HK1232288B - Method for measuring reactivity of fviii - Google Patents

Description

Method for measuring the reactivity of FVIII

Technical Field

The present invention relates to methods for measuring the reactivity of coagulation factor VIII (FVIII) in the presence of a substance that has the activity of functionally replacing FVIII (e.g., a method for measuring FVIII activity or FVIII inhibitor titer). The present invention also relates to kits, etc., for measuring the reactivity of FVIII in the presence of a substance that has the activity of functionally replacing FVIII.

Background Art

Hemophilia is a bleeding disorder caused by a congenital defect or dysfunction of either factor VIII or coagulation factor IX (FIX). The former is known as hemophilia A, and the latter as hemophilia B. Both genes are located on the X chromosome; and because they are X-chromosome-linked recessive inherited genetic abnormalities, 99% or more of those who develop the disease are male. It is known that the prevalence is approximately 1 in 10,000 male births, and the ratio between hemophilia A and hemophilia B is approximately 5:1.

The main bleeding sites in hemophilia patients include intra-articular, intramuscular, subcutaneous, intraoral, intracranial, digestive tract, and intranasal. Among them, repeated intra-articular bleeding can develop into hemophilic arthropathy with joint disorders and difficulty walking, which may eventually require joint replacement. Therefore, it is a major factor that reduces the quality of life (QOL) of hemophilia patients.

The severity of hemophilia is very relevant to the FVIII activity or FIX activity in the blood. Patients with less than 1% coagulation factor activity are classified as severe, patients with 1% or more to less than 5% activity are classified as moderate, and patients with 5% or more and less than 40% activity are classified as mild. Patients with severe symptoms (accounting for approximately half of hemophilia patients) show bleeding symptoms (if they do not accept the preventive replacement therapy described later) several times a month, and this frequency is significantly higher than the frequency of patients with moderate symptoms and patients with mild symptoms.

In addition to hemophilia and acquired hemophilia, von Willebrand's disease, which is caused by malfunction or deficiency of von Willebrand factor (vWF), is known to be a related bleeding disorder. vWF is not only required for normal platelet adhesion to the subendothelial tissue at sites of vascular wall damage, but is also required to form a complex with FVIII and maintain normal levels of FVIII in the blood. In patients with von Willebrand's disease, these functions are impaired, leading to hemostatic dysfunction.

In order to prevent and/or treat the bleeding in hemophiliac, mainly use the blood coagulation factors from plasma purification or those produced by genetic engineering technology.In serious hemophiliac, it is believed that by FVIII or FIX replacement therapy, the FVIII activity in the blood or the FIX activity are maintained at 1% or more for preventing the appearance of bleeding symptoms be effective (non-patent literature 1 and 2).On the other hand, in hemophiliac, particularly serious hemophiliac, it is possible to produce antibodies for FVIII or FIX, which are referred to as inhibitors.When producing such inhibitors, the effect of coagulation factor preparations is blocked by inhibitors.So, use the neutralization treatment of a large amount of coagulation factor preparations, or use the bypass treatment of the coagulation factor VII preparation (FVIIa preparation) of complex concentrate or activation.

Measurement of FVIII activity in hemophilia A is mainly performed by a one-stage coagulation assay based on the activated partial thromboplastin time (APTT) (Non-Patent Document 3) and a chromogenic assay, which is a system reconstituted using purified coagulation factors (Non-Patent Document 4).

Measurement of FVIII inhibitor titers in hemophilia A is mainly performed by the Bethesda assay or the Nijmegen Bethesda assay (Non-Patent Documents 5 and 6).

In recent years, such bispecific antibodies have been found: they bind to FIX and/or activated coagulation factor IX (FIXa) and coagulation factor X (FX) and/or activated blood coagulation factor X (FXa), and replace the cofactor function of FVIII, or more specifically, promote the function of FX activation of FIXa (non-patent literature 7 and 8; patent literature 1, 2 and 3). The bispecific antibody functionally replaces FVIII to improve the decline of the coagulation reaction caused by FVIII deficiency or dysfunction. For example, with respect to thrombin generation and APTT (which is an indicator of coagulation reaction), the bispecific antibody shortens the APTT (regardless of the presence of FVIII inhibitors) of plasma derived from hemophilia A patients and increases the generation of thrombin. The APTT-shortening effect of the bispecific antibody is significant compared to FVIII. This is because the FVIII in the plasma only shows cofactor activity after being activated by activated factor X (FXa) or thrombin, while the above-mentioned bispecific antibody does not require such an activation process and, for this reason, more quickly exhibits cofactor function.

Furthermore, antibodies against FIXa Fab and FX Fab of a bispecific antibody were obtained, and the concentration of the bispecific antibody in plasma samples from animal experiments was determined (Non-Patent Document 9).

The bispecific antibodies replace the cofactor function of FVIII, thereby affecting the assay system that measures the reactivity of FVIII itself. For example, when measuring plasma FVIII activity using a one-stage clotting assay based on APTT to diagnose the severity of hemophilia A or monitor the pharmacological activity of a FVIII preparation in patients taking a FVIII preparation, the effect of the bispecific antibody that promotes the shortening of the clotting time of the bispecific antibody in the presence of the bispecific antibody can strongly interfere, which can greatly impair the accuracy of the measurement. In addition, when determining plasma FVIII inhibitor titers using the Bethesda assay based on APTT, the effect of the bispecific antibody that promotes the shortening of the clotting time of the bispecific antibody in the presence of the bispecific antibody can strongly interfere, which can greatly impair the accuracy of the measurement. In other words, in patients taking bispecific antibodies, FVIII activity and FVIII inhibitor titers cannot be accurately measured. Therefore, a method that can measure FVIII activity and FVIII inhibitor titers even in the presence of bispecific antibodies is desirable.

Citation List

[Non-patent literature]

Non-patent literature 1: N Engl J Med. 2007; 357(6): 535-44

Non-patent document 2: Thromb Res. 2011; 127(Supplement 1): S14-7

Non-patent document 3: Thromb Diath Haemorrh. May 15, 1962; 7: 215-28

Non-patent document 4: Haemostasis. 1989 19: 196-204.

Non-patent document 5: Thromb Diath Haemorrh. 1975; 34(3): 869-72

Non-patent document 6: Thromb Haemost. 1995 Feb; 73(2): 247-51.

Non-patent literature 7: Nat Med. 2012; 18(10): 1570-74

Non-patent literature 8: PLoS One.2013;8(2):e57479.

Non-patent literature 9: J Thromb Haemost. 2014; 12(2): 206-13 Supporting information

[Patent Document]

Patent Document 1: WO2005/035756

Patent Document 2: WO2006/109592

Patent Document 3: WO2012/067176

[Summary of the invention]

[Problems to be Solved by the Invention]

The present invention relates to methods for measuring the reactivity of FVIII in the presence of a substance that has an activity that functionally replaces FVIII, for example, a method for measuring FVIII activity or FVIII inhibitor titer. Furthermore, an object of the present invention is to provide a kit, etc., for measuring the reactivity of FVIII (such as FVIII activity and FVIII inhibitor titer) in the presence of a substance that has an activity that functionally replaces FVIII.

[Methods of solving the problem]

In order to solve the above problems, the inventors of the present invention have produced a substance that neutralizes the activity of bispecific antibodies, and by targeting a test article for measuring the reactivity of FVIII, the measurement conditions for ensuring accuracy even in the presence of bispecific antibodies have been retrieved. Therefore, the inventors of the present invention have found that by using a neutralizing antibody for bispecific antibodies at an appropriate concentration (for example, a concentration when the bispecific antibody can be fully neutralized), the FVIII activity in the plasma of hemophilia A patients can be accurately evaluated by a one-stage coagulation assay based on APTT, and it has also been found that the FVIII inhibitor titer in the plasma of hemophilia A patients carrying FVIII inhibitors can be accurately evaluated by the Bethesda assay based on APTT. In addition, the inventors of the present invention have successfully discovered a kit for use in the measurement, which contains neutralizing antibodies for bispecific antibodies with FVIII-replacing activity. The present invention is based on these findings and provides the following:

[1] A method for measuring the reactivity of coagulation factor VIII, wherein the method comprises the steps of contacting (1) with (2):

(1) A blood-derived sample containing a substance having an activity of functionally replacing coagulation factor VIII,

(2) one or more substances, wherein the substance has the activity of functionally replacing coagulation factor VIII;

[2] The method of [1], wherein the substance having the activity of functionally replacing coagulation factor VIII is a bispecific antibody that binds to coagulation factor IX and/or activated coagulation factor IX and binds to coagulation factor X and/or activated blood coagulation factor X;

[3] The method of [1] or [2], wherein the bispecific antibody is any one of the following antibodies, wherein the first polypeptide binds to the third polypeptide and the second polypeptide binds to the fourth polypeptide:

A bispecific antibody, wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 9, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are the common L chain (Q499-z121/J327-z119/L404-k) of SEQ ID NO: 10; or

A bispecific antibody, wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 36, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 37, and the third polypeptide and the fourth polypeptide are a common L chain (Q153-G4k/J142-G4h/L180-k) of SEQ ID NO: 38;

[4] The method of any one of [1] to [3], wherein the neutralizing substance is one or more substances selected from the group consisting of peptides, polypeptides, organic compounds, aptamers, and antibodies that neutralize the substance having the activity of functionally replacing coagulation factor VIII;

[5] The method of any one of [2] to [4], wherein the neutralizing substance is one or more antibodies selected from the group consisting of: an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor IX; an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor IX; an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor X; an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor X; and a bispecific antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a Fab comprising an antigen binding site that binds to coagulation factor X and/or activated coagulation factor X;

[6] The method of any one of [1] to [5], wherein the neutralizing substance is a combination of one or more antibodies selected from the following combinations:

(a) an antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor IX; and an antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor X;

(b) an antibody that binds to a Fab comprising an antigen binding site that binds activated coagulation factor IX; and an antibody that binds to a Fab comprising an antigen binding site that binds coagulation factor X;

