Methods for detecting complexes

Abstract

To provide a method for performing detection of a complex with low affinity and measurement of the concentration and / or the amount of the complex under a condition in which the binding equilibrium of a complex is substantially maintained.SOLUTION: A method includes the steps of: (1) bringing a first conjugate and a sample including a complex into contact with each other to bind the first conjugate and the complex to each other; (2) binding the complex bound to the first conjugate to a second conjugate; and (3) detecting the second conjugate bound to the complex. The complex includes two or more components. At least one of the components has a KD value of 1 nM or more. The method is performed under a condition in which the binding equilibrium of the complex is substantially maintained.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting a complex, and further to a method for measuring the amount and / or concentration of a complex using the method. [Background technology]

[0002] In recent development of drugs using antibodies or recombinant proteins, quantitative analysis of the complex between the drug and the antigen targeted by the drug under development is important for predicting the efficacy and toxicity of the drug. Therefore, methods such as enzyme-linked immunosorbent assay (ELISA), size exclusion chromatography (SEC), and analytical ultracentrifugation (AUC) have been used to evaluate the size and quantity of antigen-antibody complexes (Non-Patent Documents 1, 2, 3). These methods have been performed to evaluate the complex formed between an antibody whose mechanism of action is to neutralize the target antigen and its target antigen, and therefore generally do not evaluate antigen-antibody reactions with high affinity (dissociation constants (K) lower than single-digit nM). D Therefore, the complex concentration to be evaluated is relatively high, and qualitative evaluation is relatively easy, but quantitative evaluation is difficult due to the influence of shifts in binding equilibrium.

[0003] Recently, because they have mechanisms of action other than neutralization, they have been recognized as having high affinity (K<1 nM). D Antibodies that do not require a K value have also been developed, and such antibodies are expected to be effective even if the binding rate to the antigen is low. The complexes that such antibodies form with physiological concentrations of antigen at antibody concentrations where efficacy is expected are extremely difficult to evaluate because the concentrations are low and the influence of shifts in binding equilibrium is large. Emicizumab, a bispecific antibody, is an example of this, and has Factor VIII replacement activity, binding to two antigens, FIXa and FX, and converting FX to FXa by bringing them into close proximity. The K value of the amount of the ternary complex formed by FIXa, FX, and emicizumab in the liquid phase is DSince there is a correlation between simulations using these values ​​and data from a thrombin generation test, which is an in vitro drug efficacy test, the amount of the ternary complex is thought to reflect the drug's potency (Non-Patent Documents 4 and 5). Meanwhile, for bispecific antibodies whose mechanism of action is neutralization, a method has been reported in which the antibody or the free antigen recognized by the antibody is measured using an anti-idiotype antibody. However, it has been reported that, compared to measurements performed by forming a complex using two types of anti-idiotype antibodies, measurements performed by forming a ternary complex using two types of antigens are subject to interference from other substances, the degree of dilution of the sample is high, and the measurement time is long, resulting in large discrepancies and making the measurement difficult (Patent Documents 1, 2, and 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2013 / 092611 [Patent Document 2] WO2013 / 113663 [Patent Document 3] WO2014 / 009474 [Non-patent literature]

[0005] [Non-Patent Document 1] MAbs, 9(4), 664-679, 2017 [Non-patent document 2] Biochemistry, 51(3), 795-806, 2012. [Non-patent document 3] Biosci Rep, 33(4), 2013. [Non-patent document 4] Nat Med, 18(10), 1570-4, 2012. [Non-Patent Document 5] Thromb Haemost, 117(7), 1348-1357, 2017. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, quantitative evaluation of the amount or concentration of the ternary complex is thought to be able to predict the efficacy of emicizumab. D The neutralizing activity of this antibody is approximately 1 μM, more than 1,000 times weaker than that of a typical antibody. As a result, the complex formed is low in concentration and easily dissociates. Therefore, existing methods such as SEC and AUC have difficulty even detecting its presence due to their lack of sensitivity and the large influence of shifts in binding equilibrium.

[0007] Furthermore, a highly sensitive protein detection method is the ligand binding assay, which utilizes an antigen-antibody reaction. However, even if an antibody that specifically recognizes a complex can be selected, it is extremely difficult to reflect the state of the complex in solution due to the shift in the binding equilibrium of the complex caused by the generally required long incubation time and the dissociation of the complex caused by the washing procedure. Furthermore, a method for measuring the affinity between proteins with high sensitivity by measuring free antigen or free antibody under conditions that make it difficult for the shift in binding equilibrium to occur, such as KinExA (registered trademark), is known. However, it was not known that such a method could be used to detect complexes with very weak affinity, or even to measure their concentration and / or amount.

[0008] Therefore, the present invention aims to realize quantitative evaluation of antigen-antibody complex formation for low-affinity antibodies in solution by utilizing a method that can minimize the effects of shifts in binding equilibrium and dissociation of the complex while taking advantage of the high sensitivity of the ligand binding assay. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have succeeded in discovering a method for detecting low affinity complexes and measuring the concentration and / or amount of the complexes under conditions in which the binding equilibrium of the complexes is substantially maintained. Furthermore, the present inventors have succeeded in discovering a method for evaluating the dynamics of the complex and a method for determining a therapeutic method using a drug based on the concentration and / or amount of the complex determined by the above-mentioned measurement method. Specifically, the present invention provides the following [1] to

[33] . [1] A method for detecting a complex in a sample, comprising: (1) contacting a first binder with a sample containing a complex to bind the first binder to the complex; (2) binding the complex bound to the first binder to a second binder; and (3) detecting a second binder bound to the complex; The complex comprises two or more components, and at least one K D The method is carried out under conditions where the binding equilibrium of the complex is substantially maintained, and the value is 1 nM or more. [2] The method according to [1], wherein the components of the complex are not labeled or immobilized on a solid phase. [3] The method according to [1] or [2], wherein the component is at least one selected from the group consisting of peptides, polypeptides, and proteins. [4] The method according to any one of [1] to [3], wherein at least two of the components are an antibody and an antigen recognized by the antibody. [5] The method according to any one of [1] to [4], wherein at least three of the components are a bispecific antibody and two antigens recognized by the antibody. [6] The method according to any one of [1] to [5], wherein the second binder is labeled. [7] The method according to any one of [1] to [6], wherein the sample is a blood sample, more preferably whole blood, serum, or plasma. [8] The method according to any one of [1] to [7], wherein the first binder is bound to a solid phase. [9] The method according to [8], wherein the solid phase is a chip, a microfluidic chip, a disk, or beads.

[10] The method according to any one of [1] to [9], wherein the measurement is performed by KinExA or Gyrolab.

[11] The method according to any one of [5] to

[10] , wherein the bispecific antibody is (a) below, and the two antigens are (b) and (c) below: (a) a bispecific antibody that recognizes blood coagulation factor IX and / or activated blood coagulation factor IX, and blood coagulation factor X; (b) Blood coagulation factor IX or activated blood coagulation factor IX (c) Blood coagulation factor X

[12] A method for determining the concentration and / or amount of a complex in a sample, comprising the steps of: (4) preparing a composite for generating a regression equation; (5) detecting the complex of step (4) by a process including steps (1) to (3); (6) simulating the concentration of the complex in step (4); (7) calculating a regression equation from the signal value detected in step (5) and the simulated concentration; and (8) applying the signal value detected in step (3) to the regression equation.

[13] The method according to

[12] , wherein at least one component is a drug.

[14] A method for determining a therapeutic method using a drug based on the concentration and / or amount of the complex determined by the method according to

[13] .

[15] A method for evaluating the dynamics of a complex based on the concentration and / or amount of the complex determined by the method according to any one of

[12] to

[14] .

[16] A method for determining the concentration and / or amount of a complex in a sample, wherein the complex contains two or more components, and at least one K D(5) detecting the complex of step (4) by a process including steps (1) to (3), (6) simulating the concentration of the complex of step (4), (7) calculating a regression equation from the signal value detected in step (5) and the simulated concentration, and (8) applying the signal value detected in step (3) to the regression equation.

[17] A method for detecting a complex in a sample, wherein the complex is a ternary complex containing, as components, a bispecific antibody and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen, and allowing the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder that recognizes an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder that has bound to the complex.

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

[17] , wherein the first binder is bound to a solid phase.

[19] The method according to

[18] , wherein the method is carried out under conditions that do not substantially generate new complexes on the solid phase and / or do not substantially cause dissociation of the complexes.

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

[19] , wherein the method is carried out under conditions where the time is short enough that theoretically the binding equilibrium does not shift.

[21] The method according to any one of

[18] to

[20] , wherein the method is carried out under conditions where the contact time between the sample and the solid phase is 10 seconds or less.

[22] The method according to any one of

[18] to

[21] , wherein the steps from the step of binding the complex to the first binder bound to the solid phase to the step immediately before the step of detecting the second binder are performed within 10 minutes.

[23] The method according to [2] or [6], wherein the label is a luminescent label, a chemiluminescent label, an electrochemiluminescent label, a fluorescent label, or a radioactive label.

[24] The method according to

[14] , wherein the determination of the treatment method is the determination of the dosage or the administration frequency of the drug.

[25] A method for determining a drug-based treatment method based on a signal value detected by the method according to

[13] .

[26] A method for determining a drug-based treatment method based on a standardized signal value calculated from a signal value detected by the method described in

[13] .

[27] A method for determining a therapeutic method using a drug based on an arbitrary unit calculated from a signal value detected by the method according to

[13] .

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

[26] , further comprising a washing step.

[29] The method according to [7], wherein the blood sample, more preferably whole blood, serum, or plasma, is prepared by adding 1 / 100 to 1 / 2 equivalent of 1 M HEPES solution.

[30] A method for preparing or producing a sample, comprising adding 1 M HEPES solution in an amount of 1 / 100 to 1 / 2 equivalent of the sample.

[31] The method according to

[30] , wherein the sample is a blood sample, more preferably whole blood, serum or plasma.

[32] A method for evaluating the effect of a drug based on the concentration and / or amount of the complex determined by the method according to

[13] .

[33] A method for evaluating the safety of a drug based on the concentration and / or amount of the complex determined by the method according to

[13] . [Effects of the Invention]

[0010] The method of the present invention is to detect weak affinity complexes, such as ternary complexes formed by bispecific antibodies, using K D For antibodies with a wide range of affinities, including antibodies with low affinity in the order of nanomolar to micromolar, the ternary complex itself can be detected in biological samples without being labeled, and the concentration and / or amount of the ternary complex can be quantitatively determined. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the complexes formed by FIX, FX and anti-FIX / FX bispecific antibodies. [Figure 2] (A) A diagram showing a method for detecting FIX captured by an anti-FIX / FX bispecific antibody using an anti-FIX antibody. (B) A diagram showing a method for detecting FX captured by an anti-FIX / FX bispecific antibody using an anti-FX antibody. [Figure 3] (A) A diagram showing the concentration-dependent detection results of FIX, (B) A diagram showing the concentration-dependent detection results of FX. [Figure 4] (A) A diagram showing a ternary complex assay format in which an anti-FIX antibody (XB12) is immobilized on beads and a fluorescently labeled anti-FX antibody (SB04) is used for detection. (B) A diagram showing a ternary complex assay format in which an anti-FX antibody (SB04) is immobilized on beads and a fluorescently labeled anti-FIX antibody (XB12) is used for detection. [Figure 5] (A) The figure shows the results of measuring samples to which FIX, FX, and bispecific antibodies were added by KinExA in the format shown in Figure 4(A). (B) The figure shows the results of detecting samples to which FIX, FX, and bispecific antibodies were added by KinExA in the format shown in Figure 4(B). [Figure 6] (A) This figure shows the relationship between antibody concentration and calculated ternary complex concentration when the dissociation constants of an anti-FIX / FX bispecific antibody for FIX and FX are the same and each dissociation constant is changed by a factor of 2. (B) This figure shows the relationship between antibody concentration and calculated ternary complex concentration when the dissociation constants of an anti-FIX / FX bispecific antibody for FIX and FX are changed by a factor of 2 and the FIX side is two-fold larger than the FX side. [Figure 7] (A) A graph showing the relationship between the concentration of the anti-FIX / FX bispecific antibody Q4 / / J3, the signal value of the ternary complex detected by KinExA, and the calculated concentration of the ternary complex. (B) A graph showing the correlation between the signal value of the ternary complex detected by the Q4 / / J3 antibody and the calculated concentration of the ternary complex. [Figure 8-1] (A) A graph showing the relationship between the concentrations of the anti-FIX / FX bispecific antibodies Q4 / / J3 antibody, Q3 / / J1 antibody, and emicizumab and the signal values ​​of the ternary complexes formed by each antibody as detected by KinExA. (B) A graph showing the regression between the signal value of the ternary complex induced by the Q4 / / J3 antibody and the calculated concentration of the ternary complex. [Figure 8-2] (C) The signal values ​​of the ternary complex in FIG. 8(A) are converted into concentrations using the regression equation in FIG. 8(B). [Figure 9-1] (A) A graph comparing the time course of signal intensity during measurement when the ternary complex formed by the anti-FIX / FX bispecific antibody Q4 / / J3 antibody and the Q3 / / J1 antibody was detected by KinExA. (B) A graph comparing the time course of signal intensity during measurement when the ternary complex formed by the anti-FIX / FX bispecific antibody Q3 / / J1 antibody and emicizumab was detected by KinExA. [Figure 9-2] (C) A graph comparing the transitions of signal values ​​during measurement when the ternary complex formed by the anti-FIX / FX bispecific antibody Q4 / / J3 antibody and emicizumab was detected by KinExA. [Figure 10] FIG. 1 shows the results of KinExA detection of the ternary complex formed over time after mixing the anti-FIX / FX bispecific antibody Q3 / / J1 antibody and emicizumab with a sample containing hFIX and hFX. [Figure 11] (A) Relationship between the concentration of the anti-FIX / FX bispecific antibody Q4 / / J3 and the response of the ternary complex formed by each antibody as detected by Gyrolab. (B) Regression of the signal value of the ternary complex induced by the Q4 / / J3 antibody with the calculated concentration of the ternary complex. [Figure 12-1] (A) A graph showing the relationship between the concentrations of the anti-FIX / FX bispecific antibodies Q4 / / J3 antibody, Q3 / / J1 antibody, and emicizumab and the signal values ​​of the ternary complexes they form in congenitally FVIII-deficient human plasma, as detected by KinExA. (B) A graph showing the regression between the signal value of the ternary complex induced by the Q4 / / J3 antibody and the calculated concentration of the ternary complex. [Figure 12-2] (C) The signal values ​​of the ternary complex in FIG. 12(A) are converted into concentrations using the regression equation in FIG. 12(B). [Figure 13] FIG. 1 shows a method for detecting the binary complex formed by tocilizumab and soluble hIL-6R using an anti-IL-6R antibody that recognizes an epitope different from that recognized by tocilizumab, and an anti-hIgG Fc antibody. [Figure 14] FIG. 14 shows the results of KinExA detection of a sample to which tocilizumab and hIL-6R had been added using the method shown in FIG. 13. [Figure 15] (A) A graph showing the relationship between tocilizumab concentration, the signal value of the binary complex formed by tocilizumab and hIL-6R detected by KinExA, and the calculated concentration of the binary complex. (B) A graph showing the correlation between the signal value of the binary complex formed by tocilizumab and hIL-6R and the calculated concentration of the binary complex. DETAILED DESCRIPTION OF THE INVENTION

[0012] In one aspect, the present invention is a method for detecting a complex in a sample. In another aspect, the present invention is a method for determining the concentration and / or amount of a complex in a sample.

