a CD45 fluorescence intensity of 4000 to 20000, where the average fluorescence intensity of CD31 for negative cells is approximately 200, the average fluorescence intensity of CD45 is approximately 200, and the staining intensity of CD31 for the most brightly stained cells is approximately 150,000 and the staining intensity of CD45 is approximately 70,000.

[0214] CD31 above high CD45 low When the expression of CD31 and CD45 is detected by flow cytometry, the cell population has a CD31 fluorescence intensity of 9,000 to 150,000 and a CD45 fluorescence intensity of 50 to 3,000, where the average fluorescence intensity of CD31 for negative cells is approximately 200, the average fluorescence intensity of CD45 is approximately 200, and the staining intensity of CD31 for the most brightly stained cells is approximately 150,000 and the staining intensity of CD45 is approximately 70,000.

[0215] If the average fluorescence intensity of the reference negative cells changes, the above range of fluorescence intensity may also change.

[0216] In a more preferred embodiment of the present invention, the organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), a cell population shown as any of P1, P2, and P3 in FIG. 2 is selected.

[0217] In step (iii), the amount of nucleic acid uptake is measured for the cell population that has been subjected to steps (i) and (ii).

[0218] The amount of nucleic acid uptake can be quantified by labeling the nucleic acid and detecting the signal intensity of the label. Such labels include those used for labeling the above-mentioned antibodies. The method for labeling nucleic acid can be a method generally used in the art. The label used for nucleic acid is one that can be distinguished from the labels used for labeling Stabilin, CD31 and CD45.

[0219] In a preferred embodiment, the amount of nucleic acid uptake is measured by labeling the nucleic acid with a fluorescent label and using a flow cytometer.

[0220] II-2. Compositions for nucleic acid uptake assays The second aspect of the present invention relates to a composition for a nucleic acid uptake assay (hereinafter also referred to as composition II of the present invention). A nucleic acid uptake assay is a test for evaluating the uptake of a nucleic acid into a cell, and in a preferred embodiment, the assay is carried out by the measurement method II of the present invention.

[0221] Composition II of the present invention includes an isolated cell population derived from an organ that expresses Stabilin. In the present invention, "isolated" refers to a state in which the cell population is separated from an organ. In one embodiment, the cell population is a crudely purified cell population.

[0222] In the present invention, the term "organ" refers to an organ in a living body that contains a cell population, including blood and bone marrow. In one embodiment, the organ is the liver, mesenteric lymph, blood, bone marrow, stomach, lung, or spleen, and preferably the liver.

[0223] The expression of Stabilin in the cell population may be detected by a method generally used in the art (eg, the method for detecting Stabilin described in II-1 above).

[0224] In one embodiment, a cell population expressing Stabilin is identified by sorting an organ-derived cell population based on the expression levels of CD31 and CD45.

[0225] In a preferred embodiment, the organ-derived cell population contained in Composition II of the present invention is a human liver non-parenchymal cell population, and is CD31 high CD45 low Cell population or CD31 low CD45 high In a more preferred embodiment, the organ-derived cell population contained in composition II of the present invention is a human liver non-parenchymal cell population, consisting of the cell population shown as P1 or P2 in FIG.

[0226] In another preferred embodiment, the organ-derived cell population contained in the composition II of the present invention is a monkey liver non-parenchymal cell population, and low CD45 high Cell population, CD31 intermediate CD45 intermediate Cell population, or CD31 high CD45 low In a more preferred embodiment, the organ-derived cell population contained in composition II of the present invention is a monkey liver non-parenchymal cell population, and consists of any of the cell populations shown as P1, P2, and P3 in FIG.

[0227] These cell populations express stabilin and are suitable for evaluating nucleic acid uptake.

[0228] Composition II of the present invention may contain a culture medium in addition to the cell population. The culture medium can be appropriately selected depending on the type of organ and the animal species. For example, a culture medium for a cell population derived from human liver may be OptiThaw Kupffer Cell Thaw / Culture Media (Sekisui XenoTech). For a cell population derived from monkey liver, a culture medium may be HCM (LONZA).

[0229] Composition II of the present invention can also be produced by a method comprising the steps of (i) preparing organ-derived cells from an organ removed from a living body, and (ii) selecting a population of organ-derived cells that express Stabilin.

[0230] Steps (i) and (ii) can be carried out as described in II-1 above.

[0231] II-3. Nucleic Acid Screening Method A third aspect of the present invention relates to a method for screening for an antibody having an antigen-removing activity (hereinafter also referred to as screening method II of the present invention).

[0232] Screening method II of the present invention comprises the following steps: (i) providing two or more nucleic acids having the same nucleotide sequence but different chemical modifications; (ii) measuring the amount of each of the two or more nucleic acids prepared in (i) above, by using the measurement method II of the present invention; and (iii) A step of comparing the amounts of nucleic acid taken up into cells measured in (ii) above, and selecting a nucleic acid that shows a desired amount of uptake.