(c) an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor IX; and an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor IX; and

(d) an antibody that binds Fab comprising an antigen binding site that binds coagulation factor IX; an antibody that binds Fab comprising an antigen binding site that binds coagulation factor X; and an antibody that binds Fab comprising an antigen binding site that binds activated coagulation factor IX;

[7] The method of any one of [1] to [6], wherein the method for measuring the reactivity of coagulation factor VIII is a method for measuring coagulation factor VIII activity or a method for measuring the titer of a coagulation factor VIII inhibitor;

[8] A kit for use in the method of any one of [1] to [7], wherein the kit comprises one or more antibodies selected from the group consisting of: an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor IX; an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor IX; an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor X; an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor X; and a bispecific antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a Fab comprising an antigen binding site that binds to coagulation factor X and/or activated coagulation factor X;

[9] [8] The kit of claim 9, wherein the kit comprises one or more antibodies selected from the group consisting of:

(a) an antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor IX; and an antibody that binds to a Fab comprising an antigen binding site that binds to coagulation factor X;

(b) an antibody that binds to a Fab comprising an antigen binding site that binds activated coagulation factor IX; and an antibody that binds to a Fab comprising an antigen binding site that binds coagulation factor X;

(c) an antibody that binds to Fab comprising an antigen binding site that binds to coagulation factor IX; and an antibody that binds to Fab comprising an antigen binding site that binds to activated coagulation factor IX; and

(d) an antibody that binds Fab comprising an antigen binding site that binds coagulation factor IX; an antibody that binds Fab comprising an antigen binding site that binds coagulation factor X; and an antibody that binds Fab comprising an antigen binding site that binds activated coagulation factor IX;

[10] A method for diagnosing the severity of a disease in a patient to whom a substance having an activity of functionally replacing coagulation factor VIII is administered, wherein the method uses the method of any one of [1] to [7];

[11] A method for diagnosing inhibitor titer in a patient to whom a substance having an activity of functionally replacing coagulation factor VIII is administered, wherein the method uses the method of any one of [1] to [7];

[12] A method for monitoring the pharmacological activity of a FVIII preparation in a patient administered with a FVIII preparation and a substance having an activity of functionally replacing coagulation factor VIII, wherein the method uses the method of any one of [1] to [7];

[13] The method of any one of [10] to [12], wherein the patient is a patient selected from the group consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand disease patient, and a patient with hemophilia A in which inhibitors to blood coagulation factor VIII and/or activated blood coagulation factor VIII appear;

[14] The kit of [8] or [9], wherein the kit is for diagnosing the severity of a disease in a patient to whom a substance having an activity of functionally replacing coagulation factor VIII is administered;

[15] The kit of [8] or [9], wherein the kit is for diagnosing the inhibitor titer in a patient to whom a substance having an activity of functionally replacing coagulation factor VIII is administered;

[16] The kit of [8] or [9], wherein the kit is for monitoring the pharmacological activity of a FVIII preparation in a patient to whom a FVIII preparation and a substance having an activity of functionally replacing coagulation factor VIII are administered; and

[17] The kit of any one of [14] to [16], wherein the patient is a patient selected from the group consisting of a hemophilia A patient, an acquired hemophilia A patient, a von Willebrand disease patient, and a patient with hemophilia A in which inhibitors to blood coagulation factor VIII and/or activated blood coagulation factor VIII appear.

[Effects of the Invention]

The present invention provides a method for measuring FVIII activity and FVIII inhibitor titer, which is not affected by the activity of substances with FVIII-replacing activity. Substances with FVIII-replacing activity include bispecific antibodies that bind to FIX and/or FIXa and FX and/or FXa.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the result of the one-stage coagulation assay performed under the neutralization of anti-FIXa/FX bi-specific antibody using rAQ8-mIgG2b and rAJ540-rbtIgG.When (it contains 10U/dL or 100U/dL recombinant FVIII with buffer dilution FVIII-deficient plasma, has supplemented anti-FIXa/FX bi-specific antibody ACE910) (#3 and #7), it is confirmed that FVIII activity is higher than the scope of calibration curve.On the other hand, when (it contains 10U/dL or 100U/dL recombinant FVIII with buffer dilution FVIII-deficient plasma containing two classes of antibodies for anti-FIXa/FX bi-specific antibody, has supplemented anti-FIXa/FX bi-specific antibody ACE910) (#4 and #8), it is confirmed that FVIII activity is similar to those activities (#1 and #5) of the group not adding anti-FIXa/FX bi-specific antibody. When FVIII-deficient plasma containing 10 U/dL or 100 U/dL of recombinant FVIII was diluted with a buffer containing only two types of antibodies against the anti-FIXa/FX bispecific antibody (#2 and #6), FVIII activity was confirmed to be similar to that of the group without added anti-FIXa/FX bispecific antibody (#1 and #5).

Fig. 2 shows the result of the one-stage coagulation assay performed under neutralization of anti-FIXa/FX bispecific antibody with AQ1 and AJ541 or AQ1 and AJ522.When FVIII-deficient plasma (it contains 10U/dL recombinant FVIII, has supplemented anti-FIXa/FX bispecific antibody ACE910) is diluted with buffer (#4), it is confirmed that FVIII activity is higher than the scope of calibration curve.On the other hand, when diluting FVIII-deficient plasma (it contains 10U/dL recombinant FVIII, has supplemented anti-FIXa/FX bispecific antibody ACE910) with buffer (#5) containing two classes of antibodies AQ1 and AJ541 for anti-FIXa/FX bispecific antibody or buffer (#6) containing two classes of antibodies AQ1 and AJ522 for anti-FIXa/FX bispecific antibody, it is confirmed that FVIII activity is similar to those activities (#1) of the group not adding anti-FIXa/FX bispecific antibody. When FVIII-deficient plasma containing 10 U/dL recombinant FVIII was diluted with a buffer containing only two types of antibodies AQ1 and AJ541 against anti-FIXa/FX bispecific antibodies (#2) or a buffer containing only two types of antibodies AQ1 and AJ522 (#3), FVIII activity was confirmed to be similar to that of the group without added anti-FIXa/FX bispecific antibodies (#1).

Fig. 3 shows the result of the one-stage coagulation assay performed under neutralization of anti-FIXa/FX bi-specific antibody with AQ512 and AJ114 or AQ512 and AJ521.When (#4) with buffer dilution FVIII-deficient plasma (it contains 10U/dL recombinant FVIII, has supplemented anti-FIXa/FX bi-specific antibody hBS23), it was confirmed that FVIII activity was higher than the scope of calibration curve.On the other hand, when (#5) with buffer containing two classes of antibodies AQ512 and AJ114 for anti-FIXa/FX bi-specific antibody or (#6) containing two classes of antibodies AQ512 and AJ521 for anti-FIXa/FX bi-specific antibody dilution FVIII-deficient plasma (it contains 10U/dL recombinant FVIII, has supplemented anti-FIXa/FX bi-specific antibody hBS23), it was confirmed that FVIII activity was similar to those activities (#1) of the group not adding anti-FIXa/FX bi-specific antibody. When FVIII-deficient plasma containing 10 U/dL recombinant FVIII was diluted with a buffer containing only two types of antibodies AQ512 and AJ114 against the anti-FIXa/FX bispecific antibody (#2) or a buffer containing only two types of antibodies AQ512 and AJ521 (#3), FVIII activity was confirmed to be similar to that of the group without the addition of the anti-FIXa/FX bispecific antibody (#1).

Fig. 4 shows the result of the Bethesda assay performed using rAQ8-mIgG2b and rAJ540-rbtIgG in the neutralization of anti-FIXa/FX bispecific antibodies.The FVIII inhibitor plasma (#3) containing only anti-FIXa/FX bispecific antibodies ACE910 shows 100% or more equivalent activity of FVIII with a calibration curve.On the other hand, the FVIII inhibitor plasma (#4) containing anti-FIXa/FX bispecific antibodies and two types of antibodies for anti-FIXa/FX bispecific antibodies shows a FVIII inhibitor titer similar to the inhibitor plasma (#1) without additives.The FVIII inhibitor plasma (#2) containing only two types of antibodies for anti-FIXa/FX bispecific antibodies shows a result similar to the result of #1.

DETAILED DESCRIPTION

The method for measuring FVIII activity of the present invention includes the steps of contacting (1) and (2) described below. Otherwise, the method can be performed according to a method generally used for measuring FVIII activity. Details will also be explained in the Examples.

(1) A blood-derived sample containing a substance having the activity of functionally replacing FVIII

(2) A substance wherein the substance having the activity of functionally replacing FVIII

Methods for measuring FVIII activity

A commonly used method for measuring FVIII activity known to those skilled in the art can be used, for example, one can use a one-stage coagulation assay (Casillas et al., (1971) Coagulation 4: 107-11) using factor VIII-deficient plasma (Sysmex, Kobe, Japan), which is based on clotting time (aPTT measurement). For example, a one-stage coagulation assay is performed as follows. Three solutions are mixed: 50 μL of 10-fold diluted test plasma, 50 μL of FVIII-deficient plasma, and 50 μL of APTT reagent; and incubated at 37° C. for 5 minutes, followed by the addition of 50 μL of calcium solution to initiate the coagulation reaction, and then the time to coagulation is measured. In addition, instead of the test plasma, a serial dilution of a normal plasma sample is measured (the FVIII activity in the 10-fold diluted normal plasma is designated as 100%), and a calibration curve is generated by plotting the FVIII activity on the horizontal axis and the clotting time on the vertical axis. The clotting time of the test plasma was converted into FVIII activity using a calibration curve and the FVIII activity in the test plasma was calculated.Herein, unless otherwise indicated, the phrase "measurement of FVIII activity" is used as a phrase that may include "measurement of activated coagulation factor VIII (FVIIIa) activity".