[0013] In one aspect, the present invention relates to a method for measuring the presence and / or amount of a complex in a sample using a ligand binding assay, which is an immunoassay. Examples of ligand binding assay methods that can be used include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance (SPR), electrochemiluminescence (ECL), and KinExA (registered trademark) Kinetic Exclusion Assay (Drake et al., 2004, Analytical Biochemistry 328:35-43).

[0014] In one embodiment, a method can be used that has a smaller spatial size of the reaction field, a larger specific surface area of ​​the solid phase surface, and a shorter assay time than methods using microtiter plates, such as, but not limited to, a method using a microfluidic chip, a disk, or beads, more specifically, a method using, for example, the KinExA (registered trademark) or Gyrolab (registered trademark) immunoassay system (Fraley et al., 2013, Bioanalysis 5: 1765-74). In another aspect, the present invention relates to a method for measuring the presence and / or amount of a complex in a sample using a Single Molecule Array method, an Alpha (Amplified Luminescence Proximity Homogeneous Assay) method, or a time-resolved fluorescence method.

[0015] In one aspect, the present invention provides a method for detecting a complex in a sample, comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components, and at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value of the binding equilibrium is 1 nM or more.

[0016] In one aspect, the present invention provides a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components and at least one K D the value is 1 nM or more, the method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and none of the components of the complex is labeled or immobilized on a solid phase.

[0017] In another aspect, the present invention provides a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components and at least one K D The value is 1 nM or more.

[0018] In another aspect, the present invention relates to a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex comprises two or more components, and none of the components of the complex is labeled or immobilized.

[0019] In another aspect, the present invention relates to a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex comprises two or more components, and the steps are carried out under conditions such that the binding equilibrium of the complex is substantially maintained.

[0020] In another aspect, the present invention provides a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a first binder with a sample containing a complex to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components, and at least one K between the components is detected. D wherein the value is 1 nM or greater and none of the components of the complex are labeled or immobilized.

[0021] In another aspect, the present invention relates to a method for detecting a complex in a sample, the method comprising the steps of: (1) contacting a sample containing a complex with a first binder to allow the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder; and (3) detecting the second binder bound to the complex, wherein the complex comprises two or more components, the method is carried out under conditions such that the binding equilibrium of the complex is substantially maintained, and none of the components of the complex is labeled or immobilized.

[0022] In one aspect, the first conjugate of the invention is bound to a solid phase.

[0023] In one embodiment, the present invention provides a method for detecting a complex in a sample, comprising the steps of: (1) contacting a first binder bound to a solid phase with a sample containing a complex to bind the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components, and at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value is 1 nM or more.

[0024] In another embodiment, the present invention provides a method for detecting a complex in a sample, comprising the steps of: (1)(a) contacting a sample containing a complex with a first binder to bind the complex to the first binder; (1)(b) binding the first binder bound to the complex to a solid phase; (2) binding the complex bound to the first binder bound to the solid phase with a second binder; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components, and at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value is 1 nM or more.

[0025] In another embodiment, the present invention provides a method for detecting a complex in a sample, comprising: (1)+(2)(a) contacting a sample containing a first binder, a second binder, and a complex, and allowing the first binder and the second binder to bind to the complex, respectively; (b) binding the first binder bound to the complex bound to the second binder to a solid phase; and (3) detecting the second binder bound to the complex, wherein the complex contains two or more components, and at least one K between the components is detected. D The method is carried out under conditions where the binding equilibrium of the complex is substantially maintained, and the value of the first binder is 1 nM or more. Here, in step (1)+(2)(a), the first binder, the second binder, and a sample containing the complex may be contacted simultaneously. Alternatively, the first binder may be contacted with the sample containing the complex, and then the second binder may be contacted. Alternatively, the second binder may be contacted with the sample containing the complex, and then the first binder may be contacted.

[0026] In another aspect, the present invention may include one or more washing steps.

[0027] In another aspect, the present invention relates to a method for determining the concentration and / or amount of a complex in a sample, further comprising the steps of: (4) preparing a composite for generating a regression equation; (5) detecting the complex of step (4) by a process including steps (1) to (3); (6) simulating the concentration of the complex in step (4); (7) calculating a regression equation from the signal value detected in step (5) and the simulated concentration; and (8) A step of applying the signal value detected in the step (3) to the regression equation.

[0028] In one embodiment, the present invention provides a method for determining the concentration and / or amount of a complex in a sample, wherein the complex comprises two or more components, and at least one K D(5) detecting the complex in step (4) by a process including steps (1) to (3) above; (6) simulating the complex concentration in step (4); (7) calculating a regression equation from the signal value detected in step (5) and the simulated concentration; and (8) applying the signal value detected in step (3) to the regression equation.

[0029] In one particular embodiment, the first conjugate of the present invention is bound to a solid phase, and the present invention provides a method for determining the concentration and / or amount of a complex in a sample, wherein the complex comprises two or more components and at least one K D (5) detecting the complex in step (4) by a process including steps (1) to (3) above; (6) simulating the concentration of the complex in step (4); (7) calculating a regression equation from the signal value detected in step (5) and the simulated concentration; and (8) applying the signal value detected in step (3) to the regression equation.

[0030] In one particular embodiment, in step (4), the K D Preferably, the complex with known values ​​is prepared at multiple concentrations.

[0031] In a specific embodiment, in step (6), the concentrations of the components of the complex and the K D The complex concentration is simulated from the values.

[0032] As used herein, a complex refers to a complex formed from two or more components. Here, components include peptides, polypeptides, proteins, organic compounds, nucleic acids, etc. A complex may be formed from two or more components, and may be formed from the same type of components or different types of components, such as a protein and a nucleic acid, or a protein and an organic compound. A complex may also contain the same components.

[0033] As used herein, the term "polypeptide" generally refers to peptides and proteins having a length of approximately 10 amino acids or more. While polypeptides are generally derived from living organisms, they are not particularly limited and may be, for example, polypeptides consisting of an artificially designed sequence. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present invention. Polypeptides also include antibodies and medium-sized peptides with molecular weights of 500 to 2000 (e.g., polypeptides having a cyclic moiety) (see WO2013100132).

[0034] In the present specification, the organic compound is, for example, a low molecular weight compound, preferably having a molecular weight of 1000 or less.

[0035] A nucleic acid herein is, for example, an antisense molecule, an siRNA molecule, an RNA aptamer or a ribozyme.

[0036] The first binder may be selected from, for example, a polypeptide, an antibody, an antibody fragment, a fusion polypeptide comprising an antibody or antibody fragment and a non-antibody polypeptide, a fusion polypeptide comprising an antibody or antibody fragment and a soluble receptor, or a fusion polypeptide comprising an antibody or antibody fragment and a peptidic binding molecule, as long as it binds to the complex. Preferably, the first binder binds to the complex without dissociating the complex.

[0037] The second binder may be selected from, for example, a polypeptide, an antibody, a fusion polypeptide comprising an antibody or antibody fragment and a non-antibody polypeptide, a fusion polypeptide comprising an antibody or antibody fragment and a soluble receptor, or a fusion polypeptide comprising an antibody or antibody fragment and a peptidic binding molecule. The second binder preferably binds to the complex without dissociating the complex and / or without inhibiting the binding of the first binder to the complex.

[0038] It is preferred that the first binder and the second binder each bind to a different component that forms the complex. In one embodiment, when the complex contains components A and B, it is preferred that the first binder binds to component A and the second binder binds to component B. In another embodiment, when the complex contains components A, B, and C, and component A binds only to component B and component B binds only to component C, it is preferred that the first binder binds to component A and the second binder binds to component C, or alternatively, the first binder binds to component C and the second binder binds to component A.

[0039] In one particular embodiment, a conjugate is provided comprising a bispecific antibody and two antigens recognized by the antibody, where a first conjugate binds to one antigen and a second conjugate binds to the other antigen.

[0040] In another embodiment, when the complex comprises component A, component B, component C, and component D, where component A binds only to component B, component B binds only to component A and component C, component C binds only to component B and component D, and component D binds only to component C, it is preferred that the first binder binds to component A and the second binder binds to component D, or alternatively, the first binder binds to component D and the second binder binds to component A.

[0041] In one embodiment, the present invention provides a method for detecting a complex in a sample, the complex comprising an antigen and an antibody that recognizes the antigen as components, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen to bind the first binder to the complex; (2) binding the complex bound to the first binder to a second binder that recognizes the antigen; and (3) detecting the second binder that has bound to the complex, wherein at least one K between the components is detected. D In a specific embodiment, the complex is a complex in which at least one of the antigen-binding sites of the antibody is bound to the antigen. In another specific embodiment, the complex is a complex in which all of the antigen-binding sites of the antibody are bound to the antigen.

[0042] In a specific embodiment, the first binder of the present invention is bound to a solid phase, and the present invention provides a method for detecting a complex in a sample, the complex comprising an antigen and an antibody recognizing the antigen as components, the method comprising: (1) contacting a sample containing the complex with a first binder recognizing the antigen bound to the solid phase, and binding the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder recognizing the antigen; and (3) detecting the second binder bound to the complex, wherein at least one K between the components is detected. DIn a specific embodiment, the complex is a complex in which at least one of the antigen-binding sites of the antibody is bound to the antigen. In another specific embodiment, the complex is a complex in which all of the antigen-binding sites of the antibody are bound to the antigen.

[0043] In one aspect, the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing, as components, a bispecific antibody and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen and allowing the first binder to bind to the complex; (2) allowing the complex bound to the first binder to bind to a second binder that recognizes an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder that has bound to the complex.

[0044] In a specific embodiment, the first binder of the present invention is bound to a solid phase, and the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing, as components, a bispecific antibody and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen bound to the solid phase, to bind the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder that recognizes an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder bound to the complex.

[0045] In one embodiment, the present invention provides a method for detecting a complex in a sample, the complex being a ternary complex containing, as components, a bispecific antibody and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen and allowing the first binder to bind to the complex; (2) binding the complex bound to the first binder with a second binder that recognizes an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder that has bound to the complex, wherein at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value of the binding equilibrium is 1 nM or more.

[0046] In a specific embodiment, the first binder of the present invention is bound to a solid phase, and the present invention also provides a method for detecting a complex in a sample, wherein the complex is a ternary complex containing, as components, a bispecific antibody and two antigens recognized by the bispecific antibody, and the method comprises the steps of: (1) contacting a sample containing the complex with a first binder that recognizes the antigen bound to the solid phase, and binding the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder that recognizes an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder bound to the complex, wherein at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value of the binding equilibrium is 1 nM or more.

[0047] That is, in one embodiment, the complex is a complex in which a bispecific antibody and both of the antigens recognized by the antibody are bound to the antibody. For example, when a bispecific antibody binds to antigen A and antigen B, the complex refers to a ternary complex in which the bispecific antibody binds to antigen A and antigen B. Here, antigen A and antigen B may be the same antigen or different antigens.

[0048] In one specific embodiment, in the case of the bispecific antibody (ACE910:Emicizumab, Q499-z121 / J327-z119 / L404-k) described below in patent document (WO 2012 / 067176), the complex comprises emicizumab bound to blood coagulation factor IX (FIX) or activated blood coagulation factor IX (FIXa), and blood coagulation factor X (FX) or activated blood coagulation factor X (FXa). Note that, in this specification, unless otherwise specified, FIX and FIXa may be used synonymously, and the term FIX(a) may be used to refer to a concept that includes both FIX and FIXa. Note that, in this specification, unless otherwise specified, FX and FXa may be used synonymously, and the term FX(a) may be used to refer to a concept that includes both FX and FXa.

[0049] A specific embodiment of the present invention provides a method for detecting a complex containing emicizumab. When an antibody that binds to FIX(a) is selected as the first binder, an antibody that binds to FX(a) can be selected as the second binder. Also, when an antibody that binds to FX(a) is selected as the first binder, an antibody that binds to FIX(a) can be selected as the second binder.

[0050] The antibody that binds to FIX(a) is not particularly limited, but preferably binds to an epitope different from the epitope in FIX(a) of emicizumab. Such antibodies can be appropriately selected from, for example, A19, A25, A31, A38, A39, A40, A41, A44, A50, A69, and XB12 described in patent document (WO 2006 / 109592). The antibody that binds to FX(a) is not particularly limited, but preferably binds to an epitope different from the epitope in FX(a) of emicizumab. Such antibodies can be appropriately selected from, for example, B2, B5, B9, B10, B11, B12, B13, B14, B15, B16, B18, B19, B20, B21, B23, B25, B26, B27, B31, B34-1, B34-2, B35, B36, B38, B42, SB04, SB15, and SB27 described in patent document (WO 2006 / 109592).

[0051] In one aspect, the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing emicizumab, FIX(a), and FX as components, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes FIX(a) or FX to allow the first binder to bind to the complex; (2) binding the complex bound to the first binder with a second binder that recognizes FIX(a) or FX, which is an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder bound to the complex.

[0052] In a specific embodiment, the first binder of the present invention is bound to a solid phase, and the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing emicizumab, FIX(a), and FX as components, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes FIX(a) or FX bound to the solid phase, and binding the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder that recognizes FIX(a) or FX, which is an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder bound to the complex.

[0053] In one aspect, the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing emicizumab, FIX(a), and FX as components, and the method comprises the steps of: (1) contacting a sample containing the complex with a first binder that recognizes FIX(a) or FX to allow the first binder to bind to the complex; (2) binding the complex bound to the first binder to a second binder that recognizes FIX(a) or FX, which is an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder that has bound to the complex, wherein the steps are carried out under conditions such that the binding equilibrium of the complex is substantially maintained.

