Antibodies that neutralize substances with functional substitution activity for coagulation factor VIII (F.VIII)

Abstract

Production was attempted for an antibody that neutralizes the activity of a bispecific antibody having F.VIII function-substituting activity, for use in a method for measuring the reactivity of F.VIII in the presence of a bispecific antibody having F.VIII function-substituting activity. As a result, it was found that use of the produced antibody made it possible to accurately evaluate F.VIII activity in blood plasma of a hemophilia A patient, in an APTT-based one-stage clotting assay with respect to a wide range of bispecific antibodies having F.VIII function-substituting activity. Furthermore, it was found that it was also possible to accurately evaluate F.VIII inhibitor titer in blood plasma of a hemophilia A patient having an F.VIII inhibitor, in an APTT-based Bethesda assay.

Description

[Technical Field]

[0001] The present invention relates to an antibody that neutralizes a substance having an activity of substituting for coagulation factor VIII (F.VIII), and also to an antibody for use in a method for measuring the reactivity of F.VIII in the presence of a substance having an activity of substituting for F.VIII. [Background technology]

[0002] Hemophilia is a bleeding disorder caused by a congenital deficiency or dysfunction of F.VIII or coagulation factor IX (F.IX). The former is called hemophilia A, and the latter is called hemophilia B. Both genes are present on the X chromosome, and the genetic abnormality is inherited in an X-linked recessive manner, so more than 99% of affected patients are male. The prevalence rate is approximately 1 in 10,000 male births, and the ratio of hemophilia A to hemophilia B is known to be approximately 5:1.

[0003] The main bleeding sites in hemophilia patients include intra-articular, intramuscular, subcutaneous, oral, intracranial, gastrointestinal, and nasal cavities. In particular, repeated bleeding into joints can progress to hemophilic arthropathy, which is accompanied by joint damage and difficulty walking, and may ultimately require joint replacement. This is a major factor that reduces the quality of life of hemophilia patients.

[0004] The severity of hemophilia correlates well with the F.VIII or F.IX activity in the blood. Patients with less than 1% clotting factor activity are classified as severe, those with activity between 1% and 5%, and those with activity between 5% and 40% are classified as moderate, and those with activity between 5% and 40%, respectively. Severe hemophilia, which accounts for approximately half of all hemophilia patients, will experience bleeding episodes several times a month without preventive replacement therapy, which is significantly more frequent than moderate and mild patients.

[0005] Related bleeding disorders include hemophilia, acquired hemophilia, and von Willebrand disease, which is caused by a functional defect or deficiency of von Willebrand factor (vWF). vWF is necessary not only for platelets to adhere normally to the subendothelial tissue at the site of injury in the blood vessel wall, but also for forming a complex with F.VIII and maintaining normal F.VIII levels in the blood. In patients with von Willebrand disease, these functions are impaired, resulting in impaired hemostasis.

[0006] Blood coagulation factors purified from plasma or produced by recombinant DNA technology are primarily used to prevent and / or treat bleeding in hemophilia patients. In severe hemophilia patients, F.VIII or F.IX replacement therapy, maintaining blood F.VIII or F.IX activity at 1% or higher, is considered effective in preventing bleeding episodes (Non-Patent Documents 1 and 2). However, hemophilia patients, particularly those with severe hemophilia, may develop antibodies against F.VIII or F.IX, known as inhibitors. When inhibitors develop, the effectiveness of coagulation factor preparations is impaired. Consequently, neutralization therapy using large amounts of coagulation factor preparations or bypass therapy using complex concentrates or activated coagulation factor VII preparations (F.VIIa preparations) are performed.

[0007] F.VIII activity measurement in hemophilia A is mainly performed using the one-stage clotting assay (Non-Patent Document 3), which is based on activated partial thromboplastin time (APTT), and the chromogenic assay, which is a reconstitution system using purified coagulation factors (Non-Patent Document 4). F.VIII inhibitor titration in hemophilia A is mainly performed by the Bethesda assay or Nijmegen Bethesda assay (Non-patent documents 5 and 6).