In addition to the one-stage coagulation assay, a thrombin generation assay (TGA), a measurement method using rotational thromboelastometry, a FVIII chromogenic assay, a coagulation waveform analysis, a thrombin and activated factor X generation assay, etc. can be used as a method for measuring FVIII activity. The method for measuring the titer of a FVIII inhibitor of the present invention comprises the steps of contacting the following (1) and (2). Otherwise, the method can be performed according to a conventional method for measuring the titer of a FVIII inhibitor. Details will also be explained in the Examples.

(1) A blood-derived sample containing a substance having the activity of functionally replacing FVIII

(2) A substance wherein the substance having the activity of functionally replacing FVIII

Method for measuring FVIII inhibitor titer

A commonly used method for measuring the FVIII inhibitor titer known to those skilled in the art can be used, for example, the Bethesda assay (Kasper et al., (1975) Thrombos Diath Haemorrh 34:869-872), the ELISA method, and the Nijmegen Bethesda assay (Nijmegen modified assay) (Verbruggen et al., (1995) Thromb Haemost 73:247-251). For example, the Bethesda assay is performed as follows. A solution produced by mixing equal amounts of normal plasma and test plasma is incubated at 37°C for 2 hours, and then the residual factor VIII activity in normal plasma is measured by a one-stage coagulation assay based on the activated partial thromboplastin time (APTT). The effect of inhibiting 50% of the factor VIII activity in normal plasma is designated as 1 Bethesda (1BU), and the FVIII inhibitor titer is thus calculated in units of Bethesda. When the FVIII inhibitor titer in the test plasma is high and the residual FVIII activity is not within the range of 25% to 75%, the Bethesda unit is recalculated using the test plasma appropriately diluted with buffer, and then, the value is multiplied by the dilution ratio to calculate the FVIII inhibitor titer in the test plasma.

FVIII

FVIII is one of a series of molecules involved in blood coagulation, exhibiting cofactor activity (when it is activated by thrombin or FXa) and promoting the FX activation reaction by FIXa.

FVIII inhibitors

FVIII inhibitors are alloantibodies directed against foreign FVIII and occur in 20% to 30% of patients with hemophilia A. Initially normal individuals may develop autoantibodies against FVIII. Typically, most FVIII inhibitor alloantibodies and autoantibodies act as neutralizing anti-FVIII antibodies and reduce or eliminate FVIII activity.

Replaces FVIII activity

The present invention has the active substance of functionally replacing FVIII and can be rephrased as the substance with FVIII-sample activity.In the present invention, phrase " functionally replacing FVIII " refers to, promotes the FX activation realized by FIXa (promoting the FXa realized by FIXa to produce).More specifically, in the present invention, phrase " functionally replacing FVIII " refers to identification FIX and/or FIXa and FX and/or FXa, and promotes the activation of FX by FIXa (promoting the FXa realized by FIXa to produce).Use the measurement system such as comprising FIXa, FX, synthetic substrate S-2222 (synthetic substrate of FXa) and phospholipid, the activity promoting FXa to produce can be evaluated. Such a measurement system showed a correlation between the severity of the disease and clinical symptoms in cases of hemophilia A (Rosen S, Andersson M, Blomback M et al ^. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985; 54: 811-23).

A preferred embodiment of the substance having the activity of functionally replacing FVIII of the present invention includes, for example, a bispecific antibody that binds to FIX and/or FIXa and binds to FX and/or FXa. Such antibodies can be obtained according to the methods described in, for example, WO2005/035756, WO2006/109592, and WO2012/067176. The bispecific antibodies of the present invention include the antibodies described in these documents.

Preferred bispecific antibodies include, for example, ACE910 (Q499-z121 / J327-z119 / L404-k) (a bispecific antibody in which the H chain consisting of the amino acid sequence of SEQ ID NO: 9 and the L chain of SEQ ID NO: 10 are bound, and the H chain consisting of the amino acid sequence of SEQ ID NO: 11 and the L chain of SEQ ID NO: 10 are bound) and hBS23 (Q153-G4k / J142-G4h / L180-k) (a bispecific antibody in which the H chain consisting of the amino acid sequence of SEQ ID NO: 36 and the L chain of SEQ ID NO: 38 are bound, and the H chain consisting of the amino acid sequence of SEQ ID NO: 37 and the L chain of SEQ ID NO: 38 are bound), which are bispecific antibodies described in patent document (WO2012/067176).

Neutralization

In the present invention, "neutralize" in the context of neutralizing a substance having an activity that functionally replaces FVIII means, for example, completely or partially inhibiting the activity of the substance having an activity that functionally replaces FVIII. For example, when the substance having an activity that functionally replaces FVIII is an antibody, complete or partial inhibition of the activity that functionally replaces FVIII can be achieved by completely or partially inhibiting the binding of the antibody to the antigen, but is not limited thereto.

Neutralizing substances

In the present invention, the term "substance" of the neutralizing substance among the substances that neutralize the substance having the activity of functionally replacing FVIII refers to, for example, peptides, polypeptides, organic compounds, aptamers, antibodies, etc. that bind to the substance having the activity of functionally replacing FVIII.

A plurality of neutralizing substances may be used in combination, and for example, an antibody and an aptamer may be used in combination.

peptides

Polypeptides in the present invention generally refer to proteins and peptides having a length of approximately 10 amino acids or longer. Typically, these are biologically derived polypeptides, but are not particularly limited to such polypeptides and may include, for example, polypeptides comprising artificially designed sequences. Furthermore, these may be any natural polypeptide, synthetic polypeptide, or recombinant polypeptide. Furthermore, the polypeptides of the present invention also include fragments of the aforementioned polypeptides.

organic compounds

The organic compound in the present invention is, for example, a low molecular weight compound, preferably having a molecular weight of 1000 or less.

Aptamer

The term "aptamer" refers to a nucleic acid molecule that specifically binds to a target molecule, such as a polypeptide. For example, the aptamer of the present invention can be an RNA aptamer that can specifically bind to a substance having FVIII-replacing activity. The production and therapeutic use of aptamers are well established in the art. For example, aptamers can be obtained by using the SELEX method (see U.S. Patent Nos. 5,475,096, 5,580,737, 5,567,588, 5,707,796, 5,763,177, 6,699,843, etc.).

Antibody

When the substance having the activity of functionally replacing FVIII is a bispecific antibody that binds to FIX and/or FIXa and binds to FX and/or FXa, examples of antibodies that bind to the substance having the activity of functionally replacing FVIII include antibodies selected from the following: antibodies that bind to Fab, the Fab containing an antigen-binding site that binds to FIX; antibodies that bind to Fab, the Fab containing an antigen-binding site that binds to FIXa; antibodies that bind to Fab, the Fab containing an antigen-binding site that binds to FX; antibodies that bind to Fab, the Fab containing an antigen-binding site that binds to FXa; and bispecific antibodies that bind to Fab containing an antigen-binding site that binds to FIX and/or FIXa and to Fab containing an antigen-binding site that binds to FX and/or FXa. The above antibodies can be used alone or in combination. For example, it is possible to use multiple antibodies that bind to Fab containing an antigen-binding site that binds to one type of antigen, for example, multiple types of antibodies that bind to Fab containing an antigen-binding site that binds to FIX. For example, when the substance having the activity of functionally replacing FVIII is a bispecific antibody that binds to FIX and/or FIXa and binds to FX and/or FXa, the following antibody combination can be used:

(a) an antibody that binds to a Fab that contains an antigen binding site that binds to FIX; and an antibody that binds to a Fab that contains an antigen binding site that binds to FX;

(b) an antibody that binds to a Fab that contains an antigen binding site that binds FIXa; and an antibody that binds to a Fab that contains an antigen binding site that binds FX;

(c) an antibody that binds to a Fab that contains an antigen binding site that binds to FIX; and an antibody that binds to a Fab that contains an antigen binding site that binds to FIXa; and

(d) an antibody that binds Fab containing an antigen binding site that binds FIX; an antibody that binds Fab containing an antigen binding site that binds FX; and an antibody that binds Fab containing an antigen binding site that binds FIXa.

Examples of antibodies that bind to a Fab containing an antigen-binding site that binds to FIX and/or FIXa include AQ8, AQ1, and AQ512 antibodies.The nucleotide sequence of the variable region and the amino acid sequence predicted therefrom were analyzed by GENETYX version 9 (GENETYX CORPORATION).

The amino acid sequence and nucleotide sequence of the H chain variable region of AQ8 are indicated by the following SEQ ID NO:

Amino acid sequence: SEQ ID NO: 1; and

Nucleotide sequence: SEQ ID NO: 5.

The amino acid sequence and nucleotide sequence of the L chain variable region of AQ8 are indicated by the following SEQ ID NO:

Amino acid sequence: SEQ ID NO: 2; and

Nucleotide sequence: SEQ ID NO: 6.

The amino acid sequence and nucleotide sequence of H-chain CDRs 1-3 of AQ8 are indicated by the following SEQ ID NOs:

CDR1 amino acid sequence: SEQ ID NO: 12;

CDR2 amino acid sequence: SEQ ID NO: 13;

CDR3 amino acid sequence: SEQ ID NO: 14;

CDR1 nucleotide sequence: SEQ ID NO: 15;

CDR2 nucleotide sequence: SEQ ID NO: 16; and

CDR3 nucleotide sequence: SEQ ID NO: 17.

The amino acid sequence and nucleotide sequence of L-chain CDRs 1-3 of AQ8 are indicated by the following SEQ ID NOs:

CDR1 amino acid sequence: SEQ ID NO: 18;

CDR2 amino acid sequence: SEQ ID NO: 19;

CDR3 amino acid sequence: SEQ ID NO: 20;

CDR1 nucleotide sequence: SEQ ID NO: 21;

CDR2 nucleotide sequence: SEQ ID NO: 22; and

CDR3 nucleotide sequence: SEQ ID NO: 23.

Examples of antibodies that bind to Fab containing an antigen-binding site that binds to FX and/or FXa include AJ540, AJ541, AJ522, AJ114, and AJ521 antibodies. The nucleotide sequence of the variable region and the amino acid sequence predicted therefrom were analyzed by GENETYX Ver. 9 (GENETYX CORPORATION).