[0054] In a specific embodiment, the first binder of the present invention is bound to a solid phase, and the present invention relates to a method for detecting a complex in a sample, wherein the complex is a ternary complex containing emicizumab, FIX(a), and FX as components, the method comprising the steps of: (1) contacting a sample containing the complex with a first binder that recognizes FIX(a) or FX bound to the solid phase, and binding the complex to the first binder; (2) binding the complex bound to the first binder bound to the solid phase with a second binder that recognizes FIX(a) or FX, which is an antigen different from the antigen recognized by the first binder; and (3) detecting the second binder bound to the complex, wherein the steps are carried out under conditions in which the binding equilibrium of the complex is substantially maintained.

[0055] In a specific embodiment, when a complex is composed of a bispecific antibody, an antigen A recognized by the antibody, and an antigen B recognized by the antibody, and the antibody binds to antigen A and antigen B, it is preferred that the first binder binds to antigen A and the second binder binds to antigen B. In a further specific embodiment, when a complex comprises emicizumab, a bispecific antibody bound to FIX(a) and FX(a), it is preferred that the first binder binds to FIX(a) and the second binder binds to FX(a), or it is preferred that the first binder binds to FX(a) and the second binder binds to FIX(a).

[0056] In one embodiment, the method of the invention is carried out without labeling or immobilizing the components of the complex to be detected. Labeling refers to modification with, but is not limited to, a luminescent label, a chemiluminescent label, an electrochemiluminescent label, a fluorescent label, digoxigenin, biotin, avidin, or a radioactive label. Immobilization refers to binding or fixing, whether directly or indirectly, to a solid phase such as, but not limited to, beads, discs, microfluidic chips, magnetic particles, or microtiter plates.

[0057] In one embodiment, the method of the present invention does not include a step of newly generating a complex by, for example, adding an excess of a component that forms a complex to a sample to be measured, except for a sample used to generate a regression line.

[0058] In one embodiment, the affinity (dissociation constant (K D ) is not particularly limited, but is 1 nM or more, preferably 10 nM or more, 100 nM or more, and particularly preferably 1 μM or more. In certain embodiments, the method of the present invention is directed to a method for detecting weak affinity (K D The present invention is a method for measuring the presence and / or amount of a complex (high or low in ATP).

[0059] When a complex is composed of three or more components and has multiple binding sites, the lowest affinity binding site is considered to be important for maintaining the complex, and therefore the affinity of the lowest affinity binding site can be used synonymously with the affinity of the complex.

[0060] In one embodiment, when the complex is a complex in which both antigen A and antigen B recognized by a bispecific antibody are bound to the antibody, K D The affinity of the neutralizing antibody is preferably 1 nM or more, more preferably 10 nM or more, even more preferably 100 nM or more, and particularly preferably 1 μM or more. Many of the neutralizing antibodies used as therapeutic agents have strong affinity, for example, K D The affinity of antibodies ranges from 0.1 nM to several tens of nM (Carter, Nat Rev Immunol. 6(5):2006 343-57(2006)). Even for antibodies used as therapeutic agents, antibodies whose primary purpose is not neutralization do not necessarily need to have as strong an affinity for the antigen as neutralizing antibodies, and in some cases, weak affinity is preferable. For example, the affinity of the bispecific antibody emicizumab described above for its antigens, FIX or FIXa and FX or FXa, is approximately 1 μM for both, which is a very weak affinity for antigens compared to general neutralizing antibodies (Kitazawa, Thromb Haemost 117(7):1348-1357(2017)).

[0061] In one particular embodiment, the present invention can be used to detect complexes of bispecific antibodies and their antigens, for example: Ozoralizumab (antigens are TNF and albumin) RG7716 (antigens are VEGF-A and angiopoietin-2) RG-7990 (antigens are IL-13 and IL-17) Lutikizumab (antigens are IL-1α and IL-1β)

[0062] In one embodiment, the method of the present invention is capable of detecting a complex comprising an antibody with weak affinity for an antigen and the antigen bound to that antibody. In one embodiment, the method of the present invention is a method for detecting a complex comprising a bispecific antibody that has weak affinity for at least one antigen and the antigen bound to that antibody. In one embodiment, the method of the present invention is a method for detecting a complex comprising a bispecific antibody that has weak affinity for both antigens and an antigen bound to the bispecific antibody.

[0063] As used herein, an antibody with weak affinity to an antigen is an antibody with a K D The value is 1 nM or more, preferably 10 nM or more, 100 nM or more, and particularly preferably 1 μM or more.

[0064] Complexes containing components with weak affinity between them (e.g., K D For complexes with a low affinity (e.g., several nM or more), the components tend to dissociate easily, making it difficult to maintain the desired complex, and the concentration of the complex formed is low. To detect such complexes and more specifically to quantitatively measure the amount and / or concentration of the complex, it is necessary to minimize the shift in the binding equilibrium of the complex due to the generally required long incubation time and the dissociation of the complex due to washing procedures.

[0065] Thus, in one aspect, the present invention provides a method for detecting a complex in a sample under conditions in which the binding equilibrium of the complex is substantially maintained, such as conditions in which substantially no new complex is formed on the solid phase and / or substantially no dissociation of the complex occurs.

[0066] These conditions can be achieved by a method that has a smaller reaction space, a larger specific surface area of ​​the solid phase surface, and a shorter assay time than methods using microtiter plates, such as a method using a microfluidic chip, disk, or beads, more specifically, a method using, for example, the KinExA (registered trademark) or Gyrolab (registered trademark) immunoassay system (Fraley et al., 2013, Bioanalysis 5: 1765-74). Here, a short assay time means the time during which the sample is in contact with the solid phase; for example, when a sample containing an antigen is added to a column packed with beads to which antibodies capable of capturing the antigen are immobilized, and the antibodies in the sample are captured, the time during which any specific antigen in the sample is in contact with any specific point in the column is preferably 10 seconds or less, more preferably 9 seconds or less, 8 seconds or less, 7 seconds or less, 6 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, 2 seconds or less, 1 second or less, 0.5 seconds or less, 0.1 seconds or less, 0.05 seconds or less, or 0.01 seconds or less, but preferably at least 0.001 seconds or more, 0.01 seconds or more.

[0067] In one embodiment, the present invention provides a method for detecting a complex in a blood sample, wherein the complex is a ternary complex containing a bispecific antibody contained in the blood sample as a component and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting a first binder with the blood sample containing the complex to bind the first binder to the complex; (2) binding the complex bound to the first binder to a second binder; and (3) detecting the second binder bound to the complex; and wherein at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value of the binding equilibrium is 1 nM or more.

[0068] In a specific embodiment, the first conjugate of the present invention is bound to a solid phase, and the present invention provides a method for detecting a complex in a blood sample, wherein the complex is a ternary complex containing a bispecific antibody contained in the blood sample as a component and two antigens recognized by the bispecific antibody, the method comprising the steps of: (1) contacting the first conjugate bound to the solid phase with a blood sample containing the complex to bind the complex to the first conjugate; (2) binding the complex bound to the first conjugate bound to the solid phase with a second conjugate; and (3) detecting the second conjugate bound to the complex, wherein at least one K between the components is detected. D The method is carried out under conditions in which the binding equilibrium of the complex is substantially maintained, and the value of the binding equilibrium is 1 nM or more.

[0069] In one embodiment, conditions under which binding equilibrium is substantially maintained are conditions under which substantially no new complexes are formed on the solid phase during the assay and / or substantially no dissociation of complexes occurs. In another embodiment, the condition under which the binding equilibrium is substantially maintained refers to a time that is so short that theoretically the binding equilibrium does not shift. "Substantially maintaining the binding equilibrium" means, for example, when the measured value is used as an index, that the difference in measured values ​​when measured multiple times is preferably 50% or less, more preferably 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or 0.1% or less. A time short enough that the binding equilibrium theoretically does not shift means the time during which the sample and the solid phase are in contact, for example, when a sample containing an antigen is added to a column packed with beads to which an antibody capable of capturing the antigen is immobilized, and the antibody in the sample is captured, the time during which any specific antigen in the sample is in contact with any specific point in the column is preferably 10 seconds or less, more preferably 9 seconds or less, 8 seconds or less, 7 seconds or less, 6 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, 2 seconds or less, 1 second or less, 0.5 seconds or less, 0.1 seconds or less, 0.05 seconds or less, or 0.01 seconds or less, and preferably at least 0.001 seconds or more, more preferably 0.01 seconds or more.

[0070] In one embodiment, the second conjugate may be labeled, which may be, but is not limited to, a luminescent label, a chemiluminescent label, an electrochemiluminescent label, a fluorescent label, digoxigenin, biotin, or a radioactive label.

[0071] The solid phase as used herein may be a bead, a disk, a microfluidic chip, a magnetic particle, or a microtiter plate, and is not limited thereto as long as the object of the present invention can be achieved.

[0072] The first binder can be bound to the solid phase by any method known to those skilled in the art. In one embodiment, when the first conjugate is a polypeptide, the binding of the first conjugate to the solid phase is carried out by chemical bonding via the N-terminal group and / or the ε-amino group (lysine) of the amino acid backbone of the polypeptide, the ε-amino group of a different lysine, a carboxyl functional group, a sulfhydryl functional group, a hydroxyl functional group, and / or a phenol functional group, and / or a sugar alcohol group of the carbohydrate structure of the polypeptide.

[0073] In one embodiment, the first binder is bound to the solid phase by passive adsorption, as described, for example, by Butler, JE, in "Solid Phases in Immunoassay" (1996) 205-225 and Diamandis, EP, and Christopoulos, TK (Editors), in "Immunoassay" (1996) Academic Press (San Diego).

[0074] In one embodiment, the first binder is bound to a solid phase via a specific binding pair. In one embodiment, such a binding pair (first component / second component) is selected from streptavidin or avidin / biotin, antibody / antigen (see, e.g., Hermanson, GT, et al., Bioconjugate Techniques, Academic Press (1996)), lectin / polysaccharide, steroid / steroid-binding protein, hormone / hormone receptor, enzyme / substrate, IgG / protein A and / or protein G, etc. In one embodiment, the first binder is linked to biotin, and binding is achieved via avidin or streptavidin immobilized on the solid phase.

[0075] As used herein, the term "sample" refers to a biological sample obtained, for example, from a human, but is not limited thereto and may also refer to a biological sample obtained from a non-human source. A biological sample may be a liquid sample obtained from a human. A "sample" may also be a sample prepared in vitro. A liquid sample is, for example, a blood sample, and includes serum, plasma, or whole blood. In the present invention, it is preferable to use a plasma sample. Methods for obtaining blood samples from humans are well known to those skilled in the art. Furthermore, liquid samples may be interstitial fluid, a solution obtained by mashing tissue, a solution obtained by solubilizing tissue, or the like, and the tissue may be fresh or frozen. Methods for obtaining tissue from humans are well known to those skilled in the art. Note that blood samples that have undergone some kind of processing may also be included in the blood sample of the present invention.

[0076] As used herein, "detection" includes quantitative and qualitative detection, and examples of qualitative detection include simply determining whether a complex is present, determining whether a certain amount of the complex is present, and comparing the amount of the complex with that of another sample (e.g., a control sample). On the other hand, examples of quantitative detection include determining the concentration of the complex and determining the amount of the complex.

[0077] "Affinity" refers to the strength of the sum of non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Unless otherwise indicated, "affinity," as used herein, refers to the intrinsic binding affinity, reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0078] In one embodiment, K D The value is measured by radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured at the lowest concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0079] According to another aspect, K DValues ​​are measured using a BIACORE® surface plasmon resonance assay. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve protein binding of approximately 10 response units (RU). After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C and a flow rate of approximately 25 μl / min. The binding rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). D ) is k off / k on The on-rate is calculated as a ratio of 10 to 10. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette). Whether or not the epitopes are different can be confirmed by, for example, a competitive assay, and means that one of the antibodies being compared inhibits the binding of the other antibody to the antigen by 50% or more.

[0080] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, including chimeric, humanized, or human antibodies, so long as they exhibit the desired antigen-binding activity. In certain embodiments, the antibody is a multispecific antibody, e.g., a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes on the same antigen. A bispecific antibody can be prepared as a full-length antibody or an antibody fragment. In one embodiment, the antibody is a bispecific antibody that specifically binds to a first antigen and a second antigen. In one embodiment, a bispecific antibody has i) a first binding specificity that specifically binds to a first antigen or a first epitope on the antigen, and ii) a second binding specificity that specifically binds to a second antigen or a second epitope on the same antigen. In one embodiment, the second epitope on the same antigen is a different epitope from the first epitope.

[0081] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0082] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0083] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. In one embodiment, the antibody includes an antibody variant, e.g., an antibody variant having one or more amino acid substitutions, insertions, or deletions, an antibody variant with modified glycosylation, or an antibody derivative conjugated with a water-soluble polymer such as PEG.

[0084] The antibody may be a recombinant antibody produced using genetic engineering technology. Recombinant antibodies can be obtained by cloning the DNA encoding the antibody from antibody-producing cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating it into a vector, and introducing this into a host (host cell) for production.

[0085] IgG-type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein C et al. Nature 1983, 305: 537-540). Alternatively, bispecific antibodies can be secreted by introducing into cells the genes for the L and H chains constituting the two types of IgG of interest, a total of four genes, and co-expressing them. Regarding L chains, since there is less diversity in the L chain variable region than in the H chain variable region, it is expected that a common L chain capable of conferring binding ability to both H chains will be obtained, and the bispecific antibodies referred to herein may be antibodies that share a common L chain.

[0086] Bispecific antibodies can also be prepared by chemically cross-linking Fab' fragments. Alternatively, leucine zippers derived from Fos, Jun, etc. can be used instead of chemical cross-linking.