[0008] Recently, a bispecific antibody, emicizumab, has been discovered that binds to both F.IX and / or activated coagulation factor IX (F.IXa) and coagulation factor X (FX) and / or activated blood coagulation factor X (F.Xa) and substitutes for the cofactor function of F.VIII, i.e., promoting FX activation by F.IXa (Non-Patent Documents 7, 8, Patent Documents 1, 2, and 3). By substituting the function of F.VIII, this bispecific antibody ameliorates the impaired coagulation response caused by F.VIII deficiency or dysfunction. For example, with regard to APTT and thrombin generation, which are indicators of the coagulation response, this bispecific antibody shortens the APTT and increases thrombin generation in plasma from hemophilia A patients, regardless of the presence of F.VIII inhibitors. The APTT-shortening effect of this bispecific antibody is more pronounced than that of F.VIII. This is because F.VIII in plasma exhibits cofactor activity only after activation by thrombin or activated factor X (F.Xa), whereas this bispecific antibody does not require such an activation process and therefore exhibits cofactor function more quickly. Furthermore, antibodies against the F.IXa Fab and FX Fab of this bispecific antibody were obtained, and the concentrations of this bispecific antibody in plasma samples from animal experiments were measured (Non-Patent Document 9).

[0009] Because this bispecific antibody substitutes for the cofactor function of F.VIII, it affects assay systems measuring the reactivity of F.VIII itself. For example, when measuring plasma F.VIII activity using an APTT-based one-stage clotting assay to diagnose the severity of hemophilia A or monitor the pharmacological activity of F.VIII preparations in patients administered F.VIII preparations, the bispecific antibody's ability to promote coagulation time strongly interferes, significantly reducing accuracy. Furthermore, when measuring plasma F.VIII inhibitor titers using the APTT-based Bethesda assay, the bispecific antibody's ability to promote coagulation time strongly interferes, significantly reducing accuracy. In other words, accurate measurements of F.VIII activity and F.VIII inhibitor titers cannot be performed in patients administered this bispecific antibody. Therefore, a method for measuring F.VIII activity and F.VIII inhibitor titers is needed, even in the presence of this bispecific antibody.

[0010] Antibodies against emicizumab, a bispecific antibody that substitutes for the function of promoting FX activation by F.IXa, and methods for measuring F.VIII activity and F.VIII inhibitor titer using the antibodies have also been developed (Patent Documents 4 and 5).

[0011] Emicizumab is not the only bispecific antibody that substitutes for the function of promoting FX activation by F.IXa; novel bispecific antibodies with improved function are also known (Patent Document 6). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2005 / 035756 [Patent Document 2] WO2006 / 109592 [Patent Document 3] WO2012 / 067176 [Patent Document 4] WO2016 / 047656 [Patent Document 5] WO2016 / 047652 [Patent Document 6] WO2019 / 065795 [Non-patent literature]

[0013] [Non-Patent Document 1] N Engl J Med. 2007;357(6):535-44 [Non-patent document 2] Thromb Res. 2011;127 (suppl1):S14-7 [Non-patent document 3] Thromb Diath Haemorrh. 1962 May 15;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 Document 7] Nat Med. 2012; 18(10):1570-74 [Non-patent document 8] PLoS One. 2013; 8(2):e57479. [Non-Patent Document 9] J Thromb Haemost. 2014; 12(2):206-13 Supporting Information Summary of the Invention [Problem to be solved by the invention]

[0014] As described above, in patients with bleeding disorders such as hemophilia who have been administered a bispecific antibody, the presence of the bispecific antibody in plasma samples makes it difficult to accurately measure F.VIII activity and F.VIII inhibitor titers. The present invention addresses this problem by providing an antibody that neutralizes a substance with F.VIII functional substituting activity, which can be used in a method for accurately measuring F.VIII reactivity even in the presence of a substance with F.VIII functional substituting activity, for example, a method for accurately measuring F.VIII activity and F.VIII inhibitor titers. Another object of the present invention is to provide a method, kit, etc. for measuring F.VIII reactivity, for example, F.VIII activity and F.VIII inhibitor titers, in the presence of a substance with F.VIII functional substituting activity. [Means for solving the problem]

[0015] To achieve the above objectives, the present inventors prepared a substance that neutralizes the activity of a bispecific antibody with F.VIII functional substituting activity, targeting a test item for measuring F.VIII reactivity, and investigated whether the accuracy of the assessment of F.VIII reactivity is maintained even in the presence of the bispecific antibody. As a result, the present inventors found that by using a neutralizing antibody against a bispecific antibody with F.VIII functional substituting activity at a concentration that sufficiently neutralizes the bispecific antibody, F.VIII activity in the plasma of hemophilia A patients can be accurately assessed using an APTT-based one-stage clotting assay. The present inventors also succeeded in developing a kit containing a neutralizing antibody against a bispecific antibody with F.VIII functional substituting activity, which can be used in this assay. Furthermore, the present inventors found that the antibody of the present invention neutralizes a wide range of bispecific antibodies. Based on these findings, the present invention provides the following.