The amino acid sequence and nucleotide sequence of the H chain variable region of AJ540 are indicated by the following SEQ ID NO:

Amino acid sequence: SEQ ID NO: 3; and

Nucleotide sequence: SEQ ID NO: 7.

The amino acid sequence and nucleotide sequence of the L chain variable region of AJ540 are indicated by the following SEQ ID NO:

Amino acid sequence: SEQ ID NO: 4; and

Nucleotide sequence: SEQ ID NO: 8.

The amino acid sequence and nucleotide sequence of H-chain CDRs 1-3 of AJ540 are indicated by the following SEQ ID NOs:

CDR1 amino acid sequence: SEQ ID NO: 24;

CDR2 amino acid sequence: SEQ ID NO: 25;

CDR3 amino acid sequence: SEQ ID NO: 26;

CDR1 nucleotide sequence: SEQ ID NO: 27;

CDR2 nucleotide sequence: SEQ ID NO: 28; and

CDR3 nucleotide sequence: SEQ ID NO: 29.

The amino acid sequence and nucleotide sequence of L-chain CDRs 1-3 of AJ540 are indicated by the following SEQ ID NOs:

CDR1 amino acid sequence: SEQ ID NO: 30;

CDR2 amino acid sequence: SEQ ID NO: 31;

CDR3 amino acid sequence: SEQ ID NO: 32;

CDR1 nucleotide sequence: SEQ ID NO: 33;

CDR2 nucleotide sequence: SEQ ID NO: 34; and

CDR3 nucleotide sequence: SEQ ID NO: 35.

The term "antibody" is used in the broadest sense and can be a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, a multispecific antibody (e.g., a bispecific antibody), an antibody-derived substance, and a modified antibody product (Miller K et al. J Immunol. 2003, 170 (9), 4854-61), as long as they exhibit the desired biological activity. The antibodies can be mouse antibodies, human antibodies, humanized antibodies, chimeric antibodies, or those derived from another species, or they can be artificially synthesized antibodies. The antibodies disclosed herein can belong to any type (e.g., IgG, IgE, IgM, IgD and IgA), species (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. The immunoglobulin can be derived from any species (e.g., human, mouse or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably in a broad sense.

The term "antibody-derived material" includes a portion of an antibody, preferably an antibody variable region, or at least the antigen-binding region of an antibody. Antibody-derived materials include, for example, Fab, Fab', F(ab')2, Fv fragments, linear antibodies and single-chain antibodies (scFv), sc(Fv) ₂ , _Fab3 , domain antibodies (dAb) (WO2004/058821, WO2003/002609), diabodies, trimers, tetrabodies, microbodies, and multispecific antibodies formed from antibody-derived materials, but are not limited thereto. Here, "Fab" is composed of the CH1 domain and variable region of a single light chain and a single heavy chain. In addition, "Fv" is a minimal antibody-derived material and includes a complete antigen recognition region and an antigen-binding region. The antibody-derived material can be, for example, a fusion between an IgG antibody and Fc. For example, one can refer to Example 2 in the specification of U.S. Patent No. 5,641,870; Zapata G et al. Protein Eng. 1995, 8(10), 1057-1062; Olafsen T et al. Protein Eng. Design & Sel. 2004, 17(4): 315-323; Holliger P et al. Nat. Biotechnol. 2005, 23(9): 1126-36; Fischer N et al. Pathobiology. 2007, 74(1): 3-14; Shen J et al. J Immunol Methods. 2007, 318, 65-74; and Wu et al. Nat Biotechnol. 2007, 25(11), 1290-7.

Examples of modified antibody products can include antibodies linked to various molecules such as polyethylene glycol (PEG). The antibodies of the present invention include such modified antibody products. There is no limitation on the substance to be linked in the modified antibody products of the present invention. In order to produce such modified antibody products, chemical modifications can be made to the resulting antibodies. Such methods are well established in the art.

" Bispecific " antibody refers to an antibody with a variable region that recognizes different epitopes, wherein the region is within the same antibody molecule. Bispecific antibodies can be antibodies that recognize two or more different antigens, or antibodies that recognize two or more different epitopes on the same antigen. Bispecific antibodies can include not only complete antibodies, but also antibody sources. The antibodies of the present invention also include bispecific antibodies. Herein, anti-FIXa/FX bispecific antibodies and bispecific antibodies in conjunction with FIXa and FX are used synonymously.

Methods for producing genetically engineered antibodies

Recombinant antibodies produced using genetic engineering techniques can be used as antibodies. Recombinant antibodies can be obtained by cloning DNA encoding the antibody from hybridomas or antibody-producing cells (such as sensitized lymphocytes that produce antibodies), inserting them into vectors, and then introducing them into hosts (host cells) to produce antibodies.

The antibodies include human antibodies, mouse antibodies and rat antibodies, and their origin is not limited. They can also be genetically modified antibodies such as chimeric antibodies and humanized antibodies.

Methods for obtaining human antibodies are known. For example, a transgenic animal carrying the entire component of a human antibody gene can be immunized with a target antigen to obtain a human antibody of interest (see International Publications WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).

Genetically modified antibodies can be produced using known methods. Specifically, for example, chimeric antibodies contain the H and L chain variable regions of an immunized animal antibody and the H and L chain constant regions of a human antibody. Chimeric antibodies can be obtained as follows: DNA encoding the variable regions of an antibody derived from an immunized animal is ligated to DNA encoding the constant regions of a human antibody, inserted into an expression vector, and then introduced into a host to produce the antibody.

Humanized antibodies are modified antibodies, which are also referred to as reshaped human antibodies. Humanized antibodies are constructed by transferring the CDRs of antibodies derived from immunized animals to the complementarity determining regions of human antibodies. Conventional gene recombination techniques for such purposes are known (see European Patent Application Publication No. EP 239400; International Publication No. WO 96/02576; Sato K et al., Cancer Research 1993, 53: 851-856; International Publication No. WO 99/51743).

Bispecific antibodies are antibodies that have specificity for two different antigens.

Although bispecific antibodies are not limited to those of the IgG class, for example, IgG-type bispecific antibodies can be secreted from hybrid hybridomas (quadromas) produced by fusing two types of IgG antibody-producing hybridomas (Milstein C. et al., Nature 1983, 305: 537-540). They can also be secreted by introducing L chain and H chain genes constituting two types of target IgG (a total of four types of genes) into cells to co-express the genes.

In this case, by introducing appropriate amino acid substitutions into the CH3 region of the H chain, IgG having a heterogeneous combination of H chains can be preferentially secreted (Ridgway JB et al. Protein Engineering 1996, 9: 617-621; Merchant AM et al. Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; Davis JH et al. Protein Eng Des Sel. 2010, 4: 195-202).

Regarding L chains, since the diversity of L chain variable regions is lower than that of H chain variable regions, it is foreseeable that a consensus L chain can be generated, which can confer binding ability to both H chains. The antibodies of the present invention can be antibodies comprising a consensus L chain. By introducing genes for the consensus L chain and two H chains into cells, bispecific IgG can be efficiently expressed.

Epitope

The antibody, which is one embodiment of a substance that neutralizes the substance having the activity of functionally replacing FVIII of the present invention, includes antibodies that bind to a specific epitope that overlaps with the epitope bound by the above-mentioned antibodies, and preferably antibodies that bind to the same epitope.

Whether an antibody recognizes the same epitope as the epitope recognized by another antibody or an epitope overlapping therewith can be confirmed by the competition between the two antibodies for the epitope. Using measures such as enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer method (FRET) and fluorescence measurement microvolume assay technology (FMAT (registered trademark)), by competitive binding assay, the competition between antibodies can be evaluated. The amount of the antibody bound to the antigen is indirectly associated with the binding ability of the candidate competing antibody (test antibody), which competitively binds to the same or overlapping epitope. In other words, as the amount or affinity of the test antibody for the same or overlapping epitope increases, the amount of the antibody bound to the antigen decreases, and the amount of the test antibody bound to the antigen increases. Specifically, the antibody and the test antibody that are appropriately labeled are added to the antigen at the same time, and the antibody bound is then detected using the label. By pre-labeling the antibody, the amount of the antibody bound to the antigen can be easily determined. The label is not particularly limited, and the labeling method is selected according to the assay technology used. The specific example of the labeling method includes fluorescent labeling, radioactive labeling and enzyme labeling.

As used herein, "antibodies that bind to overlapping epitopes" or "antibodies that bind to the same epitope" refer to test antibodies that can reduce the amount of labeled antibody binding by at least 50% at a concentration that is typically 100 times, preferably 80 times, more preferably 50 times, even more preferably 30 times, and more preferably 10 times the concentration of unlabeled antibody at which the binding of the unlabeled antibody reduces the amount of labeled antibody binding by 50% (IC ₅₀ ). The epitope recognized by the antibody can be analyzed by methods known to those skilled in the art, for example, it can be performed by Western blotting or the like.

Antibody production methods

The antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, the DNA encoding the target antibody is inserted into an expression vector. The expression vector is inserted so that expression occurs under the control of expression regulatory regions (such as enhancers and promoters). Next, the expression vector is used to express the antibody and transform the host cell. In this step, an appropriate combination of host and expression vector can be used.

Examples of vectors include M13 series vectors, pUC series vectors, pBR322, pBluescript, and pCR-Script. In addition to these vectors, for example, pGEM-T, pDIRECT, or pT7 can also be used for the purpose of cDNA subcloning and excision.

In particular, for use in vectors for the purpose of producing antibodies, expression vectors are useful. For example, when the host is Escherichia coli such as JM109, DH5α, HB101 or XL1-Blue, the expression vector must have a promoter that allows efficient expression in Escherichia coli, for example, lacZ promoter (Ward et al., Nature (1989) 341, 544-546; and FASEB J (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043) or T7 promoter. Examples of such vectors include the above-mentioned vectors as well as pGEX-5X-1 (manufactured by Pharmacia), "QIA Expression System" (manufactured by QIAGEN), pEGFP and pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase).