[0087] In addition, there are other antibodies (IDrugs 2010, 13:698-700), such as IgG-scFv (Protein Eng Des Sel. 2010 Apr;23(4):221-8), sc(Fv)2 such as BiTE (Drug Discov Today. 2005 Sep 15;10(18):1237-44), DVD-Ig (Nat Biotechnol. 2007 Nov;25(11):1290-7. Epub 2007 Oct 14., MAbs. 2009 Jul;1(4):339-47. Epub 2009 Jul 10.), and two-in-one antibodies (Science. 2009 Mar 20;323(5921):1610-4., Immunotherapy. 2009 Sep;1(5):749-51.), and bispecific antibodies such as Tri-Fab, tandem scFv, and diabodies are also known (MAbs. 2009 November;1(6):539-547.). Furthermore, even when using molecular forms such as scFv-Fc and scaffold-Fc, bispecific antibodies can be efficiently produced by preferentially secreting heterogeneous Fc combinations (Ridgway JB et al. Protein Engineering 1996,9:617-621; Merchant AM et al. Nature Biotechnology 1998,16:677-681; WO2006 / 106905; Davis JH et al. Protein Eng Des Sel. 2010,4:195-202.).

[0088] Bispecific antibodies can also be generated using diabodies, which are heterodimers of two cross-over scFv fragments.

[0089] In one specific embodiment of the present invention, the detection of a ternary complex can be carried out as follows. Here, an example is shown in which the bispecific antibody emicizumab recognizes FIX(a) and FX(a). The ternary complex to be measured is composed of FIX(a), FX(a), and emicizumab. In addition to emicizumab, a bispecific antibody that recognizes FIX(a) and FX(a) is prepared for creating a regression equation. Here, the affinity of the bispecific antibody used for creating the regression equation is not necessarily high (K D Although it is not necessary for the affinity to be low, a high affinity is preferable. As a bispecific antibody for creating a regression equation, for example, the Q4 / / J3 antibody (K value for hFIX) D The value was 17.7 nM, and the K D value was 11.5 nM), Q3 / / J1 antibody (K D The value was 1.20 μM, and the K D Although a value of 58.3 nM can be used, here we show an example using Q4 / / J3.

[0090] Prepare a FIX(a) antibody whose epitope on FIX(a) is different from that of emicizumab, and an FX(a) antibody whose epitope on FX(a) is different from that of emicizumab. Examples of the anti-FIX(a) antibody XB12 and the anti-FX antibody SB04 are shown below, but the anti-FIX(a) antibody is not limited to XB12, and the anti-FX antibody is not limited to SB04.

[0091] The ternary complex can be detected using a ligand binding assay, which is an immunoassay, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance (SPR), electrochemiluminescence (ECL), or KinExA (registered trademark) Kinetic Exclusion Assay (Drake et al., 2004, Analytical Biochemistry 328:35-43).

[0092] In one embodiment, methods that have a smaller reaction space, a larger specific surface area of ​​the solid phase, and a shorter assay time than methods using microtiter plates, such as methods using microfluidic chips, disks, or beads, can be used. More specifically, methods using the KinExA® or Gyrolab® immunoassay system (Fraley et al., 2013, Bioanalysis 5: 1765-74), for example, can be used, but are not limited to these. For example, the KinExA® can use the KinExA 3200 (Sapidyne) as a measurement device, and the Gyrolab® immunoassay system can use the Gyrolab xP (Gyros Protein Technologies) as a measurement device.

[0093] Gyrolab is a fully automated ligand binding assay system that performs measurements using a dedicated Bioaffy CD. The flow path on the CD contains an affinity bead column. By rapidly passing the sample through the column, antibodies immobilized on the beads specifically capture the analyte in the sample. The captured analyte is then detected using a fluorescently labeled antibody. This measurement principle, particularly the rapid capture of analytes in the sample and the automated washing process, are similar to those of the KinExA (Sapidyne), making it a suitable choice for this measurement instrument. In fact, because the KinExA and Gyrolab have similar measurement principles, they are used to calculate the dissociation constant of an antigen-antibody reaction in solution by measuring free antibodies in the sample (Anal Biochem. 2012 Jul 15;426(2):134-41).

[0094] As a specific embodiment, an example using KinExA 3200 (Sapidyne) will be shown below. [Preparation of beads] A 20 μg / mL solution of XB12 or SB04 was prepared in phosphate-buffered saline (pH 7.4, Sigma-Aldrich). One mL of this solution was added to each PMMA bead (Sapidyne) and incubated at room temperature for 2 hours to immobilize the antibody on the beads. After centrifuging at low speed for a few seconds to remove the supernatant, blocking buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, 1% BSA, pH 7.4) was added and incubated at room temperature for 1 hour to block the beads. Either XB12- or SB04-immobilized beads can be used; however, the following example uses XB12-immobilized beads. [Preparation of detection antibody solution] To use XB12-immobilized beads, the following example shows fluorescently labeled SB04: Fluorescently labeled SB04 (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences, according to the manufacturer's protocol) can be used as a detection antibody. A detection antibody solution containing a fluorescently labeled antibody can be prepared, for example, by preparing a fluorescently labeled (Lightning-Link® Rapid Dylight® 650, Innova Biosciences, Inc., according to the manufacturer's protocol) anti-FX antibody (in-house preparation: SB04) at 0.5 μg / mL in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). To remove aggregates of the fluorescent label during the preparation process, the solution can be first prepared at 5 μg / mL, then centrifuged at 18,000 g for 10 minutes, and the supernatant can be further diluted to 0.5 μg / mL. The fluorescent label is not limited to this. [Sample preparation] Here, an example is shown in which bispecific antibodies Q4 / / J3 antibody, Q3 / / J1 antibody, and emicizumab are measured. hFIX and hFX were fixed at 5 and 8 μg / mL (89.3 and 136 nM), respectively. The following samples were spiked: Q4 / / J3 antibody at concentrations between 0.0488 and 50 μg / mL (0.326 and 333 nM) with a common ratio of 4 (six samples), Q3 / / J1 antibody at concentrations between 1.38 and 354 μg / mL (9.20 and 2360 nM) with a common ratio of 2 (nine samples), and emicuzumab at concentrations between 6.25 and 400 μg / mL (41.7 and 2667 nM) with a common ratio of 2 (seven samples). These samples were prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). A blank sample was prepared by spiking only hFIX and hFX without the anti-FIX / FX bispecific antibody. The concentrations of hFIX and hFX were set to physiological concentrations (plasma concentrations). After sample preparation, the sample was incubated at room temperature for 1 hour before measurement to allow complex formation to reach equilibrium.

[0095] 〔measurement〕 Measurements are performed according to the vendor's recommended protocol. For example, one tube of prepared bead suspension is transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution are placed in the appropriate positions in the KinExA 3200 autosampler. All measurement processes are automated by the KinExA 3200 and autosampler. The protocol controlling the instrument is created using KinExA Pro software (Sapidyne). The protocol specifies the appropriate reagent and sample positions for each measurement process, ensuring that the appropriate reagents are sampled at the appropriate times. First, XB12-coated beads are packed into a column in a flow cell. The volume of the bead suspension applied is monitored with a camera and optimized to ensure the beads are packed to the appropriate height with consistent reproducibility. Next, the sample is passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex formed by the bispecific antibody bound to both hFIX and hFX. After the first wash with Running Buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution is passed through the column, allowing the fluorescently labeled SB04 antibody to bind to hFX in the captured ternary complex. After the second and third washes with Running Buffer (+), the KinExA signal is detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volume, time, and flow rate of the sample, the running buffer (+) in the first washing step, the detection antibody solution, the running buffer (+) in the second washing step, and the running buffer (+) in the third washing step are all varied, and measurements are performed under conditions where the signal-to-noise ratio is sufficient. For example, measurements can be performed under the following conditions. Sample: 135 μL, 32.4 s, 0.25 mL / min Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min Detection antibody solution: 800 μL, 192 s, 0.25 mL / min, Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. For example, measure the blank sample with n=5 and the other samples with n=1.

[0096] 〔simulation〕 The dissociation constant K D Simulation is performed using The concentration of the ternary complex was simulated using a method used in a literature review (Thromb Haemost, 117(7), 1348-1357, 2017). Calculations were performed using Microsoft Excel 2013. In this simulation, the FIX concentration, FX concentration, bispecific antibody concentration, and the K values ​​of the bispecific antibody for FIX and FX were calculated. D Since it is necessary to define the K values ​​for each, the concentrations of FIX and FX were set constant at physiological concentrations (plasma concentrations) of 5 and 8 μg / mL (89.3 and 136 nM), respectively, and the anti-FIX / FX bispecific antibody concentration was varied in two-fold increments over the range of 0.0122-1600 μg / mL (0.0814-10667 nM). D For values, for example, the condition that both are equal, or K for FIX D Value of K for FX D The concentration of the ternary complex under each condition is simulated, and the values ​​are plotted with the ternary complex concentration on the Y-axis and the bispecific antibody concentration on the X-axis. The following example shows the Q4 / / J antibody as a bispecific antibody. 〔analysis〕 The mean (Mean) and standard deviation (SD) of the signal values ​​of the blank sample are calculated, and the detection limit can be set, for example, as Mean + 3.29 × SD. The mean (Mean) and standard deviation (SD) of the signal values ​​can be calculated using, for example, Microsoft Excel 2013. For the signal values ​​of other samples that are above the detection limit, the mean signal value of the blank sample is subtracted to calculate the ternary complex signal value. The measured values ​​are plotted with the ternary complex signal value on the Y axis and the bispecific antibody concentration on the X axis.

[0097] The Y-axis represents the signal value of the ternary complex spiked with the Q4 / / J3 antibody, and the X-axis represents the simulated value of the ternary complex containing the Q4 / / J3 antibody. The obtained results are plotted and a correlation analysis is performed. This correlation analysis can be performed using, for example, Microsoft Excel 2013. In the correlation analysis, for example, a linear approximation curve is drawn, and the regression equation and R 2 In addition, if the relationship y = a(x / (x + b)) is observed within a specific concentration range of the Q4 / / J3 antibody, nonlinear regression is performed on the plot within this range to obtain the regression equation and R 2 The signal value of the ternary complex of the sample spiked with the bispecific antibody is converted to a concentration using the regression equation.

[0098] In one particular embodiment, Gyrolab xP (Gyros Protein Technologies) can be used as a measuring device and Bioaffy 200 CD as a measuring CD. [Preparation of capture antibody solution] While either XB12 or SB04 can be used for capture, this example uses XB12. Because the Gyrolab CD is equipped with streptavidin beads, the XB12 used for capture is biotinylated using Sulfo-NHS-LC-Biotin (Thermo Scientific) (biotin-XB12). For the protocol, refer to the product protocol. After biotinylation, the column is treated with Zeba Spin Desalting Columns (Thermo Scientific) to remove free biotin. A 100 μg / mL solution is prepared in buffer (0.01M HEPES, 0.15M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). To remove aggregates, the column is centrifuged at 18,000 g for 10 minutes, and the supernatant is used. [Preparation of detection antibody solution] Fluorescently labeled anti-FX antibody (SB04, in-house preparation) was prepared using the Alexa Fluor 647 Antibody Labeling Kit, Thermo Scientific, according to the manufacturer's protocol. A 3 μg / mL solution was prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). To remove aggregates of the fluorescently labeled antibody, the solution was centrifuged at 18,000 g for 10 minutes, and the supernatant was used. [Sample preparation] hFIX and hFX were kept constant at 5 and 8 μg / mL (89.3 and 136 nM), respectively. Five concentrations of Q4 / / J3 antibody were spiked between 0.195 and 50 μg / mL (1.30 and 333 nM) with a common ratio of 4. These samples were prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). A blank sample was prepared containing only hFIX and hFX, without the anti-FIX / FX bispecific antibody. The concentrations of hFIX and hFX were set to physiological plasma concentrations. After sample preparation, the samples were incubated at room temperature for 1 hour to allow complex formation to reach equilibrium before measurement.

[0099] 〔measurement〕 Measurements are performed according to the vendor's recommended protocol. For example, the following method is used: a Gyrolab xP workstation (Gyros Protein Technologies) is used as the measurement device, and a Bioaffy 200 is used as the measurement CD. All measurement processes are automated by Gyrolab. The protocol controlling the instrument uses the 200-3W-001 Wizard method in the Gyrolab Control software (Gyros Protein Technologies). The operation proceeds in the order of capture antibody, sample, and detection antibody, with washing steps between each step. The concentrations of capture antibody and detection antibody are set to conditions that result in a high signal-to-noise ratio. For the washing step, an assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4) is used. According to the plate design generated from the protocol, the capture antibody solution, sample, detection antibody solution, and washing assay buffer are added to a dedicated PCR plate, and the plate is then loaded into the instrument prior to measurement. During the measurement process, a biotin-XB12 solution is first transferred through the flow channel and captured by streptavidin beads. After washing, free hFIX in the sample, the binary complex of hFIX and the bispecific antibody, and the ternary complex of hFIX, hFX, and the bispecific antibody are captured by the biotin-XB12 beads. After washing, a fluorescently labeled SB04 solution is passed through the plate, and the fluorescently labeled SB04 antibody binds to hFX in the captured ternary complex. Detection is performed using a photomultiplier tube (PMT) at three fixed response amplification levels: 1, 5, and 25%, and the measured values ​​at each setting are recorded. Of the prepared samples, a blank sample (n=5) and a sample spiked with Q4 / / J3 antibody (n=3) are measured.

[0100] 〔analysis〕 It is preferable to use values ​​obtained under PMT conditions that provide a sufficiently large detected response without detector saturation for analysis. The mean and standard deviation (SD) of the signal values ​​of blank samples measured with n = 5 can be calculated, and the detection limit can be set, for example, as Mean + 3.29 × SD. The mean and standard deviation (SD) of the signal values ​​can be calculated using, for example, Microsoft Excel 2013. The mean of the blank sample response is subtracted from the signal values ​​of other samples that are above the detection limit to calculate the ternary complex response.