[0233] The two or more nucleic acids having the same nucleotide sequence and different chemical modifications in step (i) are each modified as described in II-1 above.

[0234] Step (ii) can be carried out for each of the two or more nucleic acids as described in II-1 above.

[0235] In step (iii), by selecting a nucleic acid that exhibits a desired amount of uptake into cells, it can be used for applications (such as nucleic acid medicines) according to its characteristics.

[0236] All prior art documents cited in this specification are hereby incorporated by reference.

[0237] The present invention will now be described in more detail with reference to examples. EXAMPLES

[0238] [Example 1] Preparation of monkey liver non-parenchymal cells Monkey liver non-parenchymal cells obtained by the method described below were purchased (Ina Research) and used to quantify the expression level of FcγRIIB and to evaluate cell binding and uptake of antibody-antigen complexes.

[0239] Specifically, cynomolgus monkeys (Macaca fascicularis) were intravenously administered 330 units / kg of heparin sodium (Mochida Pharmaceutical Co., Ltd.), and then anesthetized with thiopental sodium (Tanabe Mitsubishi Pharma Co., Ltd.) and killed by exsanguination. The entire liver lobe was then removed through laparotomy and stored in chilled William's E Medium (Thermo Fisher Scientific).

[0240] The liver was cut into individual lobes and perfused with Hanks-HEPES buffer containing glycol ether diamine tetraacetic acid (EGTA) for 6-10 minutes to remove blood. The liver was then perfused with 0.05% collagenase solution (Sigma Aldrich) for 6-8 minutes, transferred to a culture dish, and liver cells were dispersed and filtered using gauze.

[0241] This cell suspension was centrifuged at 50 xg for 1 minute at 4°C to precipitate parenchymal cells. The supernatant was collected and centrifuged again, and the supernatant was collected and centrifuged at 1000 xg to precipitate non-parenchymal cells. The supernatant was then removed, and the precipitate was suspended in HBSS(+) (GIBCO), and 39% OptiPrep (Alere Technologies) was layered on top. This was centrifuged at 400 xg for 15 minutes at 4°C, and the collected supernatant was centrifuged at 3000 rpm for 5 minutes at 4°C. The precipitate was suspended in HCM (LONZA), and this was used as monkey liver non-parenchymal cells.

[0242] [Example 2] Alexa488 labeling of anti-monkey FcγRIIB antibody and monkey myostatin Each protein was labeled with Alexa 488 using the Alexa flour 488 labeling kit (Thermo Fisher Scientific) according to the attached protocol. The concentration of each protein and the labeling efficiency of the fluorescent substance were calculated by measuring the absorbance using Nanodrop (Thermo Fisher Scientific) and calculating according to the formula described in the attached protocol.

[0243] [Example 3] Identification of cells with high FcγRIIB expression in monkey liver non-parenchymal cells 2x10 5 Pacific blue-labeled anti-CD31 antibody (BioLegend), APC-labeled anti-CD45 antibody (Miltenyi), and Alexa488-labeled anti-monkey FcγRIIB antibody (Sino Biological) were added to each monkey non-parenchymal liver cell to make a volume of 100 μL, and the cells were incubated on ice for 30 minutes. The cells were then centrifuged at 3000 rpm for 5 minutes at 4°C, the supernatant was removed, and the cells were washed with PBS(-). The cells were resuspended in 2% FBS-containing PBS(-), and the fluorescence of the cell solution was measured using a FACS Canto II (Becton, Dickinson and Company), and dot plots were developed using forward scatter (FSC) and side scatter (SSC) (Figure 1). All cells expanded with FSC and SSC were further expanded with Pacific blue (CD31) and APC (CD45) (Figure 2).

[0244] Non-parenchymal liver cells are a mixture of various cell populations, including vascular endothelial cells, Kupffer cells, and stellate cells. However, by using two cell markers, CD31 and CD45, which are also known as cell markers for LSECs in humans, we were able to separate them into three populations. The three cell populations that emerged were called P1 (CD31 Low CD45 High ), P2(CD31 Intermediate CD45 Intermediate ), P3(CD31 High CD45 Low) The dots seen in other areas were excluded from the analysis because they were considered to be autofluorescence from cells not expressing these markers or nonspecific fluorescence from debris. Expression of monkey FcγRIIB was evaluated by detecting the fluorescence intensity of Alexa488 in these cell populations (FITC filter detection).

[0245] The expression of monkey FcγRIIB in these cell populations was evaluated. As shown in Figure 3, no peak shift was observed in P1 and P3 by addition of anti-monkey FcγRIIB antibody compared to the negative control antibody labeled with Alexa488 (Figure 3A and Figure 3C), but a clear peak shift to the high intensity side was observed in P2 by the anti-monkey FcγRIIB antibody, demonstrating that FcγRIIB was expressed only in P2.