The vector may contain a signal sequence for polypeptide secretion. In the case of production in the periplasm of E. coli, the pelB signal sequence (Lei, S.P. et al., J. Bacteriol. (1987) 169, 4397) can be used as a signal sequence for polypeptide secretion. The vector can be transferred to the host cell using, for example, the calcium chloride method or electroporation method.

In addition to Escherichia coli expression vectors, examples of vectors for producing the antibodies of the present invention include mammalian-derived expression vectors (e.g., pcDNA3 (manufactured by Invitrogen Corp.), pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, and pCDM8), insect cell-derived expression vectors (e.g., “Bac-to-BAC Baculovirus Expression System” (manufactured by GIBCO BRL) and pBacPAK8), plant-derived expression vectors (e.g., pMH1 and pMH2), animal virus-derived expression vectors (e.g., pHSV, pMV, and pAdexLcw), retrovirus-derived expression vectors (e.g., pZIPneo), yeast-derived expression vectors (e.g., “Pichia Expression Kit” (manufactured by Invitrogen Corp.), pNV11, and SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608 and pKTH50).

For the purpose of expression in animal cells (such as CHO cells, COS cells or NIH3T3 cells), the vector must have a promoter required for intracellular expression, for example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res (1990) 18, 5322), CAG promoter (Gene (1991) 108, 193) or CMV promoter, and more preferably, a gene for selecting transformed cells (for example, a drug resistance gene that can function as a marker with drugs (neomycin, G418, etc.)). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Intent to stably express gene and increase the number of gene copies in the cell includes, with a carrier (e.g., pCHOI) transfected with nucleic acid synthesis pathway defective CHO cells with a DHFR gene that serves as its complement, and methotrexate (MTX) is used in gene amplification. Intent to transiently express gene includes, using COS cells (which have a gene expressing SV40T antigen on their chromosomes) to transform the cells with a carrier (pcD etc.) with the replication origin of SV40. The replication origin derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) etc. can also be used. The expression vector for increasing the number of gene copies in the host cell system can additionally contain selection markers such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene or dihydrofolate reductase (dhfr) gene. The antibodies of the present invention obtained by the above method can be separated from the host cell or from extracellular (culture medium etc.), and purified to pure and uniform antibodies in fact. The antibodies can be separated and purified by methods conventionally used for separating and purifying antibodies, and the types of methods are not limited. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.

The chromatographic methods include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). The chromatographic methods described above can be performed using liquid chromatography, for example, HPLC and FPLC. Columns used for affinity chromatography include protein A columns and protein G columns. Columns using protein A include, for example, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention includes antibodies highly purified using these purification methods.

The obtained antibodies can be purified to homogeneity. The separation and purification of the antibodies can be performed using separation and purification methods commonly used for protein separation and purification. For example, by appropriately selecting and combining column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis and other electric focusing, but not limited thereto, antibodies can be separated and purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns used for affinity chromatography include, for example, protein A columns and protein G columns.

Method for obtaining samples

In the present invention, the sample of blood origin is preferably the sample of blood origin collected from the test subject.Such blood origin sample can be derived from the test subject of the substance having FVIII-replacement activity.Test subject includes, for example, the patient (bleeding disease patient) with bleeding symptoms at any part in the body.The main bleeding site is intra-articular, intramuscular, subcutaneous, intraoral, intracranial, digestive tract, intranasal etc., but is not limited to this.Bleeding disease patient is preferably the patient with the bleeding disease caused by the reduction of FVIII activity and/or FVIIIa activity or lack.The patient with the bleeding disease caused by the reduction of FVIII activity and/or FVIIIa activity or lack is the patient with bleeding symptoms, and example includes the patient with any one or both of FVIII activity and FVIIIa activity before or after reducing or lacking. A reduction in the activity of FVIII and FVIIIa means that these activities in a patient are preferably less than 40% (e.g., less than 40%, less than 30%, less than 20% or less than 10%), more preferably less than 10% (e.g., less than 10%, less than 9%, less than 8%, less than 7% or less than 6%), even more preferably less than 5% (e.g., less than 5%, less than 4%, less than 3% or less than 2%), and particularly preferably less than 1%, compared to the activities in a healthy individual, without being limited thereto.

More specifically, examples of such diseases include, but are not limited to, hemophilia (hemophilia A and hemophilia B), acquired hemophilia, and von Willebrand factor (vWF) dysfunction or deficiency-induced vascular dysplasia (BD). Blood-derived samples include serum, plasma, or whole blood. In the present invention, the use of plasma samples is preferred. Methods for obtaining blood-derived samples from test subjects are well known to those skilled in the art.

Reagent test kit

Different types of reagents such as the buffer required for the reactive method for measuring FVIII of the present invention can be pre-packaged and provided as a test kit. Except buffer, the test kit of the present invention can also include a plasma sample separated from people (the FVIII activity and FIX activity in their blood are normal), a substance with FVIII-replacement activity, and any article that can be used in FVIII activity measurement, or any article that can be used in FVIII inhibitor titer measurement. In addition, the different types of reagents included in the test kit can be made into powder or liquid form according to their usage pattern. In addition, they can be stored in a suitable container and used at the appropriate time.

For example, the method of the present invention can be used to diagnose the severity of a disease in a patient who has been administered a substance that functionally replaces FVIII. The method of the present invention can be used to measure the reactivity of FVIII, and based on the measurement results, the severity of the disease and/or inhibitor titer of the patient can be diagnosed/assessed. The diagnostic and assessment methods can be performed by methods known to those skilled in the art.

For example, the pharmacological activity of a FVIII preparation in a patient administered a FVIII preparation and a substance having the activity of functionally replacing FVIII can be monitored by using the method of the present invention.Monitoring can be performed by methods known to those skilled in the art.

The kit of the present invention can be used as a kit for diagnosing the severity of a disease in a patient who has been administered a substance having an activity that functionally replaces FVIII. The kit of the present invention can be used to measure the reactivity of FVIII, and based on the measurement results, the severity of the disease in the patient can be diagnosed/assessed. The diagnostic and assessment methods can be performed by methods known to those skilled in the art.

The kit of the present invention can be used as, for example, a kit for monitoring the pharmacological activity of a FVIII preparation in a patient administered a FVIII preparation and a substance having an activity that functionally replaces FVIII. Monitoring can be performed by methods known to those skilled in the art.

For example, one may use a method for treating a patient comprising the steps of:

(a) administering a first dose of a substance having the activity of functionally replacing FVIII;

(b) monitoring the responsiveness of FVIII in the patient;

(c) determining, based on the observed reactivity of FVIII, that the second agent has the activity of functionally replacing FVIII; and

(d) administering to said patient a second dose of a substance having activity that functionally replaces FVIII.

Additionally, one can use, for example, a method for treating a patient comprising the steps of:

(a) administering a substance having the activity of functionally replacing FVIII after the first administration interval;

(b) monitoring the responsiveness of FVIII in said patient;

(c) determining a second administration interval for the substance having the activity of functionally replacing FVIII based on the observed reactivity of FVIII; and

(d) administering to the patient a substance having an activity of functionally replacing FVIII after the second administration interval.

One can also use, for example, a method for treating a patient, the method comprising: monitoring the reactivity of FVIII, and varying the dosage and/or interval of administration of the substance having the activity of functionally replacing coagulation factor VIII according to the reactivity of FVIII.

The substance having the activity of functionally replacing FVIII is preferably a bispecific antibody that binds to FIX and/or FIXa and binds to FX and/or FXa. It is more preferably a bispecific antibody in which a first polypeptide binds to a third polypeptide and a second polypeptide binds to a fourth polypeptide.

A bispecific antibody, wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 9, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, and the third polypeptide and the fourth polypeptide are the common L chain (Q499-z121/J327-z119/L404-k) of SEQ ID NO: 10, or

A bispecific antibody, wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 36, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 37, and the third polypeptide and the fourth polypeptide are the common L chain (Q153-G4k/J142-G4h/L180-k) of SEQ ID NO: 38.

For the aforementioned bispecific antibodies, the dosage is, for example, 0.001 mg/kg to 100 mg/kg. It is preferably about 0.001 mg/kg, about 0.003 mg/kg, about 0.005 mg/kg, about 0.01 mg/kg, about 0.03 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg and about 100 mg/kg. The dosage before and after the monitoring step can be the same or different. In the case of the aforementioned bispecific antibodies, the administration interval is, for example, at least 1 day or longer. The interval is preferably 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year. The dosage intervals before and after the monitoring step can be the same or different.

Target patients for the method or kit of the present invention are, for example, hemophilia A patients, acquired hemophilia A patients, von Willebrand disease patients, and hemophilia A patients who have developed inhibitors to FVIII and/or FVIIIa.

As used herein, embodiments expressed by the expression "comprising..." include embodiments expressed by the expression "consisting essentially of..." and embodiments expressed by the expression "consisting of..."

All patents and references explicitly cited herein are incorporated by reference into this specification in their entirety.

The examples will further illustrate the present invention but should not be construed as limiting thereto.

[Example]

Hereinafter, examples will specifically describe the present invention, but should not be construed as being limited thereto.

[Example 1] Production of Antibodies Against Anti-FIXa/FX Bispecific Antibodies and Sequencing of Variable Regions

An attempt was made to produce an antibody against ACE910 (Q499-z121/J327-z119/L404-k) (a bispecific antibody in which an H chain consisting of an amino acid sequence of SEQ ID NO: 9 is bound to an L chain of SEQ ID NO: 10, and an H chain consisting of an amino acid sequence of SEQ ID NO: 11 is bound to an L chain of SEQ ID NO: 10), which is a bispecific antibody described in Patent Document 3 (WO 2012/067176). F(ab')2 consisting of each Fab of the anti-FIXa side and the anti-FX side was produced using gene recombination technology and pepsin digestion.