[0101] In one particular embodiment, when human plasma is used as a sample, the method can be carried out, for example, as follows: Here, an example using KinExA 3200 (Sapidyne) is shown. [Preparation of plasma with buffering and anticoagulant properties] For example, it can be prepared as follows, but is not limited to this. Congenital Factor VIII-deficient human plasma (George King Bio-Medical) is mixed with, for example, 1 / 9 equivalent of 1 M HEPES buffer solution (pH 7.1-7.5, Nacalai Tesque), followed by 10,000 units / 10 mL of Heparin Sodium (Mochida Pharmaceutical) to a final concentration of 10 units / mL. The amount of 1 M HEPES buffer solution added here is, for example, 1 / 100 to 1 / 2 equivalent. More specifically, it is 1 / 99, 1 / 49, 1 / 19, 1 / 9, 1 / 4, or 1 / 2 equivalent. Diluting the plasma for measurement, as in conventional ELISA, would result in dissociation of the complex, making it impossible to measure the complex in plasma. However, using plasma directly would result in pH changes due to changes in plasma carbonate ion concentration, which would not reflect physiological conditions. Therefore, to prevent this, a HEPES buffer solution is used to buffer the plasma. In addition, heparin sodium is added to further suppress the coagulation reaction. The plasma prepared in this manner will be referred to as FVIIId(++) plasma hereinafter. Whole blood and serum can be prepared in the same manner as above. Preparation of beads and detection antibody solution are carried out by the methods already described. [Sample preparation] Prepare FVIIId(++) plasma samples spiked with Q4 / / J3 antibody at concentrations ranging from 0.0488 to 50 μg / mL (0.326 to 333 nM) with a common ratio of 4 (six samples total), Q3 / / J1 antibody at concentrations ranging from 1.38 to 354 μg / mL (9.20 to 2360 nM) with a common ratio of 2 (nine samples total), and emicuzumab at concentrations ranging from 6.25 to 400 μg / mL (41.7 to 2667 nM) with a common ratio of 2 (seven samples total). Use FVIIId(++) plasma without anti-FIX / FX bispecific antibody as a blank sample. After sample preparation, incubate at room temperature for 1 hour before measurement to allow complex formation to reach equilibrium. Measurement, simulation and analysis are carried out by the methods already described.

[0102] In one embodiment, at least one of the components constituting the complex contained in the sample is a drug that can be used for treatment, and a method for determining a treatment method based on the concentration and / or amount of the complex in the sample is provided. In certain embodiments, determining a treatment regimen is determining the dosage of a drug, determining the frequency of administration.

[0103] In one embodiment, at least one of the components constituting the complex contained in the sample is a drug that can be used for treatment, and the treatment is determined by detecting the complex. In another embodiment, treatment decisions are made based on the signal value obtained by detecting the complex. In another embodiment, the treatment is determined based on a normalized signal value calculated based on the signal value obtained by detecting the complex. In another embodiment, the treatment is determined based on an arbitrary unit calculated based on the signal value obtained by detecting the complex.

[0104] Specific embodiments of the present invention are described below based on emicizumab. Emicizumab is a bispecific antibody that recognizes FIX(a) and FX and is used to treat hemophilia A. When emicizumab is administered to a patient, it binds to FIX(a) and / or FX in the blood, forming a binary complex of emicizumab and FIX(a), a binary complex of emicizumab and FX, and a ternary complex of emicizumab, FIX(a), and FX. The mechanism of action of emicizumab is that emicizumab binds to FIX(a) and FX, bringing them into physical proximity, thereby promoting the conversion of FX to FXa by FIXa. Therefore, the formation of the ternary complex is necessary for emicizumab to exert its therapeutic effect. Therefore, detecting the ternary complex and determining its concentration and amount are important for determining treatment methods, particularly for determining dose and dosing frequency.

[0105] Furthermore, when a bispecific antibody is used in combination with a coagulation factor concentrate containing FIX(a) and FX, such as FEIBA, the amount of the ternary complex may potentially increase, resulting in an enhanced pharmacological effect. Measurement of the ternary complex may be useful for assessing the enhanced pharmacological effect.

[0106] In one specific embodiment of the present invention, treatment decisions can be made based on the concentration or amount of the ternary complex. First, known concentrations of the bispecific antibody are spiked into plasma containing known concentrations of FIX and FX to prepare a calibration curve sample. The signal values ​​obtained by measuring the ternary complex and the simulated concentrations are used to calculate a regression equation. Plasma is prepared by centrifugation from a blood sample collected after administration of the bispecific antibody, and the ternary complex is detected. The signal values ​​obtained are used to calculate the concentration converted using the regression equation. Measurement of samples over time after administration of the bispecific antibody allows for the determination of the concentration of the ternary complex over time. The simulated concentration is the equilibrium concentration of the ternary complex, calculated using the antibody concentration, FIX concentration, FX concentration, and the dissociation constants for FIX and FX, assuming independent binding. Methods for calculating the regression equation include, but are not limited to, linear regression and nonlinear regression.

[0107] In another specific embodiment of the present invention, a treatment can be determined by evaluating the amount of the ternary complex based on the signal value obtained by detecting the complex. Plasma is prepared by centrifugation from a blood sample collected after administration of the bispecific antibody, and the signal value is obtained by measuring the ternary complex. By measuring the sample over time after administration of the bispecific antibody, the time course of the signal value of the ternary complex can be obtained.

[0108] In another specific embodiment of the present invention, a treatment can be determined by evaluating the amount of the ternary complex based on a standardized signal value. For example, a standardization sample can be prepared by spiking a bispecific antibody at a known concentration into pooled plasma, measuring the ternary complex, and using the signal value obtained as the standardization signal value. Plasma is prepared by centrifugation from a blood sample collected after administration of the bispecific antibody, and the signal value obtained by measuring the ternary complex is divided by the standardization signal value to obtain a standardized signal value. Time-dependent sample measurements after administration of the bispecific antibody can provide a time course of the standardized signal value of the ternary complex. Here, pooled plasma refers to, for example, plasma obtained by mixing human plasma from multiple individuals, dispensing, and freezing the mixture, or plasma obtained by dispensing and freezing human plasma spiked with a bispecific antibody at a known concentration.

[0109] In another specific embodiment of the present invention, a treatment can be determined by evaluating the amount of the ternary complex based on an arbitrary unit. First, the correspondence between the standardized signal value obtained by the above method and the arbitrary unit (AU) of the amount of the ternary complex is determined. Plasma is prepared by centrifugation from a blood sample collected after administration of the bispecific antibody, and the signal value obtained by measuring the ternary complex is divided by the standardization signal value to obtain a standardized signal value. The standardized signal value is then converted to AU. By measuring samples over time after administration of the bispecific antibody, the time course of the AU of the ternary complex can be obtained.

[0110] In one embodiment, at least one of the components constituting the complex contained in the sample is a drug that can be used for treatment, and a method for evaluating the kinetics of the drug based on the concentration and / or amount of the complex in the sample is provided.

[0111] In a specific embodiment, a method for evaluating the pharmacokinetics of a complex containing a bispecific antibody is provided. The pharmacokinetic properties of a bispecific antibody can potentially differ between the antibody alone, when it binds to one antigen to form a binary complex, and when it binds to two antigens to form a ternary complex, depending on the properties of the antigen itself, as well as structural and physicochemical changes that occur when the complex is formed. Therefore, measuring the ternary complex allows for evaluation of the antigen-dependent pharmacokinetic properties of the bispecific antibody, and also allows for highly accurate pharmacokinetic prediction using modeling and simulation.

[0112] In one embodiment, at least one of the components constituting the complex contained in the sample is a drug that can be used for treatment, and a method is provided for evaluating the efficacy and safety of the drug based on the concentration and / or amount of the complex in the sample. In certain embodiments, evaluation of drug efficacy and safety is performed in the same manner as the treatment decisions described above. [Example]

[0113] Example 1: Examination of detection of ternary complex To specifically detect the ternary complex formed by the binding of an anti-FIX / FX bispecific antibody to FIX and FX, it is necessary to recognize only the ternary complex, distinguishing it from binary complexes formed by antibody and FIX, antibody and FX, uncomplexed (free) antibody, free FIX, and free FX (Figure 1). Therefore, we devised a method to specifically detect the ternary complex by sandwiching the complex between two antibodies, one of which is immobilized and the other is fluorescently labeled (Figure 4(A, B)). Since the ternary complex in solution is thought to be in equilibrium between binding and dissociation, we considered that incubating the sample with the solid phase for a long time, such as 1 hour, as in conventional ELISA, would shift the equilibrium and result in an inaccurate measurement of the state of the complex in solution. Furthermore, because the affinity between the antigen and antibody in the complex is not sufficiently high, the complex easily dissociates during long incubations with the detection antibody solution or multiple washes with large amounts of buffer, as in conventional ELISA. Therefore, we adopted the KinExA (Sapidyne) measurement device, which allows for a sufficiently short reaction time between the sample and the antibody on the solid phase and rapid washing with a small amount of buffer. In Example 11, we also adopted the Gyrolab, which we believed could achieve measurements under similar conditions. Example 2: Selection of anti-FIX and anti-FX antibodies In this example, to select anti-FIX and anti-FX antibodies suitable for implementing the assay, the bispecific antibodies were immobilized on beads, and candidate anti-FIX and anti-FX antibodies were fluorescently labeled and used as detection antibodies. Using a KinExA 3200 (Sapidyne), we confirmed whether hFIX and hFX could be detected in a concentration-dependent manner by a sandwich assay. We also examined whether hFIXa and hFXa, the activated forms of each coagulation factor, could be detected in a similar manner.

[0114] [Preparation of beads] A 20 μg / mL solution of anti-FIX / FX bispecific antibody (Q4 / / J3) was prepared in phosphate-buffered saline (pH 7.4, Sigma-Aldrich). One mL of this solution was added to each PMMA bead (Sapidyne) and incubated at room temperature for 2 hours to immobilize the antibody on the beads. After centrifuging at low speed for a few seconds to remove the supernatant, blocking buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, 1% BSA, pH 7.4) was added and the beads were blocked by incubating at room temperature for 1 hour.

[0115] [Preparation of detection antibody solution] Fluorescently labeled anti-FIX antibody (in-house preparation: XB12) and anti-FX antibody (in-house preparation: SB04) solutions (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences, according to the manufacturer's protocol) were diluted to 0.5 μg / mL in assay buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, 0.1% BSA, pH 7.4). To remove aggregates of the fluorescently labeled antibody, the solution was first diluted to 5 μg / mL, then centrifuged at 18,000 xg for 10 minutes, and the supernatant was further diluted to 0.5 μg / mL.

[0116] [Sample preparation] Next, serial dilution samples of hFIX, hFIXa, hFX, and hFXa were prepared. hFIX (Enzyme Research Laboratories) was available at 10, 5, 2.5, 1.25, and 0 μg / mL (179, 89.3, 44.6, 22.3, and 0 nM), hFIXa (Enzyme Research Laboratories) was available at 8, 4, 2, 1, and 0 μg / mL (178, 88.9, 44.4, 22.2, and 0 nM), hFX (Enzyme Research Laboratories) was available at 16, 8, 4, 2, and 0 μg / mL (272, 136, 68.0, 34.0, and 0 nM), and hFXa (Enzyme Research Laboratories) was available at 12.5, 6.25, 3.13, 1.56, and 0 μg / mL (272, 136, 68.0, The antibody was diluted to 34.0, 0 nM with assay buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, 0.1% BSA, pH 7.4).

[0117] [Measurement of FIX (FIXa)] A KinExA 3200 (Sapidyne) was used as the measurement device. Two tubes of the prepared bead suspension were transferred to a bead bottle (Sapidyne) filled with 24 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the autosampler of the KinExA 3200. All measurement processes were performed automatically using the KinExA 3200 and autosampler. Protocols to control the instrument were created using KinExA Pro software (Sapidyne). The protocols were created to specify the appropriate reagent and sample locations for each measurement process, ensuring that the appropriate reagents were sampled at the appropriate times (as in the following examples). First, beads were packed into a column in a flow cell. The volume of the bead suspension applied was monitored with a camera and optimized to ensure that the beads were packed to the appropriate height with consistent reproducibility. Next, a dilution series of hFIX (hFIXa) samples was passed through the column, allowing hFIX (hFIXa) to be captured by the antibody on the beads. After the first wash step with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled anti-FIX antibody solution was passed through the column, allowing the fluorescently labeled anti-FIX antibody to bind to the captured hFIX (hFIXa) (Figure 2 (A)). After the second and third wash steps with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volume, time, and flow rate of the sample, running buffer (+) in the first washing step, detection antibody solution, running buffer (+) in the second washing step, and running buffer (+) in the third washing step were varied to obtain a sufficient signal-to-noise ratio. The measurement conditions were as follows: Sample: 20 μL, 4.8 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Each sample was measured in duplicate.

[0118] [FX (FXa) measurement] A KinExA 3200 (Sapidyne) was used as the measurement device. Two tubes of the prepared bead suspension were transferred to a bead bottle (Sapidyne) filled with 24 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. First, beads were packed into a column in a flow cell. The volume of the bead suspension applied was monitored with a camera and optimized to ensure that the beads were packed to the appropriate height with consistent reproducibility. Next, a sample containing hFX (hFXa) was passed through the column, allowing hFX (hFXa) to be captured by the antibody on the beads. After the first wash step with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled anti-FX antibody solution was passed through the column, allowing the fluorescently labeled anti-FX antibody to bind to the captured hFX (hFXa) (Figure 2(B)). After the second and third wash steps with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volume, time, and flow rate of the sample, running buffer (+) in the first washing step, detection antibody solution, running buffer (+) in the second washing step, and running buffer (+) in the third washing step were varied to obtain a sufficient signal-to-noise ratio. The measurement conditions were as follows: Sample: 10 μL, 2.4 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 400 μL, 96 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Each sample was measured in duplicate. The anti-FIX antibody (XB12) and the anti-FX antibody (SB04) were prepared with reference to a patent document (WO 2005 / 35756).

[0119] [Results and antibody selection] In the measurement of FIX (FIXa), signal intensity was obtained in a concentration-dependent manner with hFIX. Therefore, XB12 was selected as an anti-FIX antibody that recognizes a different epitope from the anti-FIX / FX bispecific antibody and was therefore selected for the complex assay of the present invention (Figure 3(A)). Similarly, in the measurement of FX (FXa), signal intensity was obtained in a concentration-dependent manner with hFX, so SB04 was selected as the anti-FX antibody (Figure 3(B)). Furthermore, XB12 detected hFIX and hFIXa with similar signal intensity, suggesting that their binding affinity to each antibody is not significantly different. On the other hand, SB04 did not detect a concentration-dependent signal intensity with hFXa. Therefore, using these antibodies, the detectable ternary complex was considered to be formed by FIX or FIXa, the anti-FIX / FX bispecific antibody, and FX.