[0246] Because the proportion of FcγRIIB expressing cells to the total number of cells is very small, it is difficult to identify FcγRIIB expressing cells without separation by cell markers. However, by staining with the cell markers CD31 and CD45 and separating each cell population, it became possible to identify FcγRIIB expressing cells.

[0247] In addition, this method does not use anti-FcγRIIB antibodies for gating cell populations, so the expression level of FcγRIIB in the target cell population can be quantified. TM Monkey FcγRIIB expression was quantified using MESF (Bangs Laboratories) according to the attached protocol, and the expression level was 1.8 x 10 4 Expression of the molecule was confirmed.

[0248] [Example 4] Evaluation of cellular uptake of antibody-antigen complexes in cell population P2 of monkey liver nonparenchymal cells (4-1) Characteristics of the Fc of the antibody used to evaluate the uptake of antibody-antigen complexes We used SG1, SG141, SG143 (also referred to as FS154), and SG145 (also referred to as FS156), which are anti-myostatin antibodies having Fc described in WO 2016 / 117346 A1 (Patent Document 4) and WO 2016 / 098357 A1 (Patent Document 5).

[0249] When the affinity of SG1 for monkey FcγRIIB is taken as 1.0, SG143 shows 5-10 times higher binding activity, and SG145 shows 1-2 times higher binding activity (WO 2016 / 117346 A1). SG141 is an antibody with a positively charged Fc with a high pI, and shows 1.64 times higher binding activity than SG1 in a Biacore IC binding test for monkey FcγRIIB (WO 2016 / 098357 A1).

[0250] (4-2) Evaluation of cellular uptake of antibody-antigen complexes 2x10 5 Alexa488-labeled salmiostatin and each antibody were added to 50 μL of cell solution containing 10 cells at a final concentration of 0.3 μg / mL and 0 to 40 μg / mL to prepare 100 μL of reaction solution. This solution was reacted at 37°C for 15 minutes while stirring. The cells were then cooled on ice, and cold 2% FBS-containing PBS was added to wash the cells. The solution was then removed by centrifugation (3000 rpm, 5 min). The cells were further stained with anti-CD31 antibody and anti-CD45 antibody, washed, and the fluorescence intensity of Alexa488 at P1, P2, and P3 was measured using FACS Canto II.

[0251] As a result, in P1 and P3, the fluorescence peak of Alexa488-labeled myostatin did not shift significantly under antibody-added conditions compared to the absence of antibody. On the other hand, in P2, the fluorescence peak of Alexa488-labeled myostatin shifted to higher intensity in the following order at all antibody concentrations: no antibody, SG1, SG145, SG141, SG143 (Figure 4). This suggests that the antibody-antigen complex binds to FcγRIIB on the cell surface and is taken up into the cell according to the Fc characteristics of each.

[0252] (4-3) Quantification of antigen uptake Quantum TM The fluorescence intensity of the fluorescently labeled standard beads was measured using MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard according to the attached protocol, and the amount of salmyostatin taken up for each antibody and each concentration was calculated from the geometric mean fluorescence intensity of the salmyostatin-taken sample.

[0253] The results are shown in Figure 5. At all concentrations evaluated, uptake was highest when SG143 was used, followed by SG141, SG145, and SG1 in that order. Furthermore, for each antibody, uptake at higher concentrations was comparable to or higher than that at an antibody addition concentration of 0.5 μg / mL.

[0254] [Example 5] Correlation between monkey myostatin uptake in non-parenchymal cells of the liver in vitro and antigen reduction rate in vivo (5-1) Evaluation of antigen reduction rate in in vivo monkeys When monkeys are administered SG1, SG141, SG143, or SG145, which are antibodies against salmyostatin, salmyostatin and the antibody form an immune complex. The immune complex is thought to be taken up by cells that strongly express FcγRIIB and removed from the blood. Using a previously reported method (WO 2016 / 098357 A1), the blood concentration of salmyostatin 14 days after antibody administration was measured, and the reduction rate at the time of administration of each antibody was calculated, with the myostatin concentration at the time of administration of SG1 being set to 1. The specific method is as follows.

[0255] Accumulation of endogenous myostatin following administration of anti-latent myostatin antibodies was evaluated in vivo in 2-4 year-old Cambodian Macaca fascicularis (cynomolgus monkeys) (Shin Nippon Biomedical Laboratories Ltd., Japan). A dose level of 30 mg / kg was infused into the cephalic vein of the forearm using a disposable syringe, extension tube, indwelling needle, and infusion pump. The administration speed was 30 min per animal. Blood samples were collected either before the start of administration, and 5 min, 7 h, and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days after the end of administration, or 5 min, 2, 4, and 7 h, and 1, 2, 3, 7, 14, 21, 28, 35, 42, 49, and 56 days after the end of administration. Blood was collected from the femoral vein using a syringe containing sodium heparin. The blood was immediately cooled on ice and centrifuged at 1700 × g for 10 min at 4 °C to obtain plasma. Plasma samples were stored in an ultra-low temperature freezer (allowable range: -70 °C or lower) until measurement. Myostatin concentrations in the plasma samples were measured by electrochemiluminescence (ECL).