With anti-FIXa-F (ab ') 2 or anti-FX-F (ab ') 2 immune mice and rats.According to the general method, the spleen obtained from the mouse or rat or the cell obtained from the rat lymph node is fused with the mouse myeloma cell to produce hybridoma.The culture supernatant of hybridoma is evaluated by ELISA, and the ELISA detects the combination of ACE910 with anti-FIXa-arm or anti-FX-arm, and finally selects mouse antibody AQ8 and AQ1 and rat antibody AQ512 (which only binds to anti-FIXa-arm, but does not bind to the anti-FX-arm of ACE910) and rat antibody AJ540, AJ114, AJ521, AJ522 and AJ541 (which only binds to anti-FX-arm, but does not bind to the anti-FIXa-arm of ACE910).In addition, the nucleotide sequence of the variable region of AQ8 antibody or AJ540 antibody is analyzed. The nucleotide sequences of the variable regions of AQ8 and AJ540 and the amino acid sequences predicted therefrom were analyzed using GENETYX Ver. 9 (GENETYX CORPORATION).

[Example 2] Production of expression vectors for recombinant mouse antibody AQ8 and recombinant rat-rabbit chimeric antibody AJ540.

The recombinant mouse antibody AQ8 was prepared as follows: the variable region sequence of the AQ8 antibody obtained in Example 1 was combined with a known mouse IgG2b constant region sequence (heavy chain: EMBL accession number J00461; light chain: EMBL accession number V00807) to generate a full-length antibody gene, which was then inserted into an expression vector. Similarly, the recombinant rat-rabbit chimeric antibody AJ540 was generated by combining a known rabbit IgG (heavy chain: EMBL accession number L29172, light chain: EMBL accession number X00231) with the variable region of the AJ540 antibody. The resulting expression clone plasmid was introduced into HEK293 cells, and large-scale culture and purification using protein A and gel filtration were performed to produce recombinant mouse antibody AQ8 (rAQ8-mIgG2b) and recombinant rat-rabbit chimeric antibody AJ540 (rAJ540-rbtIgG).

[Example 3] One-step coagulation assay using rAQ8-mIgG2b and rAJ540-rbtIgG under neutralization of anti-FIXa/FX bispecific antibody

To FVIII-deficient plasma (George King) containing 10U/dL or 100U/dL recombinant FVIII (Kogenate FS, Bayer Yakuhin, Ltd.), 0μg/mL or 300μg/mL of anti-FIXa/FX bispecific antibody ACE910 was added. In addition, each prepared plasma sample was divided into the following two groups to prepare a measurement sample solution: one group was diluted 10 times using imidazole buffer (Kyowa Medex); and one group was diluted 10 times using imidazole buffer supplemented with 300μg/mL of rAQ8-mIgG2b and rAJ540-rbtIgG. The amount of rAQ8-mIgG2b and rAJ540-rbtIgG required to fully neutralize ACE910 was added. The details of the combination are shown below.

[Table 1]

In addition, in order to produce the calibration curve for converting clotting time into FVIII activity, by using imidazole buffer to perform 10 times, 20 times, 40 times, 80 times and 160 times dilution (the FVIII activity of various calibration curve solutions is designated as 93%, 46.5%, 23.3%, 11.6% and 5.81%), the solution Coagtrol N (Sysmex) of standard plasma is prepared. 50 microlitres of measurement sample solution or calibration curve solution, 50 μL factor VIII-deficient human plasma (Sysmex) and 50 μL Thrombocheck APTT-SLA (Sysmex) are mixed and incubated at 37°C for 5 minutes. After incubation, 50 μL 0.02mol/L calcium chloride solution (Sysmex) is added to start clotting, and the clotting time is measured using an automated blood coagulation analyzer KC4Delta (Stago).

The clotting time of the measurement sample was converted into FVIII activity based on the clotting time at each FVIII activity of the calibration curve solution.

result

The results are shown in Figure 1. When the FVIII-deficient plasma of anti-FIXa/FX bispecific antibody, containing 10U/dL or 100U/dL recombinant FVIII was supplemented with buffer dilution (#3, #7), it was confirmed that FVIII activity was higher than the scope of the calibration curve, and could not be accurately measured. On the other hand, when the FVIII-deficient plasma of anti-FIXa/FX bispecific antibody, containing 10U/dL or 100U/dL recombinant FVIII was supplemented with buffer dilution containing two types of antibodies for anti-FIXa/FX bispecific antibody (#4, #8), it was confirmed that FVIII activity was similar to those activities (#1, #5) of the group not adding anti-FIXa/FX bispecific antibody. Therefore, this shows that the antibody for anti-FIXa/FX bispecific antibody has completely neutralized the activity of bispecific antibody to realize the accurate measurement of the FVIII activity in blood plasma, even in the presence of bispecific antibody. When FVIII-deficient plasma containing 10 U/dL or 100 U/dL of recombinant FVIII was diluted with a buffer containing only two types of antibodies against the anti-FIXa/FX bispecific antibody (#2, #6), FVIII activity was confirmed to be similar to that of the group to which no anti-FIXa/FX bispecific antibody was added (#1, #5); therefore, it was found that the antibodies against the anti-FIXa/FX bispecific antibody had a neutralizing effect specific for the bispecific antibody.

[Example 4] One-step coagulation assay using AQ1 and AJ541 or AQ1 and AJ522 under neutralization of anti-FIXa/FX bispecific antibodies

To FVIII-deficient plasma (George King) containing 10U/dL recombinant FVIII (Kogenate FS, Bayer Yakuhin, Ltd.), 0μg/mL or 10μg/mL of anti-FIXa/FX bispecific antibody ACE910 was added. In addition, each prepared plasma was divided into three groups to prepare a measurement sample solution: one group was diluted 10 times using imidazole buffer (Kyowa Medex); one group was diluted 10 times using imidazole buffer supplemented with 100μg/mL of AQ1 and AJ541; and one group was diluted 10 times using imidazole buffer supplemented with 100μg/mL of AQ1 and AJ522. The amount of AQ1, AJ541 and AJ522 required to fully neutralize ACE910 was added. The details of the combination are shown below.

[Table 2]

In addition, in order to generate a calibration curve for converting clotting time into FVIII activity, a solution of standard plasma, Coagtrol N (Sysmex), was prepared by performing 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold, and 640-fold dilutions using imidazole buffer (the FVIII activities of the various calibration curve solutions were designated as 102%, 51.0%, 25.5%, 12.8%, 6.38%, 3.19%, and 1.59%). 50 microliters of the measurement sample solution or calibration curve solution, 50 μL of factor VIII-deficient human plasma (Sysmex), and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated at 37°C for 5 minutes. After incubation, 50 μL of a 0.02 mol/L calcium chloride solution (Sysmex) was added to initiate coagulation, and the coagulation time was measured using an automated blood coagulation analyzer KC4 Delta (Stago).

The clotting time of the measurement sample was converted into FVIII activity based on the clotting time at each FVIII activity of the calibration curve solution.

result

The results are shown in Figure 2. When the FVIII-deficient plasma supplemented with anti-FIXa/FX bispecific antibody ACE910, containing 10U/dL recombinant FVIII was diluted with buffer (#4), it was confirmed that FVIII activity was higher than the range of the calibration curve and could not be accurately measured. On the other hand, when the FVIII-deficient plasma supplemented with anti-FIXa/FX bispecific antibody ACE910, containing 10U/dL recombinant FVIII was diluted with a buffer (#5) containing two types of antibodies AQ1 and AJ541 for anti-FIXa/FX bispecific antibodies or a buffer (#6) containing two types of antibodies AQ1 and AJ522 for anti-FIXa/FX bispecific antibodies, it was confirmed that FVIII activity was similar to the activity of the group without adding anti-FIXa/FX bispecific antibody (#1). Therefore, this suggests that not only rAQ8-mIgG2b and rAJ540-rbtIgG, but also other antibody combinations, as antibodies against the anti-FIXa/FX bispecific antibody, can also effectively and completely neutralize the activity of the bispecific antibody ACE910.

[Example 5] One-step coagulation assay using AQ512 and AJ114 or AQ512 and AJ521 under neutralization of anti-FIXa/FX bispecific antibodies

To FVIII-deficient plasma (George King) containing 10U/dL recombinant FVIII (Kogenate FS, Bayer Yakuhin, Ltd.), 0μg/mL or 10μg/mL of anti-FIXa/FX bispecific antibody hBS23 was added. In addition, each prepared plasma was divided into three groups to prepare a measurement sample solution: one group was diluted 10 times using imidazole buffer (Kyowa Medex); one group was diluted 10 times using imidazole buffer supplemented with 100μg/mL of AQ512 and AJ114; and one group was diluted 10 times using imidazole buffer supplemented with 100μg/mL of AQ512 and AJ521. The amount of AQ512, AJ114, and AJ521 required to fully neutralize hBS23 was added. The details of the combination are shown below.

[Table 3]

In addition, in order to generate a calibration curve for converting clotting time into FVIII activity, a solution of standard plasma, Coagtrol N (Sysmex), was prepared by performing 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold, and 640-fold dilutions using imidazole buffer (the FVIII activities of the various calibration curve solutions were designated as 102%, 51.0%, 25.5%, 12.8%, 6.38%, 3.19%, and 1.59%). 50 microliters of the measurement sample solution or calibration curve solution, 50 μL of factor VIII-deficient human plasma (Sysmex), and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated at 37°C for 5 minutes. After incubation, 50 μL of a 0.02 mol/L calcium chloride solution (Sysmex) was added to initiate coagulation, and the coagulation time was measured using an automated blood coagulation analyzer KC4 Delta (Stago).

The clotting time of the measurement sample was converted into FVIII activity based on the clotting time at each FVIII activity of the calibration curve solution.

result

The results are shown in Figure 3. When FVIII-deficient plasma supplemented with anti-FIXa/FX bispecific antibody hBS23, containing 10U/dL recombinant FVIII, was diluted with buffer (#4), it was confirmed that FVIII activity was higher than the range of the calibration curve and could not be accurately measured. On the other hand, when FVIII-deficient plasma supplemented with anti-FIXa/FX bispecific antibody hBS23, containing 10U/dL recombinant FVIII, was diluted with a buffer (#5) containing two types of antibodies AQ512 and AJ114 for anti-FIXa/FX bispecific antibodies or a buffer (#6) containing two types of antibodies AQ512 and AJ521 for anti-FIXa/FX bispecific antibodies, it was confirmed that FVIII activity was similar to that of the group without the addition of anti-FIXa/FX bispecific antibody (#1). These indicate that even with hBS23 (a bispecific antibody different from ACE910), FVIII activity in plasma can be accurately measured despite the presence of the bispecific antibody (by completely neutralizing its activity), and thus the approach of the present invention is effective for various bispecific antibodies with FVIII-replacing activity.