[0120] Example 3: Evaluation of affinity of anti-FIX / FX bispecific antibodies Kinetic analysis of antigen binding of the anti-FIX / FX bispecific antibodies Q4 / / J3 and Q3 / / J1 was performed using a Biacore T200 (GE Healthcare) in a buffer containing 10 mM HEPES, 150 mM NaCl, 0.05% Surfactant P20, and 2.5 mM CaCl2, pH 7.4. The Q4 / / J3 or Q3 / / J1 antibodies were captured on a Series S Sensor Chip CM4 (GE Healthcare) with immobilized sure Protein A (GE Healthcare) by amine coupling. Human FIX (Enzyme Research Laboratories) at concentrations of 8–128 nM or human FX (Enzyme Research Laboratories) at concentrations of 10–160 nM was injected as an analyte onto the captured Q4 / / J3 antibody. For the Q3 / / J1 antibody, 80-1280 nM human FIX or 10-160 nM human FX was injected. All measurements were performed at 25°C. The association rate constant ka (1 / Ms) and dissociation rate constant kd (1 / s) were calculated using 1:1 binding model fitting using Biacore Evaluation Software, and the dissociation constant KD (M) was calculated from these values ​​(Table 1).

[0121] [Table 1] The affinity of emicizumab for FIX and FX was determined based on literature values ​​(Kitazawa, Thromb Haemost 117(7):1348-1357(2017)). The Q4 / / J3 antibody, Q3 / / J1 antibody, and emicizumab were obtained according to the methods described in WO2005 / 035756, WO2006 / 109592, and WO2012 / 067176.

[0122] <Example 4> Verification of the specificity of complex measurement The anti-FIX antibody (XB12) and the anti-FX antibody (SB04), which recognize different epitopes from the anti-FIX / FX bispecific antibody selected in Example 2, were used for capture on beads and detection with fluorescent labels, or vice versa. We verified whether the ternary complex could be specifically detected in two measurement formats, 1 and 2 (Figure 4(A, B)).

[0123] [Preparation of beads] XB12 and SB04 were immobilized on PMMA beads (Sapidyne) and blocked using the method described in Example 2.

[0124] [Preparation of detection antibody solution] Solutions of fluorescently labeled (Lightning-Link® Rapid Dylight® 650, Innova Biosciences, Inc., prepared according to the manufacturer's protocol) anti-FIX antibody (in-house preparation: XB12) and anti-FX antibody (in-house preparation: SB04) were prepared according to Example 2.

[0125] [Sample preparation] Samples spiked with hFIX, hFX, and the bispecific antibody (Q4 / / J3) at concentrations of 5, 8, and 12.5 μg / mL (89.3, 136, and 83.3 nM), respectively, and samples lacking one or both of the three components were prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). The following samples were prepared: a sample spiked with all three components (Sample No. 1), a sample spiked with bispecific antibody and hFIX (Sample No. 2), a sample spiked with bispecific antibody and hFX (Sample No. 3), a sample spiked with hFIX and hFX (Sample No. 4), a sample spiked with bispecific antibody only (Sample No. 5), a sample spiked with hFIX only (Sample No. 6), a sample spiked with hFX only (Sample No. 7), and a blank sample (Sample No. 8) (Table 2). The concentrations of hFIX and hFX were set to physiological concentrations (plasma concentrations). After sample preparation, the samples were incubated at room temperature for 1 hour to allow complex formation to reach equilibrium before measurement.

[0126] [Table 2]

[0127] 〔measurement〕 A KinExA 3200 (Sapidyne) was used as the measurement instrument. One tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. In Format 1, XB12-coated beads were first loaded into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were reproducibly packed to the appropriate height. Next, the prepared sample was passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex formed by the bispecific antibody binding to both hFIX and hFX. After the first wash with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution was passed through the column, allowing the fluorescently labeled SB04 antibody to bind to the captured hFX in the ternary complex. After the second and third washes with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement (Figure 5 (A)). In Format 2, SB04-immobilized beads were first packed into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were packed to the appropriate height with good reproducibility. Next, the sample was passed through the column, allowing the beads to capture free hFX, the bispecific antibody-hFX bicomplex, and the bispecific antibody-hFX-hFX ternary complex. After the first wash with running buffer (+), a fluorescently labeled XB12 solution was passed through the column, allowing the fluorescently labeled XB12 antibody to bind to the hFIX in the captured ternary complex. After the second and third washes with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement (Figure 5 (B)).The volume, time, and flow rate of the sample, running buffer (+) in the first washing step, detection antibody solution, running buffer (+) in the second washing step, and running buffer (+) in the third washing step were varied to obtain a sufficient signal-to-noise ratio. The measurement conditions were as follows: Sample: 50 μL, 12 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Each sample was measured in duplicate.

[0128] 〔result〕 In both formats, high signal values ​​were obtained only in the sample containing all three components (Sample No. 1), while samples lacking any one component showed lower signal values. Therefore, both formats demonstrated the detection of the ternary complex. On the other hand, in Format 2, for samples other than Sample No. 1, high signal values ​​were observed in the sample spiked with bispecific antibody and hFIX (Sample No. 2), the sample spiked with hFIX and hFX (Sample No. 4), and the sample spiked with hFIX alone (Sample No. 6), suggesting that the signal may have been detected by hFIX. In Format 1, except for Sample No. 1, the sample spiked with hFIX and hFX (Sample No. 4) tended to have higher signal values ​​than the blank sample (Sample No. 8) (Figure 5 (A, B)). Therefore, in subsequent studies, it was considered appropriate to use Format 1 and consider the difference between the signal value of a sample containing the three components and the signal value of a sample containing only FIX and FX as the signal value derived from the ternary complex.

[0129] Example 5: Simulation of ternary complex formation The dissociation constant K D This was investigated through simulations using

[0130] 〔method〕 The concentration of the ternary complex was simulated using a method described in a literature review (Thromb Haemost, 117(7), 1348-1357, 2017). Calculations were performed using Microsoft Excel 2013. In this simulation, the FIX concentration, FX concentration, bispecific antibody concentration, and the K values ​​of the bispecific antibody for FIX and FX were used. D Because it is necessary to define the K values ​​for each, the concentrations of FIX and FX were set constant at physiological concentrations (plasma concentrations) of 5 and 8 μg / mL (89.3 and 136 nM), respectively, and the anti-FIX / FX bispecific antibody concentration was varied in two-fold increments over the range of 0.0122–1600 μg / mL (0.0814–10667 nM). D For values, the condition for both to be equal and the condition for K for FIX D Value of K for FX D K for FIX under the condition that the value is doubled D The concentration of the ternary complex was varied in increments of 2-fold in the range of 5-2560 nM. The ternary complex concentration under each condition was simulated, and each value was plotted with the ternary complex concentration on the Y-axis and the bispecific antibody concentration on the X-axis.

[0131] 〔result〕 K for FIX, FX D In simulations where the values ​​are equal, the relationship between the simulated bispecific antibody concentration and the ternary complex concentration takes the form of a bell, and the shape is D The bispecific antibody concentration at which the ternary complex concentration peaked varied according to the K values ​​for FIX and FX. D The lower the value, the lower the concentration shift.D Within the range of 5-40 nM, the peak bispecific antibody concentrations were unchanged at 83.3-167 nM. D The ternary complex concentration at which the value changes is K D The smaller the value, the higher the increase. D The smaller the value, the smaller the rate of increase (Fig. 6(A)). D Value of K for FX D The above tendency was also observed in the simulation where the value was doubled (Figure 6(B)).

[0132] Example 6: Comparison of measured and simulated values From the simulation results of Example 5, it is found that the ATP has a relatively high affinity (K D For bispecific antibodies, the K D The change in K value had little effect on the concentration of the ternary complex. Therefore, for high-affinity bispecific antibodies, the K D Even if there was a measurement error in the values, it would not have a significant effect on the simulation of the ternary complex concentration, and so it was thought that the measured and simulated values ​​would likely agree. Therefore, using the Q4 / / J3 antibody, which has a relatively high affinity, we compared the measured ternary complex signal values ​​with the simulated values.

[0133] [Preparation of beads] XB12 was immobilized on PMMA beads (Sapidyne) and blocked using the method described in Example 2.

[0134] [Preparation of detection antibody solution] Fluorescently labeled (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences according to the manufacturer's protocol) anti-FX antibody (in-house preparation: SB04) was prepared according to Example 2.

[0135] [Sample preparation] hFIX and hFX were kept constant at 5 and 8 μg / mL (89.3 and 136 nM), respectively. The Q4 / / J3 antibody was spiked at 11 concentrations between 0.195 and 200 μg / mL (1.30 and 1333 nM) with a common ratio of 2. The samples were prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA pH 7.4). A blank sample was prepared containing only hFIX and hFX, without the Q4 / / J3 antibody. The concentrations of hFIX and hFX were set to physiological plasma concentrations. After sample preparation, the samples were incubated at room temperature for 1 hour to allow complex formation to reach equilibrium before measurement.

[0136] 〔measurement〕 A KinExA 3200 (Sapidyne) was used as the measurement instrument. One tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. First, XB12-coated beads were loaded into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were loaded to the appropriate height with good reproducibility. Next, the sample was passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex formed by the bispecific antibody binding to both hFIX and hFX. After the first wash step with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution was passed through the column, allowing the fluorescently labeled SB04 antibody to bind to hFX in the captured ternary complex. After the second and third wash steps with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volumes, times, and flow rates of the sample, running buffer (+) in the first wash step, detection antibody solution, running buffer (+) in the second wash step, and running buffer (+) in the third wash step were varied to ensure a sufficient signal-to-noise ratio. The measurement conditions are shown below. Sample: 50 μL, 12 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min.The blank sample was measured at n=5, and the other samples were measured at n=1.

[0137] 〔analysis〕 The mean and standard deviation (SD) of the signal values ​​of blank samples measured in n = 5 were calculated (Microsoft Excel 2013), and the detection limit was set as Mean + 3.29 × SD. For the other samples whose signal values ​​were above the detection limit, the mean signal value of the blank sample was subtracted to calculate the signal value of the ternary complex. At the same time, the concentrations of hFIX, hFX, and Q4 / / J3 antibodies in the prepared samples and K D From the values ​​(see Example 3), the ternary complex concentration in each sample was simulated (see Example 5). To compare the obtained ternary complex signal values ​​with the simulated values, a plot was created with these values ​​on the left and right Y axes (signal values ​​are black circles, simulation values ​​are dotted lines) and the Q4 / / J3 antibody concentration on the X axis (Figure 7 (A)). In addition, the obtained results were plotted with the ternary complex signal values ​​on the Y axis and the simulation values ​​on the X axis, and a correlation analysis was performed using Microsoft Excel 2013. In the correlation analysis, a linear approximation curve was drawn and R 2 The values ​​were calculated (Figure 7(B)).

[0138] 〔result〕 The ternary complex signal value took on a bell-shaped shape depending on the concentration of the Q4 / / J3 antibody, which was consistent with the shape predicted by the simulation. Furthermore, a linear relationship was obtained in Figure 7(B), where the X-axis represents the simulated value and the Y-axis represents the ternary complex signal value. These results indicate that the actual measured values ​​of the ternary complex formed by the Q4 / / J3 antibody can be explained by the simulation. Therefore, assuming that the simulation is correct, by obtaining a correspondence between the ternary complex signal values ​​and the simulated values ​​for three or more samples prepared with varying concentrations of the Q4 / / J3 antibody, it is possible to calculate the ternary complex concentration by regression from the obtained ternary complex signal values ​​when measuring a sample containing an unknown concentration of bispecific antibody.

[0139] <Example 7> Evaluation of ternary complexes with antibodies of different affinities The concentrations of the ternary complexes formed by three anti-FIX / FX bispecific antibodies with different affinities (Q4 / / J3, Q3 / / J1, and emicizumab) were compared.

[0140] [Preparation of beads] Prepared as described in Example 6.

[0141] [Preparation of detection antibody solution] Fluorescently labeled (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences according to the manufacturer's protocol) anti-FX antibody (in-house preparation: SB04) was prepared according to Example 2.

[0142] [Sample preparation] hFIX and hFX were fixed at 5 and 8 μg / mL (89.3 and 136 nM), respectively. The Q4 / / J3 antibody was spiked at six concentrations (0.0488–50 μg / mL, 0.326–333 nM) with a common ratio of 4. The Q3 / / J1 antibody was spiked at nine concentrations (1.38–354 μg / mL, 9.20–2360 nM) with a common ratio of 2. Emicuzumab was spiked at seven concentrations (6.25–400 μg / mL, 41.7–2667 nM) with a common ratio of 2. The assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4) was used. A blank sample was prepared by spiking only hFIX and hFX without the anti-FIX / FX bispecific antibody. The concentrations of hFIX and hFX were set at physiological concentrations (plasma concentrations). After sample preparation, the samples were incubated at room temperature for 1 hour before measurement to allow complex formation to reach equilibrium.

[0143] 〔measurement〕 A KinExA 3200 (Sapidyne) was used as the measurement instrument. One tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. First, XB12-coated beads were loaded into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were loaded to the appropriate height with good reproducibility. Next, the sample was passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex formed by the bispecific antibody binding to both hFIX and hFX. After the first wash step with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution was passed through the column, allowing the fluorescently labeled SB04 antibody to bind to hFX in the captured ternary complex. After the second and third wash steps with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volumes, times, and flow rates of the sample, running buffer (+) in the first wash step, detection antibody solution, running buffer (+) in the second wash step, and running buffer (+) in the third wash step were varied to ensure a sufficient signal-to-noise ratio. The measurement conditions are shown below. Sample: 135 μL, 32.4 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min.The blank sample was measured at n=5, and the other samples were measured at n=1.

[0144] 〔analysis〕 The mean (Mean) and standard deviation (SD) of the signal values ​​of blank samples measured at n=5 were calculated (Microsoft Excel 2013), and the detection limit was set as Mean + 3.29 × SD. For the other samples with signal values ​​above the detection limit, the Mean signal value of the blank sample was subtracted to calculate the ternary complex signal value. The ternary complex signal value was plotted on the Y-axis and the anti-FIX / FX bispecific antibody concentration on the X-axis (Figure 8(A)). Simultaneously, the ternary complex concentration in the sample spiked with the Q4 / / J3 antibody was simulated (see Example 6). The obtained results were plotted with the ternary complex signal value of the sample spiked with the Q4 / / J3 antibody on the Y-axis and the simulated value on the X-axis. A linear regression was performed using Microsoft Excel 2013 to obtain the regression equation and R 2 The signal values ​​of the ternary complex of the samples spiked with Q3 / / J1 antibody and emicizumab were then converted to concentrations using a regression equation. The converted concentrations were plotted on the Y-axis against the anti-FIX / FX bispecific antibody concentration on the X-axis (Figure 8(C)).