[0256] (5-2) Correlation between in vivo salmiostatin reduction rate and in vitro cellular uptake clearance The values ​​obtained by converting the amount of salmyostatin cellular uptake in vitro into the uptake clearance and the in vivo reduction rate of salmyostatin from plasma were plotted for each antibody (Figure 6). As a result, a positive correlation was observed between the reduction rate of salmyostatin in plasma for each sample relative to SG1 in vivo and the amount of salmyostatin uptake by monkey liver non-parenchymal cells in vitro. SG143, which had the highest cellular uptake, was able to reduce myostatin the most from plasma in vivo, while SG1 had the smallest amount of cellular uptake and reduction rate from plasma. The ranking of cellular uptake among antibodies correlated well with the reduction rate of myostatin in plasma. Furthermore, even for antibodies that enhance the reduction rate of myostatin through different mechanisms, such as antibodies with Fc with enhanced affinity for FcγRIIB and antibodies with positively charged Fc, the ranking of cellular uptake reflected the results of the reduction rate in vivo.

[0257] These results demonstrate that the antigen reduction rate in vivo in monkeys by various antibodies that increase the myostatin reduction rate from monkey plasma through different mechanisms, such as antibodies with Fc that have enhanced affinity for FcγRIIB or antibodies with Fc that have modified isoelectric points (pI), can be predicted by the cellular uptake evaluation system using monkey liver non-parenchymal cells according to the present invention.

[0258] [Example 6] Preparation of human liver non-parenchymal cells Human liver non-parenchymal cells were purchased from Sekisui Xenotech. The preparation procedure is as follows: Human liver was perfused with collagenase solution for tissue digestion. After filtration, the cell suspension was diluted with DMEM. The diluted cell suspension was centrifuged at 100 x g for 5 min to precipitate parenchymal cells, and the supernatant was collected. The supernatant was further centrifuged at 350 x g for 10 min to precipitate non-parenchymal cells. The supernatant was then removed and resuspended in OptiThaw Kupffer Cell Thaw / Culture Media (Sekisui XenoTech).

[0259] [Example 7] Alexa647 or Alexa488 labeling of anti-human FcγRIIB antibody and human myostatin Using the Alexa flour 647 labeling kit (Thermo Fisher Scientific) or the Alexa flour 488 labeling kit (Thermo Fisher Scientific), each protein was labeled with Alexa 647 or Alexa 488 according to the attached protocol. The concentration of each protein and the labeling efficiency of the fluorescent substance were calculated by measuring the absorbance using a Nanodrop (Thermo Fisher Scientific) and calculating according to the formula described in the attached protocol.

[0260] [Example 8] Identification of cells with high FcγRIIB expression in human non-parenchymal liver cells 5x10 5 FITC-labeled anti-CD31 antibody (Miltenyi), VioBlue-labeled anti-CD45 antibody (Miltenyi), anti-human FcγRIIB human IgG antibody (Clone 2B6), and Alexa647-labeled anti-human antibody (Southern Biotech) were added to human liver non-parenchymal cells to make 100 μL, and the cells were incubated on ice for 60 minutes. The cells were then centrifuged at 600 xg for 3 minutes at 4°C, the supernatant was removed, and the cells were washed with PBS(-). The cells were resuspended in 2% FBS-containing PBS(-), and the fluorescence of the cell solution was measured using a FACS Canto II (Becton, Dickinson and Company), and a dot plot was developed using forward scatter (FSC) and side scatter (SSC) (Figure 7). The cells in the area indicated by the square were further developed using FITC (CD31; FITC filter detection) and VioBlue (CD45; Pacific Blue filter detection) (Figure 8).

[0261] Liver non-parenchymal cells are a mixture of various cell populations, including vascular endothelial cells, Kupffer cells, and stellate cells, but they could be separated into two populations by using two cell markers, CD31 and CD45, which are known to be cell markers for LSECs in humans. The two cell populations that emerged were designated P1 (CD31LowCD45High) and P2 (CD31HighCD45Low), respectively. Note that dots seen in other areas were likely to be cells that do not express these markers or nonspecific fluorescence due to autofluorescence or debris, and were therefore excluded from the analysis.