[Example 6] Bethesda assay using rAQ8-mIgG2b and rAJ540-rbtIgG under neutralization of anti-FIXa/FX bispecific antibody

To factor VIII-deficient human plasma (containing FVIII inhibitor) (George King Bio-Medical), 0 μg/mL or 300 μg/mL of anti-FIXa/FX bispecific antibody ACE910 was added. In addition, the plasma sample of each preparation was diluted 25 times or 30 times using an imidazole buffer (Kyowa Medex) (hereinafter referred to as BSA-imidazole) containing 0.25% (w/v) bovine serum albumin (Sigma-Aldrich). To Coagtrol N (Sysmex) (which is standard plasma), rAQ8-mIgG2b and rAJ540-rbtIgG were not added, or they were each added at 300 μg/mL.

The plasma samples prepared in both categories were mixed in equal amounts in the following combinations (8 categories in total), and then incubated at 37°C for 2 hours.

[Table 4]

After incubation, the mixed solution was further diluted 10-fold with BSA-imidazole to prepare a measurement sample solution. In addition, in order to prepare a calibration curve for converting clotting time into a FVIII activity value, solutions were prepared by diluting Coagtrol N with BSA-imidazole at 20-fold, 40-fold, 80-fold, 160-fold, and 320-fold dilutions (the FVIII activities of the various calibration curve solutions were designated as 100%, 50%, 25%, 12.5%, and 6.25%).

50 microliters of the measurement sample solution or calibration curve solution, 50 μL of factor VIII-deficient human plasma (Sysmex) and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated for 3 minutes at 37° C. After incubation, 50 μL of 0.02 mol/L calcium chloride solution (Sysmex) was added to initiate coagulation, and the coagulation time was measured using an automated blood coagulation analyzer KC4 Delta (Stago).

The clotting time of the measurement sample was converted into FVIII activity based on the clotting time at each FVIII activity of the calibration curve solution. In addition, when the residual FVIII activity was 50%, it was designated as 1 Bethesda, and after calculating the Bethesda value of the measurement sample, the average value calculated by multiplying the value by 25 or 30 was determined as the inhibitor titer in each original sample solution.

result

The results are shown in Figure 4. FVIII inhibitor plasma containing only the anti-FIXa/FX bispecific antibody (#3) exhibited an activity of 100% or more of FVIII of the calibration curve; therefore, the FVIII inhibitor titer could not be determined.

On the other hand, FVIII inhibitor plasma (#4) containing anti-FIXa/FX bispecific antibodies and two types of antibodies for anti-FIXa/FX bispecific antibodies showed FVIII inhibitor titers similar to those of inhibitor plasma (#1) without additives. Therefore, this shows that antibodies for anti-FIXa/FX bispecific antibodies completely neutralize the activity of bispecific antibodies to achieve accurate measurement of FVIII inhibitor titers in plasma, even in the presence of bispecific antibodies. FVIII inhibitor plasma (#2) containing only two types of antibodies for anti-FIXa/FX bispecific antibodies showed results similar to those of #1; Therefore, it was found that antibodies for anti-FIXa/FX bispecific antibodies have a neutralizing effect specific to bispecific antibodies.

Industrial Applicability

The present invention provides a method for measuring the reactivity of FVIII in the presence of a bispecific antibody having an activity that functionally replaces FVIII, for example, a method for measuring FVIII activity or FVIII inhibitor titer. By using bispecific antibodies, the application of the methods of the present invention can accurately measure the reactivity of FVIII in patients in the treatment of bleeding disorders (such as hemophilia).

Sequence Listing

<110> Nara Prefectural Medical University

Chugai Pharmaceutical Co., Ltd.

<120> Method for measuring the reactivity of FVIII

<130> C1-A1406P

<150> JP 2014-196974

<151> 2014-09-26

<160> 38

<170> PatentIn version 3.5

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Glu Val Asn Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Met Ser Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

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Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Ser Ala Thr Tyr

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Tyr Cys Ala Arg Asp Ser Tyr Tyr Ser Tyr Asp Gly Tyr Ala Met Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ala Ala Ser Val Gly

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Glu Thr Ile Thr Ile Thr Cys Gln Ala Ser Glu Asn Ile Tyr Phe Ser

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Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile

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Tyr Asn Thr Asp Ser Leu Lys Asp Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Gln Tyr Ser Met Lys Ile Asn Ser Met Gln Pro

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Glu Asp Thr Ala Thr Tyr Phe Cys Arg Gln Ser Tyr Asp Phe Pro Trp

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

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Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

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Ala Met His Trp Val Lys Gln Val Pro Gly Lys Gly Leu Lys Trp Met

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Gly Trp Ile Asn Thr Tyr Thr Gly Lys Pro Thr Tyr Ala Asp Asp Phe

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Lys Gly Arg Phe Val Phe Ser Leu Glu Ala Ser Ala Ser Thr Ala Asn

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Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Asn Val Phe Cys

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Ala Arg Glu Gly Gly Gly Tyr Tyr Trp Tyr Phe Asp Phe Trp Gly Pro

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Val Asp Trp Tyr Gln Gln Lys Thr Gly Gln Ser Pro Lys Leu Leu Ile

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Tyr Lys Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

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Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ala

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Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser Asn Ser Phe Pro Leu

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gaagtgaacc tggtggaaag cggcggaggc ctggtgcagc ccggaggcag catgtctctg 60

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cctggcaagg ctctggaatg gctggctctg atcagaaaca aggccaacgg ctacaccacc 180

gagtacagcg ccagcgtgaa gggccggttc accatcagcc gggacaacag ccagagcatc 240

ctgtacctgc agatgaacgc cctgcgggcc gaggactccg ccacctacta ctgcgccaga 300

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ggcaagagcc cccagctgct gatctacaac accgacagcc tgaaggacgg cgtgcccagc 180

agattcagcg gcagcggctc cggcacccag tacagcatga agatcaacag catgcagccc 240

gaggacaccg ctacctacttttgccggcag agctacgact tcccctggac cttcggcgga 300

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<212> DNA

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<220>

<223> Nucleic acid sequence of AJ540 VH

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cagatccagc tggtgcagag cggccctgag ctgaagaaac ccggcgagag cgtgaagatc 60

agctgcaagg ccagcggcta caccttcacc gactacgcca tgcactgggt gaaacaggtg 120

cccggcaagg gcctgaagtg gatgggctgg atcaacacct acaccggcaa gcccacctac 180

gccgacgact tcaagggcag attcgtgttc agcctggaag ccagcgccag caccgccaac 240

ctgcagatca gcaacctgaa gaacgaggac accgccaacg tgttctgcgc cagagagggc 300

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<223> Nucleic acid sequence of AJ540 VL

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gacatcgtga tgacccagag ccccaccagc atgagcatca gcgtgggcga cagagtgacc 60

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ggccagtccc ccaagctgct gatctacaag gccagcaacc ggtacacagg cgtgcccgac 180

agattcacag gcagcggcag cggcaccgac ttcaccttca ccatcagcaa catgcaggcc 240

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr

20 25 30

Asp Ile Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Pro Ser Gly Gln Ser Thr Tyr Tyr Tyr Arg Arg Glu Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Thr Gly Arg Glu Tyr Gly Gly Gly Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Gln Lys Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

<210> 10

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<212> PRT

<213> Artificial

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg Gln

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Gln Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asp Pro Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 444

<212> PRT

<213> Artificial

<220>

<223> Artificial sequence

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Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe

50 55 60

Gln Asp Arg Val Ile Met Thr Val Asp Lys Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr His Cys

85 90 95

Ala Arg Arg Lys Ser Tyr Gly Tyr Tyr Leu Asp Glu Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Glu Ser Leu Ser Leu Ser Pro

435 440

<210> 12

<211> 5

<212> PRT

<213> Artificial

<220>

<223> AQ8 heavy chain CDR1

<400> 12

Asp Tyr Tyr Met Ser

1 5

<210> 13

<211> 19

<212> PRT

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<220>

<223> AQ8 heavy chain CDR2

<400> 13

Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 14

<211> 13

<212> PRT

<213> Artificial

<220>

<223> AQ8 heavy chain CDR3

<400> 14

Asp Ser Tyr Tyr Ser Tyr Asp Gly Tyr Ala Met Asp Tyr

1 5 10

<210> 15

<211> 15

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 heavy chain CDR1

<400> 15

gactactaca tgagc 15

<210> 16

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<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 heavy chain CDR2

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ctgatcagaa acaaggccaa cggctacacc accgagtaca gcgccagcgt gaagggc 57

<210> 17

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 heavy chain CDR3

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gacagctact acagctacga cggctacgcc atggactac 39

<210> 18

<211> 11

<212> PRT

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<220>

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<400> 18

Gln Ala Ser Glu Asn Ile Tyr Phe Ser Leu Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial

<220>

<223> AQ8 light chain CDR2

<400> 19

Asn Thr Asp Ser Leu Lys Asp

1 5

<210> 20

<211> 9

<212> PRT

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<220>

<223> AQ8 light chain CDR3

<400> 20

Arg Gln Ser Tyr Asp Phe Pro Trp Thr

1 5

<210> 21

<211> 33

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 light chain CDR1

<400> 21

caggccagcg agaacatcta cttcagcctg gcc 33

<210> 22

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 light chain CDR2

<400> 22

aacaccgaca gcctgaagga c 21

<210> 23

<211> 27

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AQ8 light chain CDR3

<400> 23

cggcagagct acgacttccc ctggacc 27

<210> 24

<211> 5

<212> PRT

<213> Artificial

<220>

<223> AJ540 heavy chain CDR1

<400> 24

Asp Tyr Ala Met His

1 5

<210> 25

<211> 17

<212> PRT

<213> Artificial

<220>

<223> AJ540 heavy chain CDR2

<400> 25

Trp Ile Asn Thr Tyr Thr Gly Lys Pro Thr Tyr Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210> 26