[0016] [1] An antibody that neutralizes a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated blood coagulation factor X, wherein the bispecific antibody is selected from the group consisting of the following (a) to (t): (a) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 83, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 88, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; (b) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; (c) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (d) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (e) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 102, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113; (f) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 103, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 114; (g) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (h) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 92, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (i) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (j) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (k) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (l) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116; (m) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (n) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116; (o) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 96, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117; (p) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 97, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 107, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 118; (q) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117; (r) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 108, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 119; (s) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 96, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 109, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 120; and (t) A bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 122, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 110, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 121. [2] The bispecific antibody (1) a first antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 139 and a first antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 140; and (2) A second antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 141 and a second antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 142. The neutralizing antibody of [1], further comprising: [3] An antibody that neutralizes a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated blood coagulation factor X, wherein the bispecific antibody is selected from the group consisting of the following (a) to (t): (a) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 143, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 149, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 161, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 172; (b) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 161, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 172; (c) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 145, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 151, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (d) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 145, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 152, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (e) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 163, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 174; (f) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 164, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 175; (g) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (h) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 153, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (i) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 154, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (j) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 154, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (k) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 155, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (l) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 155, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 166, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 177; (m) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 156, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (n) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 156, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 166, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 177; (o) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 157, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 178; (p) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 158, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 168, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 179; (q) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 159, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 178; (r) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 159, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 180; (s) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 157, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 181; and (t) A bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 148, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 160, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 182. [4] An antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, or an antibody that binds to an epitope that overlaps with or is the same epitope as the epitope bound by said antibody. [5] An antibody comprising an antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 131 and an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 132, or an antibody that binds to an epitope that overlaps with or is the same epitope as the epitope bound by said antibody. [6] An antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4, or an antibody that binds to an epitope that overlaps with or is the same epitope as the epitope bound by said antibody. [7] An antibody comprising an antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 135 and an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 136, or an antibody that binds to an epitope that overlaps with or is the same epitope as the epitope bound by said antibody. [8] A nucleic acid encoding the antibody according to any one of [1] to [7]. [9] A vector into which the nucleic acid according to [8] is inserted.

[10] A cell comprising the nucleic acid of [8] or the vector of [9].

[11] A method for producing an antibody, comprising a step of culturing the cell according to

[10] .

[12] A composition comprising the antibody of [4] and / or the antibody of [6].

[13] A composition comprising the antibody of [5] and / or the antibody of [7].

[14] A method for measuring the reactivity of coagulation factor VIII, comprising the step of contacting the following (1) with (2): (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated coagulation factor X; (2) The antibody according to [4] or [5] and / or the antibody according to [6] or [7].

[15] A method for measuring the reactivity of coagulation factor VIII, comprising the step of contacting the following (1) with (2): (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated coagulation factor X; (2) The composition according to

[12] or

[13] .

[16] A kit for use in the method according to

[15] , comprising the composition according to

[12] or

[13] .

[0017] The present invention also provides the following items

[101] to

[106] .

[101] An antibody that binds to a Fab comprising an antigen-binding site that binds to coagulation factor IX and / or activated coagulation factor IX, a) the heavy chain variable region comprises CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 12, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 14; b) An antibody whose light chain variable region comprises CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 18, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[102] An antibody that binds to a Fab comprising an antigen-binding site that binds to coagulation factor X and / or activated coagulation factor X, a) the heavy chain variable region comprises CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 24, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26; b) An antibody whose light chain variable region comprises CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 30, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 32.

[103] A nucleic acid encoding the antibody according to any one of

[101] to

[102] .

[104] A vector into which the nucleic acid described in

[103] is inserted.

[105] A cell comprising the nucleic acid described in

[103] or the vector described in

[104] .