[0262] The expression of human FcγRIIB in these cell populations was evaluated. As shown in Figure 9, no peak shift was observed in P1 by the addition of anti-human FcγRIIB antibody, as in the case of the negative control antibody (Figure 9A), but a clear peak shift to the high intensity side was observed in P2 by the anti-human FcγRIIB antibody, revealing that FcγRIIB was expressed only in P2 (Figure 9B). Since the ratio of FcγRIIB-expressing cells to the total cells is very small, it is difficult to identify FcγRIIB-expressing cells without gating by cell markers. However, by staining with cell markers CD31 and CD45 as in this method and separating each cell population, it became possible to identify FcγRIIB-expressing cells.

[0263] Furthermore, this method does not use anti-FcγRIIB antibodies for gating cell populations, so the expression level of FcγRIIB in the target cell population can be quantified. TM The expression of human FcγRIIB was quantified using MESF (Bangs Laboratories) according to the attached protocol. The expression level was 2.0x10 per cell. 5 Expression of the molecule was confirmed.

[0264] [Example 9] Evaluation of cellular uptake of antibody-antigen complexes in cell population P2 of human nonparenchymal liver cells (9-1) Characteristics of the Fc of the antibody used to evaluate the uptake of antibody-antigen complexes The anti-myostatin antibody TT91 described in WO2016 / 098357 A1 was used.

[0265] When the affinity of SG1 for human FcγRIIB is taken as 1.0, TT91 shows a 14.7-fold increase in binding activity (WO2016 / 098357 A1).

[0266] (9-2) Evaluation of cellular uptake of immune complexes 5x10 5 Alexa488-labeled human myostatin was added to a final concentration of 0.3 μg / mL and each antibody was added to a final concentration of 0.1, 1, 10, or 100 μg / mL to prepare a 100 μL reaction solution. This solution was reacted at 37°C for 60 minutes while stirring. After the reaction, the cells were cooled on ice, and cold 2% FBS-containing PBS was added to wash the cells. The solution was then removed by centrifugation (600 xg, 3 min). The cells were then stained with anti-CD31 and anti-CD45 antibodies, washed, and the fluorescence intensity of Alexa488 at P2 was measured using a FACS Canto II.

[0267] As a result, the fluorescence peak of Alexa488-labeled myostatin shifted to the higher intensity side in the order of no antibody, 0.1, 1, and 10 μg / mL TT91 antibody (Figure 10), suggesting that the antibody-antigen complex binds to FcγRIIB on the cell surface and is taken up into the cells in an antibody concentration-dependent manner. A peak shift to the higher intensity side was also observed at 100 μg / mL, but was comparable to the condition of 10 μg / mL antibody.

[0268] [Example 10] Quantification of antigen uptake Quantum TM The fluorescence intensity of the fluorescently labeled standard beads was measured using MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard according to the attached protocol, and the amount of human myostatin taken up for each antibody and each concentration was calculated from the geometric mean fluorescence intensity of the human myostatin-taken sample.

[0269] The results are shown in Figure 11. Compared to the group without antibody addition, the amount of uptake in the antibody-added group increased in an antibody concentration-dependent manner, reaching a plateau at 10 μg / mL. This is thought to be because as the antibody concentration increased, the proportion of TT91 antibodies that bound to Alexa488-labeled human myostatin increased, and they bound to the cells and were taken up. On the other hand, the amount of uptake decreased at 100 μg / mL compared to 10 μg / mL, which is thought to be due to an increase in antibodies that do not bind to human myostatin in the high concentration range, which then bind to FcγRIIB and are taken up, resulting in an apparent decrease in the amount of uptake of human myostatin-bound TT91.

[0270] [Example 11] Nucleic acid uptake in monkey liver non-parenchymal cells The monkey used was a cynomolgus monkey (Macaca fascicularis). The test substance was a FITC-labeled oligodeoxynucleotide nucleic acid described in Non-Patent Document 25 (J Hepatol., 2006. 44(5):939-46) purchased from Hokkaido System Science. The sequence was 5'-FITC-T*C*C*-A*TG-ACG-TTC-CTGA*T*G*-C*T-3'. The asterisk (*) indicates that it is a phosphorothioate nucleotide.

[0271] (11-1) Evaluation of Stabilin1 and Stabilin2 expression in monkey liver nonparenchymal cells 2x10 5Pacific blue-labeled anti-CD31 antibody (BioLegend), APC-labeled anti-CD45 antibody (Miltenyi), and Alexa488-labeled anti-Stabilin1 antibody or anti-Stabilin2 antibody (Thermo Fisher Scientific) were added to each of the monkey liver non-parenchymal cells to make a volume of 100 μL, and the cells were incubated on ice for 30 minutes. The cells were then centrifuged at 3000 rpm for 5 minutes at 4°C, the supernatant was removed, and the cells were washed with PBS(-). The cells were resuspended in 2% FBS-containing PBS(-), and the fluorescence of the cell solution was measured using a FACS Canto II (Becton, Dickinson and Company), and dot plots were developed using forward scatter (FSC) and side scatter (SSC). All cells expanded with FSC and SSC were further expanded with Pacific blue (CD31) and APC (CD45).