<211> 11

<212> PRT

<213> Artificial

<220>

<223> AJ540 heavy chain CDR3

<400> 26

Glu Gly Gly Gly Tyr Tyr Trp Tyr Phe Asp Phe

1 5 10

<210> 27

<211> 15

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 heavy chain CDR1

<400> 27

gactacgcca tgcac 15

<210> 28

<211> 51

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 heavy chain CDR2

<400> 28

tggatcaaca cctacaccgg caagcccacc tacgccgacg acttcaaggg c 51

<210> 29

<211> 33

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 heavy chain CDR3

<400> 29

gagggcggag gctactactg gtacttcgac ttc 33

<210> 30

<211> 11

<212> PRT

<213> Artificial

<220>

<223> AJ540 light chain CDR1

<400> 30

Lys Ala Asn Gln Asn Val Asp Phe Asn Val Asp

1 5 10

<210> 31

<211> 7

<212> PRT

<213> Artificial

<220>

<223> AJ540 light chain CDR2

<400> 31

Lys Ala Ser Asn Arg Tyr Thr

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial

<220>

<223> AJ540 light chain CDR3

<400> 32

Met Gln Ser Asn Ser Phe Pro Leu Thr

1 5

<210> 33

<211> 33

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 light chain CDR1

<400> 33

aaggccaacc agaacgtgga cttcaacgtg gac 33

<210> 34

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 light chain CDR2

<400> 34

aaggccagca accggtacac a 21

<210> 35

<211> 27

<212> DNA

<213> Artificial

<220>

<223> Nucleic acid sequence of AJ540 light chain CDR3

<400> 35

atgcagagca acagcttccc cctgacc 27

<210> 36

<211> 448

<212> PRT

<213> Artificial

<220>

<223> Artificial sequence

<400> 36

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Arg Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Arg Ala Gly His Asn Tyr Gly Ala Gly Trp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys

210 215 220

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

260 265 270

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440 445

<210> 37

<211> 444

<212> PRT

<213> Artificial

<220>

<223> Artificial sequence

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Asn

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile Asn Thr Arg Ser Gly Gly Ser Ile Tyr Asn Glu Glu Phe

50 55 60

Gln Asp Arg Val Thr Met Thr Ile Asp Lys Ser Thr Gly Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Arg Ser Tyr Gly Tyr Tyr His Asp Glu Trp Gly Glu Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Cys Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

435 440

<210> 38

<211> 214

<212> PRT

<213> Artificial

<220>

<223> Artificial sequence

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Arg Asn Ile Glu Arg Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Glu Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Arg Lys Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Arg Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Pro Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims (31)

1. The use of one or more neutralizing substances in the preparation of a reagent for measuring the reactivity of coagulation factor VIII, wherein the one or more neutralizing substances neutralize a substance having activity that functionally replaces coagulation factor VIII, wherein the one or more neutralizing substances are one or more antibodies and for contacting a blood-derived sample containing a substance having activity that functionally replaces coagulation factor VIII, wherein the substance having activity that functionally replaces coagulation factor VIII is a bispecific antibody that binds coagulation factor IX and/or activated coagulation factor IX and binds coagulation factor X and/or activated blood coagulation factor X, wherein the reactivity of coagulation factor VIII is measured by a method for measuring coagulation factor VIII activity or by a method for measuring the titer of coagulation factor VIII inhibitors. 2. The application according to claim 1, wherein the bispecific antibody is any one of the following antibodies, wherein the first polypeptide binds to the third polypeptide and the second polypeptide binds to the fourth polypeptide: A bispecific antibody, wherein the first polypeptide is an H-chain composed of the amino acid sequence of SEQ ID NO:9, the second polypeptide is an H-chain composed of the amino acid sequence of SEQ ID NO:11, and the third polypeptide and the fourth polypeptide are a common L-chain of SEQ ID NO:10; or A bispecific antibody, wherein the first polypeptide is an H chain composed of the amino acid sequence of SEQ ID NO:36, the second polypeptide is an H chain composed of the amino acid sequence of SEQ ID NO:37, and the third polypeptide and the fourth polypeptide are a common L chain of SEQ ID NO:38. 3. The application according to claim 2, wherein the one or more neutralizing substances are one or more antibodies selected from the group consisting of: an antibody binding to Fab, the Fab comprising an antigen-binding site for binding coagulation factor IX; an antibody binding to Fab, the Fab comprising an antigen-binding site for binding activated coagulation factor IX; an antibody binding to Fab, the Fab comprising an antigen-binding site for binding coagulation factor X; an antibody binding to Fab, the Fab comprising an antigen-binding site for binding activated coagulation factor X; and a bispecific antibody that binds to a Fab comprising an antigen-binding site for binding coagulation factor IX and/or activated coagulation factor IX and a Fab comprising an antigen-binding site for binding coagulation factor X and/or activated coagulation factor X. 4. The application according to claim 3, wherein the one or more neutralizing substances are a combination of one or more selected from the group consisting of: (a) An antibody that binds to Fab, the Fab comprising an antigen-binding site for binding coagulation factor IX, and an antibody that binds to Fab, the Fab comprising an antigen-binding site for binding coagulation factor X. (b) An antibody that binds to Fab, said Fab comprising an antigen-binding site for binding activated coagulation factor IX, and an antibody that binds to Fab, said Fab comprising an antigen-binding site for binding coagulation factor X. (c) An antibody that binds to Fab, said Fab comprising an antigen-binding site for binding coagulation factor IX, and an antibody that binds to Fab, said Fab comprising an antigen-binding site for binding activated coagulation factor IX, and (d) An antibody that binds to Fab, the Fab comprising an antigen-binding site for binding coagulation factor IX; an antibody that binds to Fab, the Fab comprising an antigen-binding site for binding coagulation factor X; and an antibody that binds to Fab, the Fab comprising an antigen-binding site for binding activated coagulation factor IX. 5. Use of one or more neutralizing substances having the activity of functionally replacing coagulation factor VIII in the preparation of a diagnostic agent or kit for diagnosing the severity of hemophilia in a patient, the patient being administered the substance having the activity of functionally replacing coagulation factor VIII, wherein the one or more neutralizing substances are one or more antibodies and for contacting a blood-derived sample containing the substance having the activity of functionally replacing coagulation factor VIII, wherein the substance having the activity of functionally replacing coagulation factor VIII is a bispecific antibody binding to coagulation factor IX and/or activated coagulation factor IX and coagulation factor X and/or activated coagulation factor X. 6. The use of claim 5, wherein the substance having the activity of functionally replacing coagulation factor VIII is as defined in claim 2. 7. The use of claim 5 or 6, wherein the one or more neutralizing substances are defined as in any one of claims 3-4. 8. Use of one or more neutralizing substances having activity that functionally replaces coagulation factor VIII in the preparation of a diagnostic agent or kit for diagnosing inhibitory titers of coagulation factor VIII in patients, said patients being administered substances having activity that functionally replaces coagulation factor VIII, wherein said one or more neutralizing substances are one or more antibodies and for contacting a blood-derived sample containing said substance having activity that functionally replaces coagulation factor VIII, wherein the substance having activity that functionally replaces coagulation factor VIII is a bispecific antibody binding to coagulation factor IX and/or activated coagulation factor IX and coagulation factor X and/or activated coagulation factor X. 9. The use of claim 8, wherein the substance having the activity of functionally replacing coagulation factor VIII is as defined in claim 2. 10. The use of claim 8 or 9, wherein the one or more neutralizing substances are as defined in any one of claims 3-4. 11. Use of one or more neutralizing substances having activity that functionally replaces coagulation factor VIII in the preparation of a method monitoring agent or kit for monitoring the pharmacological activity of an FVIII formulation in a patient who is administered an FVIII formulation and a substance having activity that functionally replaces coagulation factor VIII, wherein the one or more neutralizing substances are one or more antibodies and for contacting a blood-derived sample containing the substance having activity that functionally replaces coagulation factor VIII, wherein the substance having activity that functionally replaces coagulation factor VIII is a bispecific antibody binding to coagulation factor IX and/or activated coagulation factor IX and coagulation factor X and/or activated coagulation factor X. 12. The use of claim 11, wherein the substance having the activity of functionally replacing coagulation factor VIII is as defined in claim 2. 13. The use of claim 11 or 12, wherein the one or more neutralizing substances are as defined in any one of claims 3-4. 14. The use according to claim 5, wherein the patient administering the substance is selected from the group consisting of patients with hemophilia A and patients with von Willebrand disease. 15. The use according to claim 5, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 16. The use according to claim 6, wherein the patient to whom the substance is administered is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 17. The use according to claim 6, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 18. The use according to claim 7, wherein the patient to whom the substance is administered is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 19. The use according to claim 7, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 20. The use according to claim 8, wherein the patient administering the substance is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 21. The use according to claim 8, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 22. The use according to claim 9, wherein the patient to whom the substance is administered is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 23. The use according to claim 9, wherein the patient to whom the substance is administered is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 24. The use according to claim 10, wherein the patient to whom the substance is administered is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 25. The use according to claim 10, wherein the patient to whom the substance is administered is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 26. The use according to claim 11, wherein the patient administering the substance is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 27. The use according to claim 11, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 28. The use according to claim 12, wherein the patient administering the substance is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 29. The use according to claim 12, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present. 30. The use according to claim 13, wherein the patient administering the substance is a patient selected from the group consisting of: hemophilia A patients and von Willebrand disease patients. 31. The use according to claim 13, wherein the patient administering the substance is selected from the group consisting of: patients with acquired hemophilia A and patients with hemophilia A in which an inhibitor of blood clotting factor VIII and/or activated blood clotting factor VIII is present.