[0145] 〔result〕 The relationship between the signal intensity of the ternary complex and the concentration of the anti-FIX / FX bispecific antibody showed a bell-shaped pattern, with the signal intensity reaching a maximum at a certain concentration, for all three antibodies with different affinities. Furthermore, the intensity of the signal intensity of the ternary complex varied depending on the affinity of the antibody, with the highest affinity (K DThe smaller the value, the lower the antibody concentration at which the signal was observed, and the higher the maximum signal intensity. The ternary complex concentration formed by the Q3 / / J1 antibody reached a maximum of 20.4 nM at an antibody concentration of 44.3 μg / mL (295 nM) (Figure 8(C)). At this point, 6.90%, 22.8%, and 15.0% of the antibody, hFIX, and hFX in the sample formed ternary complexes, respectively. The ternary complex concentration formed by emicizumab reached a maximum of 10.4 nM at an antibody concentration of 100 μg / mL (667 nM) (Figure 8(C)). At this point, 1.56%, 11.6%, and 7.65% of the antibody, hFIX, and hFX in the sample formed ternary complexes, respectively. Therefore, it was confirmed that the proportion of ternary complexes formed by both antibodies was low relative to each component.

[0146] Example 8: Examination of the effect of complex dissociation during the assay In the process of measuring ternary complexes formed by anti-FIX / FX bispecific antibodies with different affinities, the degree of dissociation of the ternary complex during washing after the complex is captured by beads may differ depending on the affinity. Therefore, we evaluated the effect of ternary complex dissociation on signal values ​​by examining whether signal value behavior during washing differs between anti-FIX / FX bispecific antibodies with different affinities.

[0147] 〔analysis〕 In the measurements performed in Example 7, the signal values ​​of the ternary complex were similar in each sample spiked with 3.13 μg / mL (20.8 nM) of Q4 / / J3 antibody, 11.1 μg / mL (74.0 nM) of Q3 / / J1 antibody, and 100 μg / mL (667 nM) of emicizumab. This suggests that ternary complexes with different affinities were captured and detected to the same extent on the beads in these sample measurements. Therefore, the behavior of signal values ​​during washing in these sample measurements was compared to compare the degree of dissociation of the ternary complex. To compare the behavior of signal values ​​during washing, the signal values ​​during the measurement process for each sample were output using KinExA Pro software and plotted on a graph with signal value on the Y axis and time on the X axis. The differences between each of the three types of samples were simultaneously calculated and plotted in the same manner (Figure 9 (AC)). In Figure 9, the KinExA signal remains constant for approximately 250 seconds from the start of measurement due to the bead loading, sample capture, and washing steps. Thereafter, the signal increases significantly as the fluorescently labeled detection antibody passes through the column. The signal then decreases as the washing step begins, ultimately capturing the signal value as the increase from baseline. If dissociation of the ternary complex occurs during the washing step, dissociation of the complex captured on the beads may be observed as a decrease in signal value at the same time as the free detection antibody is washed away during the detection antibody washing step. Therefore, differences in dissociation between complexes with different affinities should be observed as a time-dependent change in the difference in signal value between samples.

[0148] 〔result〕 Comparison of all samples showed similar signal transitions, and the signal difference between samples during the detection antibody washing step did not vary. These results suggest that the degree of dissociation of the ternary complex during measurement between anti-FIX / FX bispecific antibodies with different affinities is not significant enough to affect the signal value.

[0149] <Example 9> Measurement of time-dependent complex formation When measuring the ternary complex formed by the anti-FIX / FX bispecific antibody, it is possible that FIX, FX, and the anti-FIX / FX bispecific antibody, which were not present in the sample, may form new complexes on the beads during the process of capturing the complex on beads. Therefore, we evaluated time-dependent signal changes by adding the anti-FIX / FX bispecific antibody to a sample containing hFIX and hFX and detecting the signal value of the ternary complex over time at multiple time points after mixing. If the signal value increases over time after mixing, it can be confirmed that the signal value reflects the complex formation that occurred in the solution. Furthermore, by determining the time point at which the ternary complex signal value becomes constant, the time until equilibrium is reached can be determined. The Q3 / / J1 antibody and emicizumab (see Example 7) were used.

[0150] [Preparation of beads] Prepared as described in Example 6.

[0151] [Preparation of detection antibody solution] Fluorescently labeled (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences according to the manufacturer's protocol) anti-FX antibody (in-house preparation: SB04) was prepared according to Example 2.

[0152] [Sample preparation] First, hFIX and hFX were spiked into assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4) at 5 and 8 μg / mL (89.3 and 136 nM), respectively, to prepare samples. The Q3 / / J1 antibody was added to the sample immediately before measurement to a final concentration of 5.54 μg / mL (36.9 nM). Emicizumab was added to the sample immediately before measurement to a final concentration of 100 μg / mL (667 nM). A blank sample was prepared by spiking only hFIX and hFX without the anti-FIX / FX bispecific antibody.

[0153] 〔measurement〕 A KinExA 3200 (Sapidyne) was used as the measurement instrument. One tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. First, XB12-coated beads were loaded into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were loaded to the appropriate height with good reproducibility. Next, the sample was passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex formed by the bispecific antibody binding to both hFIX and hFX. After the first wash step with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution was passed through the column, allowing the fluorescently labeled SB04 antibody to bind to hFX in the captured ternary complex. After the second and third wash steps with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volumes, times, and flow rates of the sample, running buffer (+) in the first wash step, detection antibody solution, running buffer (+) in the second wash step, and running buffer (+) in the third wash step were varied to ensure a sufficient signal-to-noise ratio. The measurement conditions are shown below. Sample: 135 μL, 32.4 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Of the prepared samples, the sample that had been mixed with Q3 / / J1 antibody immediately before measurement was measured in one run, while the sample that had been mixed with emicizumab was measured in a separate run. Using the KinExA Pro software (Sapidyne)'s built-in Kinetics mode, the mixed sample was repeatedly measured over time, and the KinExA signal and the elapsed time from mixing to measurement were recorded at each time point. Repeated measurements were performed at approximately 12-minute intervals, yielding a total of 10 data points. Blank samples were also measured (n=5).

[0154] 〔analysis〕 The signal value of the ternary complex was calculated by subtracting the average signal value of the blank sample from each KinExA signal value. The measured values ​​were plotted with the time elapsed from antibody addition and mixing to measurement on the Y axis and the signal value of the ternary complex on the X axis (Figure 10).

[0155] 〔result〕 For both the Q3 / / J1 antibody and emicizumab, signal values ​​increased in a time-dependent manner and reached a plateau approximately 60 minutes after mixing, suggesting that equilibrium had been reached at this point. Furthermore, as the sample passed through the beads, new binding of FIX to the bispecific antibody and bispecific antibody to FX occurred on the beads, potentially contributing to the formation of new ternary complexes. In this case, complex signal values ​​could be obtained even at 0 minutes after mixing. Due to the nature of the measurement instrument, it is not possible to obtain signal values ​​at 0 minutes after mixing. Considering that the signal value at the first measurement time point, approximately 0.5 minutes, was approximately 30% of the value at equilibrium, and the signal increase was greatest immediately after mixing, the contribution of complex formation on the beads is unlikely. Therefore, the majority of the measured ternary complex signal values ​​were likely derived from the complex formed in solution, which was the subject of interest in this measurement.

[0156] Example 10: Evaluation of the ternary complex using Gyrolab The ternary complex measurements performed in other examples using KinExA (Sapidyne) were also performed using Gyrolab (Gyros Protein Technologies). Gyrolab is a fully automated ligand-binding assay system, and measurements are performed using a dedicated Bioaffy CD. The CD contains an affinity bead column. By passing the sample through the column in a short time, the analyte in the sample is specifically captured by antibodies immobilized on the beads. The captured analyte is then detected using a fluorescently labeled specific antibody. Gyrolab was selected as the measurement device because it has similar properties to KinExA, particularly in terms of capturing the analyte in the sample in a short time and automatically controlling the washing volume and time of the washing process. In fact, because the measurement principles of KinExA and Gyrolab are similar, they are used to calculate the dissociation constant of antigen-antibody reactions in solution by measuring free antibodies in samples (Anal Biochem. 15;426(2):134-41, 2012).

[0157] [Preparation of capture antibody solution] Since the Gyrolab CD is equipped with streptavidin beads, the XB12 used for capture was biotinylated using Sulfo-NHS-LC-Biotin (Thermo Scientific) (biotin-XB12). The protocol was based on the product's protocol. After biotinylation, free biotin was removed by desalting with Zeba Spin Desalting Columns (Thermo Scientific). A 100 μg / mL solution was prepared using buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). To remove aggregates, the column was centrifuged at 18,000 g for 10 minutes, and the supernatant was used.

[0158] [Preparation of detection antibody solution] Fluorescently labeled anti-FX antibody (SB04, in-house preparation) was prepared using the Alexa Fluor 647 Antibody Labeling Kit (Thermo Scientific) according to the manufacturer's protocol. A 3 μg / mL solution was prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). To remove aggregates of the fluorescently labeled antibody, the solution was centrifuged at 18,000 g for 10 minutes, and the supernatant was used.

[0159] [Sample preparation] hFIX and hFX were kept constant at 5 and 8 μg / mL (89.3 and 136 nM), respectively. Five concentrations of Q4 / / J3 antibody were spiked between 0.195 and 50 μg / mL (1.30 and 333 nM) with a common ratio of 4. These samples were prepared in assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4). A blank sample was prepared containing only hFIX and hFX, without the anti-FIX / FX bispecific antibody. The concentrations of hFIX and hFX were set to physiological plasma concentrations. After sample preparation, the samples were incubated at room temperature for 1 hour to allow complex formation to reach equilibrium before measurement.

[0160] 〔measurement〕 A Gyrolab xP workstation (Gyros Protein Technologies) was used as the measurement instrument, and a Bioaffy 200 was used as the CD for the measurement. All measurement processes were automated by the Gyrolab. The protocol controlling the instrument was the 200-3W-001 Wizard method in the Gyrolab Control software (Gyros Protein Technologies). The capture antibody, sample, and detection antibody were applied in this order, with wash steps between each step. The capture antibody and detection antibody concentrations were set to achieve a high signal-to-noise ratio. For the wash step, an assay buffer (0.01 M HEPES, 0.15 M NaCl, 1.2 mM CaCl2, 0.05% Surfactant P20, 0.02% NaN3, 0.1% BSA, pH 7.4) was used. The capture antibody solution, sample, detection antibody solution, and wash assay buffer were added to a dedicated PCR plate according to the plate design generated from the protocol, and the plate was then loaded into the instrument prior to measurement. In the measurement process, biotin-XB12 solution was first passed through the flow channel and captured on streptavidin beads. After washing, free hFIX, hFIX-bispecific antibody binary complexes, and hFIX, hFX, and bispecific antibody ternary complexes in the sample were captured by the biotin-XB12 beads. After washing, a fluorescently labeled SB04 solution was passed through the sample, and the fluorescently labeled SB04 antibody bound to hFX in the captured ternary complex. Detection was performed using a photomultiplier tube (PMT) at three fixed response amplification levels (1, 5, and 25%), and the measured values ​​at each setting were recorded. Of the prepared samples, five blank samples were measured, and triplicates of samples spiked with Q4 / / J3 antibody were measured.

[0161] 〔analysis〕 The response detected under the 1% PMT condition was sufficiently large and did not cause detector saturation, so it was used for analysis. The mean and standard deviation (SD) of the responses of the blank samples measured with n = 5 were calculated (Microsoft Excel 2013), and the detection limit was set as Mean + 3.29 × SD. For the responses of other samples that were above the detection limit, the mean of the blank sample response was subtracted to calculate the ternary complex response. The measured values ​​were plotted with the ternary complex response on the Y-axis and the anti-FIX / FX bispecific antibody concentration on the X-axis (Figure 11(A)). Simultaneously, the ternary complex concentration in the sample spiked with the Q4 / / J3 antibody was simulated (see Example 6). The obtained results were plotted with the ternary complex signal value of the sample spiked with the Q4 / / J3 antibody on the Y-axis and the simulated value on the X-axis. A linear regression was performed using Microsoft Excel 2013 to obtain the regression equation and R 2 The values ​​were calculated (Figure 11 (B)).

[0162] 〔result〕 The relationship between the ternary complex response and the Q4 / / J3 antibody concentration exhibited a bell-shaped pattern, with the response maximizing at a Q4 / / J3 antibody concentration of 83.3 nM (Figure 11(A)). Furthermore, a linear relationship was obtained in a plot where the X-axis represented the simulation values ​​and the Y-axis represented the ternary complex response (Figure 11(B)). These results were comparable to those obtained using KinExA in Examples 6 and 7, suggesting that Gyrolab can also measure the ternary complex in a similar manner to KinExA.

[0163] Example 11: Evaluation of ternary complexes with antibodies of different affinities in human plasma The concentrations of ternary complexes formed by three anti-FIX / FX bispecific antibodies (Q4 / / J3, Q3 / / J1, and emicizumab) with different affinities in human plasma with congenital Factor VIII deficiency were compared using a KinExA 3200 (Sapidyne) measuring device.

[0164] [Preparation of beads] Prepared as described in Example 6

[0165] [Preparation of detection antibody solution] Fluorescently labeled (prepared using Lightning-Link® Rapid Dylight® 650, Innova Biosciences according to the manufacturer's protocol) anti-FX antibody (in-house preparation: SB04) was prepared according to Example 2.

[0166] [Preparation of plasma with buffering and anticoagulant properties] Congenitally Factor VIII-deficient human plasma (George King Bio-Medical) was mixed with one-ninth equivalent of 1M HEPES buffer solution (pH 7.1-7.5, Nacalai Tesque), and then 10,000 units / 10 mL of heparin sodium (Mochida Pharmaceutical) was added to a final concentration of 10 units / mL. In this experiment, diluting the plasma as in a conventional ELISA would cause dissociation of the complex, making it impossible to measure the complex in plasma. However, using plasma directly would cause pH changes due to changes in the carbonate ion concentration in the plasma, which would not reflect physiological conditions. Therefore, to prevent this, a buffering effect was added using HEPES buffer solution. Heparin sodium was also added to further suppress the coagulation reaction. The plasma prepared in this manner is referred to as FVIIId(++) plasma below.

[0167] [Sample preparation] FVIIId(++) plasma samples were prepared by spiking the Q4 / / J3 antibody at concentrations ranging from 0.0488 to 50 μg / mL (0.326 to 333 nM) with a common ratio of 4 (six samples), the Q3 / / J1 antibody at concentrations ranging from 1.38 to 354 μg / mL (9.20 to 2360 nM) with a common ratio of 2 (nine samples), and emicuzumab at concentrations ranging from 6.25 to 400 μg / mL (41.7 to 2667 nM) with a common ratio of 2 (seven samples). FVIIId(++) plasma samples without anti-FIX / FX bispecific antibody spikes served as blank samples. After sample preparation, the samples were incubated at room temperature for 1 hour to allow complex formation to reach equilibrium before measurement.