[0272] The three cell populations that emerged were classified as P1 (CD31 Low CD45 High ), P2(CD31 Intermediate CD45 Intermediate ), P3(CD31 High CD45 Low ) plots were used. The plots in other areas were excluded from the analysis because they were considered to be cells not expressing these markers, or nonspecific fluorescence due to autofluorescence or debris. Expression of Stabilin1 and Stabilin2 was evaluated by detecting the fluorescence intensity of Alexa488 in these three cell populations (FITC filter detection). As shown in Figure 12, although there were differences in the degree, a shift to higher fluorescence intensity peaks was observed in all cell populations P1 to P3 when anti-Stabilin1 or anti-Stabilin2 antibodies were added compared to the negative control antibody labeled with Alexa488 (Figures 12A to C).

[0273] (11-2) Evaluation of cellular uptake of nucleic acids in monkey liver non-parenchymal cells 2x10 5FITC-labeled nucleic acid was added to 50 μL of cell solution containing 10 cells to a final concentration of 0 to 40 μg / mL to prepare a reaction solution of 100 μL. This solution was reacted at 37°C for 30 minutes while stirring. The solution was then cooled on ice, and cold 2% FBS-containing PBS was added to wash the cells. The solution was then removed by centrifugation (3000 rpm, 5 min). The cells were then stained with anti-CD31 antibody and anti-CD45 antibody, washed, and the fluorescence intensity of FITC at P1, P2, and P3 was measured using a FACS Canto II.

[0274] As a result, in all cell populations P1 to P3, a fluorescence peak was observed on the higher intensity side when nucleic acid was added compared to the condition without nucleic acid addition, regardless of the nucleic acid concentration. In particular, a peak shift dependent on the added concentration was observed in P2 and P3.

[0275] (11-3) Correlation between nucleic acid concentration and nucleic acid uptake The concentration-dependent cellular uptake of nucleic acids was evaluated by plotting the concentration of nucleic acids added versus the average peak fluorescence intensity shown in Figure 13. As a result, it was found that in all cell populations P1 to P3, an increase in FITC fluorescence intensity was observed depending on the concentration of added nucleic acids, and that it reached a saturated state at high concentrations. This saturability is thought to suggest that nucleic acids are taken up by a specific receptor.

[0276] [Example 12] Evaluation of cellular uptake of antibodies in cell population P2 of monkey liver nonparenchymal cells (12-1) Characteristics of the Fc of the antibody used for uptake evaluation The anti-myostatin antibodies SG1, SG141, SG143 (also referred to as FS154), and SG1081 having Fc described in WO 2016 / 117346 A1 (Patent Document 4) and WO 2016 / 098357 A1 (Patent Document 5) were used.

[0277] When the affinity of SG1 for monkey FcγRIIB is taken as 1.0, the affinity of SG143 is 5-10 times higher (WO 2016 / 117346 A1). SG141 is an antibody with an Fc that has a positive charge under neutral pH conditions due to a high pI, and shows a binding ability 1.64 times higher than SG1 in a binding test of IC for monkey FcγRIIB using Biacore (WO 2016 / 098357 A1). SG1081 has a 5-10 times higher affinity for monkey FcγRIIB than SG1 (WO 2016 / 117346 A1).

[0278] (12-2) Evaluation of cellular uptake of antibodies 2x10 prepared in the same manner as in Example 1 5 Alexa488-labeled antibody was added to 50 μL of cell solution containing monkey liver non-parenchymal cells to a final concentration of 10 to 200 μg / mL to prepare 100 μL of reaction solution. This solution was reacted at 37°C for 15 minutes while stirring. After that, the cells were cooled on ice, and 10 mM citric acid-containing Hepatocyte culture medium (pH 4.5, LONZA) was added to wash the cells. The solution was then removed by centrifugation (3000 rpm, 3 min). Furthermore, the cells were stained with anti-CD31 antibody and anti-CD45 antibody, and after washing, the fluorescence intensity of Alexa488 at P1, P2, and P3 was measured using FACS Canto II.

[0279] As a result, in the P2 cell population, fluorescence peaks of SG1, SG141, SG143, and SG1081 were confirmed at all antibody concentrations, and the higher the antibody concentration, the higher the fluorescence peak shifted to the higher intensity side (Figure 15). In addition, SG141, SG143, and SG1081 showed stronger fluorescence than SG1. This suggests that the antibodies bind to FcγRIIB on the cell surface and are taken up into the cells according to their respective Fc characteristics.

[0280] (12-3) Quantification of antibody uptake Quantum TMThe fluorescence intensity of the fluorescently labeled standard beads was measured using MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard according to the attached protocol, and the amount of antibody uptake for each antibody and each concentration was calculated from the geometric mean fluorescence intensity of the antibody-uptake sample.

[0281] The results are shown in Figure 16. At 10 μg / mL, the uptake of SG1, SG1081, and SG141 was comparable, and SG143 showed a higher uptake. At 30 μg / mL, the uptake of SG1 and SG1081 was comparable, followed by SG141 and SG143 in that order. At 100 μg / mL, the uptake of SG1 was the lowest, followed by SG1081, SG141, and SG143 in that order. At 200 μg / mL, the uptake of SG1 was the lowest, followed by SG1081, SG143, and SG141 in that order.

[0282] Furthermore, the correlation between the plasma clearance of each antibody in vivo in monkeys and the amount of uptake of each antibody into cells in this example was examined. The clearance of the antibody in monkey plasma was measured by the following method. Each antibody was administered to 2-4 year old Macaca fascicularis (cynomolgus monkeys) from Cambodia (Shin Nippon Biomedical Laboratories Ltd., Japan) at 30 mg / kg for SG1, SG141, and SG143, and at 2 mg / kg for SG1081. Blood was collected continuously up to 56 days after administration, and plasma was obtained by centrifugation. The antibody concentration in the plasma was measured by electrochemiluminescence (ECL). Plasma clearance was calculated by non-compartment model analysis. In addition, the amount of uptake into cells in monkey liver non-parenchymal cells was plotted based on the amount of uptake into cells calculated from the fluorescence intensity of Alexa488-labeled antibody when the antibody was added at 200 μg / mL.

[0283] As shown in Figure 17, a correlation was observed between in vivo plasma clearance of the antibody and in vitro cellular uptake of the antibody (R 2 =0.602).

[0284] [Example 13] Confirmation of IL-6R expression in FcγRIIB-high expressing monkey liver non-parenchymal cells First, 2x10 5 A 50μL cell solution containing monkey liver non-parenchymal cells was prepared. An antibody solution containing Alexa488-labeled tocilizumab (humanized anti-IL-6R antibody) or Alexa488-labeled hIgG as a negative control at a final concentration of 3μg / mL was also prepared. To confirm specific binding, an antibody solution containing Alexa488-labeled tocilizumab and unlabeled tocilizumab at a final concentration of 1mg / mL was also prepared as an excess amount relative to the labeled form. To each of these cell solutions and antibody solutions, Fc Receptor Blocking Solution (Human TruStain FcX, Biolegend) was added at a dilution ratio of 20 times to block FcγR. The cell solution and antibody solution prepared in this way were mixed to make a reaction solution of 100μL. The reaction solution was reacted at 4℃ for 120 minutes. After that, the cells were washed by adding cold 2% FBS-containing PBS, centrifuging (3000 rpm, 3 min), and removing the supernatant. Next, the cells were stained with anti-CD31 and anti-CD45 antibodies, and after washing, the fluorescence intensity of Alexa488 was measured using a FACS Canto II.

[0285] As in Example 3, the cell population was developed on the Y-axis and the X-axis for the expression levels of CD45 and CD31, respectively, and the cell population was divided and evaluated based on the expression level pattern. As in Example 3, the expression of FcγRIIB was confirmed for the cell populations P1 to P3, and high expression of FcγRIIB was confirmed in P2, and then the expression of IL-6R was evaluated. As a result, as shown in FIG. 18, a clear peak shift to the high intensity side was observed in P2 by Alexa488-labeled tocilizumab compared to the negative control. In addition, the peak shift was inhibited by an excess amount of unlabeled tocilizumab. Therefore, it was revealed that IL-6R was expressed in P2.

[0286] [Example 14] Evaluation of tocilizumab uptake in cell population P2 of monkey liver nonparenchymal cells 2x10 prepared in the same manner as in Example 1 5A cell solution containing 50 μL of monkey liver non-parenchymal cells and an antibody solution containing Alexa488-labeled tocilizumab at a final concentration of 3 μg / mL were prepared. In addition, to confirm specific binding, an antibody solution was prepared in which unlabeled tocilizumab was added in excess of the labeled form to a final concentration of 1 mg / mL in addition to Alexa488-labeled tocilizumab. To each of these cell solutions and antibody solutions, Fc Receptor Blocking Solution (Human TruStain FcX, Biolegend) was added at a dilution ratio of 20 times to block FcγR. The cell solution and antibody solution prepared in this way were mixed to make a reaction solution of 100 μL. This solution was reacted at 37 °C for 2, 5, 10, 15, or 30 minutes with stirring. The cells were then cooled on ice, and 10 mM citric acid-containing Hepatocyte culture medium (pH 3.0, LONZA) was added, followed by centrifugation (3000 rpm, 3 min) and removal of the supernatant. This procedure was repeated to remove the antibodies bound to the cell surface. The cells were then washed with cold 2% FBS-containing PBS. The solution was then removed by centrifugation (3000 rpm, 5 min), and the cells were stained with anti-CD31 and anti-CD45 antibodies. After washing, the fluorescence intensity of Alexa488 at P2 was measured using a FACS Canto II.