[0168] 〔measurement〕 The KinExA 3200 (Sapidyne) was used as the measurement device. One tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. All measurement processes were performed automatically by the KinExA 3200 and autosampler. The protocol controlling the instrument was created using KinExA Pro software (Sapidyne). The protocol specified the appropriate reagent and sample positions for each measurement process, ensuring that the appropriate reagents were sampled at the appropriate times. First, the XB12-immobilized beads were packed into a column in the flow cell. The volume of the bead suspension applied was optimized by monitoring with a camera to ensure that the beads were packed to the appropriate height reproducibly. The sample was then passed through the column, capturing free hFIX, the bispecific antibody-hFIX complex, and the ternary complex of the bispecific antibody bound to both hFIX and hFX. After the first wash with running buffer (+) (0.01 M HEPES, 0.15 M NaCl, 0.05% Surfactant P20, 1.2 mM CaCl2, 0.02% NaN3, pH 7.4), a fluorescently labeled SB04 solution was passed through the column, allowing the fluorescently labeled SB04 antibody to bind to hFX in the captured ternary complex. After the second and third washes with running buffer (+), the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement. The volume, time, and flow rate of the sample, the running buffer (+) in the first washing step, the detection antibody solution, the running buffer (+) in the second washing step, and the running buffer (+) in the third washing step were all varied, and measurements were taken under conditions that provided a sufficient signal-to-noise ratio. The conditions used in this measurement are shown below.Sample: 135 μL, 32.4 s, 0.25 mL / min; Running buffer (+) in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 800 μL, 192 s, 0.25 mL / min; Running buffer (+) in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer (+) in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Blank samples were measured in n=5, and the other samples were measured in n=1.

[0169] 〔analysis〕 Analysis was performed using Microsoft Excel 2013 and GraphPad Prism (GraphPad Software). The mean (Mean) and standard deviation (SD) of the signal values ​​of blank samples measured in five sets were calculated, and the detection limit was set as Mean + 3.29 × SD. For the remaining samples whose signal values ​​were above the detection limit, the mean signal value of the blank sample was subtracted to calculate the ternary complex signal value. The measured values ​​were plotted with the ternary complex signal value on the Y-axis and the anti-FIX / FX bispecific antibody concentration on the X-axis (Figure 12(A)). Simultaneously, the ternary complex concentration in a sample spiked with the Q4 / / J3 antibody was simulated (see Example 6). The FIX and FX concentrations were assumed to be physiological concentrations of 5 and 8 μg / mL (89.3 and 136 nM), respectively. The results were then plotted with the ternary complex signal value on the Y-axis and the simulated value on the X-axis. The relationship y = a(x / (x + b)) was observed in the spiked antibody concentration range of 0.0488-12.5 μg / mL (0.326-83.3 nM). Therefore, nonlinear regression was performed on the plot in this range to obtain the regression equation and R 2 The signal values ​​of the ternary complex from the samples spiked with Q3 / / J1 antibody and emicizumab were converted to concentrations using the regression equation in Figure 12(B). The converted concentrations were plotted on the Y-axis against the anti-FIX / FX bispecific antibody concentration on the X-axis (Figure 12(C)).

[0170] 〔result〕 The intensity of the signal from the ternary complex varied depending on the affinity of the anti-FIX / FX bispecific antibody, indicating that the affinity was high (K D The smaller the value, the lower the signal value observed at the antibody concentration. For the Q4 / / J3 and Q3 / / J1 antibodies, signal saturation was observed within the concentration range of the prepared samples. For the Q4 / / J3 antibody, signal saturation occurred at antibody concentrations of 12.5-50 μg / mL (83.3-333 nM), while for the Q3 / / J1 antibody, signal saturation tended to occur at antibody concentrations of 177-354 μg / mL (1180-2360 nM). For emicizumab, signal saturation did not occur up to an antibody concentration of 400 μg / mL (2667 nM).

[0171] <Example 12> Verification of the specificity of measuring the complex formed by anti-IL-6R antibody and soluble IL-6R To detect the binary complex formed by the anti-IL-6R antibody (Tocilizumab) and soluble hIL-6R, we devised a method to specifically recognize the complex using an anti-IL-6R antibody that recognizes an epitope different from that recognized by Tocilizumab, and an anti-hIgG Fc antibody (Figure 13), and verified this specificity.

[0172] [Preparation of beads] In the same manner as described in Example 2, an anti-IL-6R antibody (Clone # 17506, R&D Systems) that recognizes an epitope different from that recognized by tocilizumab was immobilized on PMMA beads (Sapidyne) and blocked.

[0173] [Preparation of detection antibody solution] Fluorescent labeling (Alexa Fluor TM A solution of anti-hIgG Fc antibody (prepared using 647 Antibody Labeling Kit, Thermo Fisher Scientific, according to the manufacturer's protocol) was prepared in the same manner as in Example 2.

[0174] [Sample preparation] Samples spiked with tocilizumab and IL-6R at concentrations of 1 and 0.1 μg / mL, respectively, samples spiked with only one of the two, and blank samples were prepared in assay buffer (0.01 M phosphate-buffered saline, 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20, 0.02% NaN3, 0.1% BSA, pH 7.4) (Table 3). The hIL-6R concentration was set to a physiological concentration. After sample preparation, the samples were incubated at room temperature for 3 hours to allow complex formation to reach equilibrium before measurement.

[0175] [Table 3]

[0176] 〔measurement〕 To minimize shifts in the binding equilibrium of the binary complex, the contact time between the sample and the solid-phase antibody was short (less than 0.5 seconds). A single tube of the prepared bead suspension was transferred to a bead bottle (Sapidyne) filled with 25 mL of running buffer (0.01 M phosphate-buffered saline, 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20, 0.02% NaN3, pH 7.4). The bead bottle, sample, and detection antibody solution were placed in each position in the KinExA 3200 autosampler. In the measurement, beads bearing an immobilized anti-IL-6R antibody that recognizes a different epitope from that recognized by tocilizumab were first loaded into a column in a flow cell. The prepared sample was then passed through the column, capturing the free hIL-6R and the hIL-6R-tocilizumab binary complex on the beads. After the first wash step with running buffer, a fluorescently labeled anti-hIgG Fc antibody solution was passed through and bound to the tocilizumab in the binary complex captured on the beads. After the second and third wash steps with running buffer, the KinExA signal was detected as an increase in fluorescence intensity from the baseline at the start of the measurement (Figure 14). The volume, time, and flow rate of the sample, running buffer in the first wash step, detection antibody solution, running buffer in the second wash step, and running buffer in the third wash step were all varied to ensure a sufficient signal-to-noise ratio. The measurement conditions are listed below. Sample: 50 μL, 12 s, 0.25 mL / min; running buffer in the first washing step: 125 μL, 30 s, 0.25 mL / min; detection antibody solution: 500 μL, 120 s, 0.25 mL / min; running buffer in the second washing step: 125 μL, 30 s, 0.25 mL / min; running buffer in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Each sample was measured in duplicate.

[0177] 〔result〕 A high signal value was obtained only in the sample containing both components (Sample No. 1), while the signal values ​​in the samples spiked with only one component (Sample Nos. 2 and 3) were comparable to that of the unspiked blank sample (Sample No. 4). This confirmed that this assay can specifically detect the binary complex. Furthermore, it is considered reasonable to consider the difference between the signal value of the sample containing both components and the signal value of the blank sample as the signal value derived from the binary complex.

[0178] <Example 13> Comparison of measured and simulated values ​​in measuring the complex formed by anti-IL-6R antibody and soluble IL-6R As in Example 6, the measured values ​​and the simulated values ​​were also compared for the measurement of the complex formed by the anti-IL-6R antibody and soluble IL-6R. D It was confirmed that the complex was formed according to the values ​​and that the binding equilibrium was not significantly shifted during measurement.

[0179] [Preparation of beads] The same procedure as in Example 12 was carried out.

[0180] [Preparation of detection antibody solution] The same procedure as in Example 12 was carried out.

[0181] [Sample preparation] IL-6R was kept constant at 0.103 μg / mL (2 nM) and tocilizumab was spiked at eight concentrations (0, 0.003, 0.009, 0.03, 0.09, 0.3, 0.9, and 3 μg / mL (0, 0.04, 0.12, 0.4, 1.2, 4, 12, and 40 nM)). Samples were prepared in assay buffer (0.01 M phosphate-buffered saline, 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20, 0.02% NaN3, 0.1% BSA, pH 7.4). An unspiked sample served as a blank. The hIL-6R concentration was set to a physiological concentration. After sample preparation, the samples were incubated at room temperature for 3 hours before measurement to allow the complex formation to reach equilibrium.

[0182] 〔measurement〕 Measurements were performed using a KinExA 3200 (Sapidyne) as the measurement device, following the same procedure as in Example 12. The volumes, times, and flow rates of the sample, running buffer in the first washing step, detection antibody solution, running buffer in the second washing step, and running buffer in the third washing step were varied to ensure a sufficient signal-to-noise ratio. The measurement conditions were as follows: Sample: 150 μL, 36 s, 0.25 mL / min; Running buffer in the first washing step: 125 μL, 30 s, 0.25 mL / min; Detection antibody solution: 500 μL, 120 s, 0.25 mL / min; Running buffer in the second washing step: 125 μL, 30 s, 0.25 mL / min; Running buffer in the third washing step: 1500 μL, 90 s, 1.00 mL / min. Each sample was measured at n=1, and the blank sample was measured at n=5.

[0183] 〔analysis〕 The mean and standard deviation (SD) of the signal values ​​of blank samples measured in n = 5 were calculated (Microsoft Excel 2013), and the detection limit was set as Mean + 3.29 × SD. For the other samples whose signal values ​​were above the detection limit, the mean signal value of the blank sample was subtracted to calculate the signal value of the binary complex. At the same time, the concentrations of tocilizumab and IL-6R in the prepared samples and K D The binary complex concentration in each sample was simulated based on the concentration (0.11 μg / mL, J Pharmacokinet Pharmacodyn. 2012 Feb;39(1):5-16.). Because tocilizumab binds divalently, the concentration of the tocilizumab molecule multiplied by 2 was used as the binding site concentration. The concentration was calculated by considering the state in which one binding site binds to hIL-6R as a binary complex. This means that the states in which one tocilizumab molecule binds to two hIL-6R molecules and two tocilizumab molecules bind to one hIL-6R molecule each were not distinguished. To compare the binary complex signal values ​​obtained with the simulated values, a plot was created with the signal values ​​on the left and right Y axes (black circles, dotted lines) and the tocilizumab binding site concentration on the X axis (Figure 15A). In addition, the obtained results were plotted with the binary complex signal value on the Y axis and the simulation value on the X axis, and correlation analysis was performed using Microsoft Excel 2013. In the correlation analysis, a linear approximation curve was drawn and R 2 The values ​​were calculated (Figure 15B).

[0184] 〔result〕 All samples spiked with tocilizumab and hIL-6 showed signals above the detection limit. Depending on the concentration of tocilizumab added, the binary complex signal value took on a sigmoid shape, which was consistent with the shape predicted from the simulation. Furthermore, in Figure 15B, where the X axis is the simulated value and the Y axis is the binary complex signal value, a linear relationship was obtained. From these findings, it can be concluded that the K D It was thought that the complex was formed according to the values, and that the measurement was performed without a significant shift in the binding equilibrium. Furthermore, by obtaining this relationship, it was thought that when a sample containing unknown concentrations of tocilizumab and hIL-6R was measured, the binary complex concentration could be calculated by regression from the binary complex signal value obtained, assuming that the simulation values ​​were correct.

Claims

1. 1. A method for detecting a complex in a sample, comprising: the complex comprises two or more components, at least two of which are an antibody and an antigen recognized by the antibody; (1) contacting a sample containing a complex with a first binder that recognizes the antigen, thereby binding the first binder to the complex; (2) binding the complex bound to the first binder to a second binder; and (3) detecting a second binder bound to the complex; and at least one K D The method is carried out under conditions where the binding equilibrium of the complex is substantially maintained, and the value is 1 nM or more.

2. The method of claim 1 , wherein the components of the complex are not labeled or immobilized on a solid phase.

3. The method of claim 1 or 2, wherein the second conjugate is labeled.

4. The method according to any one of claims 1 to 3, wherein the sample is a blood sample.

5. The method described in claim 4, wherein the blood sample is whole blood, serum or plasma.

6. The method according to any one of claims 1 to 5, wherein the first binder is bound to a solid phase.

7. The method of claim 6 , wherein the solid phase is a chip, a microfluidic chip, a disc, or a bead.

8. A method according to any one of claims 1 to 7, carried out by a method using a microfluidic chip, disc, or bead.

9. A method for determining the concentration and / or amount of a complex in a sample, comprising the steps of: (4) preparing a composite for creating a regression equation; (5) detecting the complex of the step (4) by a process including the steps (1) to (3); (6) simulating the concentration of the complex in the step (4); (7) calculating a regression equation from the signal value detected in the step (5) and the simulated concentration; and (8) A step of applying the signal value detected in the step (3) to the regression equation.

10. The method according to claim 9, wherein at least one of the components constituting the complex in the sample is a drug.

11. A method for determining a therapeutic method using a drug based on the concentration and / or amount of the complex determined by the method of claim 10.

12. A method for evaluating the dynamics of a complex based on the concentration and / or amount of the complex determined by the method according to any one of claims 9 to 11.

13. 1. A method for determining the concentration and / or amount of a complex in a sample, comprising: The complex contains two or more components, at least two of which are an antibody and an antigen recognized by the antibody, and at least one K D a value of 1 nM or more, and the binding equilibrium of the complex is substantially maintained; (1) contacting a sample containing a complex with a first binder that recognizes the antigen, thereby binding the first binder to the complex; (2) binding the complex bound to the first binder to a second binder; (3) detecting a second binder bound to the complex; (4) preparing a composite for creating a regression equation; (5) detecting the complex of the step (4) by a process including the steps (1) to (3); (6) simulating the concentration of the complex in the step (4); (7) calculating a regression equation from the signal value detected in the step (5) and the simulated concentration; and (8) applying the signal value detected in the step (3) to the regression equation; The method comprising:

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