[0287] As a result, in P2, the fluorescence peak of Alexa488-labeled tocilizumab shifted to a higher intensity side with increasing reaction time (Figure 19A). Moreover, the peak shift was inhibited by an excess of unlabeled tocilizumab (Figure 19B). This suggests that tocilizumab specifically binds to IL-6R on the cell surface and is taken up into the cells in a time-dependent manner.

[0288] Furthermore, the amount of tocilizumab taken up into the cells was quantified. TMThe fluorescence intensity of the fluorescently labeled standard beads was measured using MESF (Bangs Laboratories) according to the attached protocol. A calibration curve was drawn from the geometric mean fluorescence intensity of each standard according to the attached protocol, and the amount of Alexa488-labeled tocilizumab taken up at each reaction time was calculated from the geometric mean fluorescence intensity of the Alexa488-labeled tocilizumab-taken sample. The results are shown in Figure 19C. The amount of take-up increased almost linearly with time, and the take-up rate was 2.19x10 -6 pmol / min / 2x10 5 cells was calculated. [Industrial Applicability]

[0289] The present invention makes it possible to predict, by in vitro testing, the in vivo dynamics of a drug candidate molecule in humans and monkeys.The present invention makes it possible to predict, by in vitro testing, the antigen removal activity of a drug candidate antibody from blood in humans and monkeys. [Sequence List Free Text]

[0290] SEQ ID NO: 1: Synthetic oligonucleotide

Claims

1. 1. A method for measuring the amount of uptake of a molecule into a cell, comprising: (i) adding the molecule to a population of cells derived from an organ and incubating the mixture; (ii) after step (i), selecting a cell population derived from an organ expressing CD31 and CD45 based on the expression levels of CD31 and CD45 from the cell population derived from the organ incubated with the molecule; and (iii) measuring the uptake of the molecule into the cell population after step (ii); Including, The molecule is taken up into the cell via a receptor present on the cell surface; and (1) The organ-derived cell population is a human liver non-parenchymal cell population, and in step (ii), 【Chemistry 1】 a cell population indicated by P2 of the method of the present invention, wherein the cell population indicated by P2 is a cell population with a higher expression level of CD31 when compared at the highest cell density point of two CD31 + CD45 + cell populations that are distinguished from each other based on cell density in a development diagram in which the expression levels of CD31 and CD45 are detected by flow cytometry and plotted on the X-axis and Y-axis, respectively; or (2) The organ-derived cell population is a monkey liver non-parenchymal cell population, and in step (ii), 【Chemistry 2】 and selecting a cell population indicated by P2, wherein the cell population indicated by P2 is a cell population having the second highest CD31 expression level when compared at the highest cell density point among three CD31 + CD45 + cell populations that are distinguished from each other based on cell density in a development diagram in which the expression levels of CD31 and CD45 are detected by flow cytometry and plotted on the X-axis and Y-axis, respectively. The method.

2. The method of claim 1 , wherein the molecule is an immune complex or an antibody and the receptor is an Fc receptor.

3. The method of claim 1 , wherein the molecule is an anti-IL-6R antibody and the receptor is IL-6R.

4. The method of claim 1 , wherein the molecule is a nucleic acid and the receptor is Stabilin.

5. A composition for use in an assay for uptake of a molecule, comprising an isolated, organ-derived cell population expressing CD31 and CD45, the molecule being taken up into the cell via a receptor present on the cell surface, The assay is carried out according to the method of any one of claims 1 to 4, The composition.

6. The composition of claim 5 , wherein the cell population is a crudely purified cell population.

7. A method for producing the composition of claim 5 or 6, comprising the steps of: (1) preparing organ-derived cells from an organ removed from a living body; and (2) selecting a population of organ-derived cells based on the expression levels of CD31 and CD45; The method comprising:

8. 1. A method for predicting the in vivo clearance of a molecule, comprising: (1) measuring the amount of the molecule taken up into cells by using the method according to any one of claims 1 to 4; and (2) predicting the in vivo clearance of the molecule when the molecule is administered to a living body from the amount of uptake measured in (1) above; Including, The method, wherein the molecule is taken up into the cell via a receptor present on the cell surface.

9. 1. A method for screening a molecule, comprising: (1) providing two or more different molecules that bind to the same receptor; (2) measuring the amount of each of the molecules prepared in (1) above taken up into cells by using the method according to any one of claims 1 to 4; and (3) comparing the amounts of the molecules taken up into cells measured in (2) above with each other and selecting the molecule with the highest amount of uptake; Including, The method, wherein the molecule is taken up into the cell via a receptor present on the cell surface.

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