[106] A method for producing an antibody, comprising the step of culturing the cell described in

[105] . [Effects of the Invention]

[0018] The present invention provides antibodies that can be used to measure F.VIII activity and F.VIII inhibitor titers without being affected by the activity of substances that have F.VIII functional substituting activity. Substances that have F.VIII functional substituting activity include bispecific antibodies that bind to F.IX and / or F.IXa and FX and / or F.Xa. By using the antibodies provided by the present invention, it is possible to accurately measure F.VIII activity and F.VIII inhibitor titers even in the presence of substances that have F.VIII functional substituting activity in a sample. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of a one-stage clotting assay under anti-F.IXa / FX bispecific antibody neutralization using IDA0288 and IDA0339 or rAQ8 and rAJ540. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to an antibody that neutralizes a substance having a functionally substituting activity for F.VIII and a nucleic acid encoding the antibody. The antibody of the present invention can be used to measure F.VIII activity and F.VIII inhibitor titer in a sample when measuring F.VIII reactivity in the presence of a substance having a functionally substituting activity for F.VIII, without being affected by the activity of the substance. The nucleic acid in the present invention includes both DNA and mRNA forms.

[0021] F.VIII functional alternative activity F.VIII is one of a series of molecules involved in blood coagulation, and when activated by thrombin or F.Xa, it exhibits cofactor activity and promotes the FX activation reaction by F.IXa. Substances having F.VIII functional substitution activity of the present invention can also be referred to as substances having F.VIII-like activity. In the present invention, "substituting the function of F.VIII" means promoting the activation of FX by F.IXa (promoting F.Xa production by F.IXa). More specifically, in the present invention, "substituting the function of F.VIII" means recognizing F.IX and / or F.IXa and FX and / or F.Xa and promoting the activation of FX by F.IXa (promoting F.Xa production by F.IXa). The F.Xa production-promoting activity can be evaluated, for example, using an assay system consisting of F.IXa, FX, the synthetic substrate S-2222 (a synthetic substrate for F.Xa), and phospholipids. Such assays correlate with disease severity and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomback M et al. Clinical applications of a chromogenic substrate method for determination of F.VIII activity. Thromb Haemost 1985; 54: 811-23).

[0022] A preferred embodiment of the substance of the present invention having an activity that substitutes for the function of F.VIII is a bispecific antibody that binds to F.IX and / or F.IXa and FX and / or F.Xa. Such antibodies can be obtained, for example, according to the methods described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, WO2019 / 065795, etc. Bispecific antibodies of the present invention include the antibodies described in these documents.

[0023] Preferred bispecific antibodies include emicizumab (Q499-z121 / J327-z119 / L404-k) (a bispecific antibody in which a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 is associated with a light chain set forth in SEQ ID NO: 10, and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 is associated with a light chain set forth in SEQ ID NO: 10), which is a bispecific antibody described in patent document (WO 2012 / 067176); hBS23 (Q153-G4k / J142-G4h / L180-k) (a bispecific antibody in which a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 36 is associated with a light chain set forth in SEQ ID NO: 38, and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 37 is associated with a light chain set forth in SEQ ID NO: 38); and any of the bispecific antibodies (a) to (t) below, which are described in patent document (WO 2019 / 065795). (a) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 83, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 88, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; (b) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; (c) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 90, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (d) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 85, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 91, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (e) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 102, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113; (f) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 103, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 114; (g) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 84, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 89, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (h) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 92, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (i) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 112; (j) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 93, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (k) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (l) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 94, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116; (m) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 104, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115; (n) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 95, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116; (o) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 96, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117; (p) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 86, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 97, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 107, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 118; (q) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 106, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117; (r) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 98, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 108, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 119; (s) a bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 96, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 109, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 120; (t) A bispecific antibody comprising a first antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 122, a first antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 99, a second antibody heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 110, and a second antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 121.

[0024] These bispecific antibodies described in (a) to (t) are (1) a first antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 139 and / or a first antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 140, and / or (2) A second antibody heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 141 and / or a second antibody light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 142. It may further include:

[0025] Furthermore, preferred examples of bispecific antibodies of the present invention include any of the antibodies described in (a1) to (t1) below, which are bispecific antibodies described in Patent Document (WO2019 / 065795). Note that in the following antibodies, a first antibody heavy chain and a first antibody light chain are associated with each other, and a second antibody heavy chain and a second antibody light chain are associated with each other. (a1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 143, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 149, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 161, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 172; (b1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 161, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 172; (c1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 145, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 151, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (d1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 145, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 152, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (e1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 163, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 174; (f1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 164, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 175; (g1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 144, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 150, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (h1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 153, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (i1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 154, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 162, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 173; (j1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 154, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (k1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 155, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (l1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 155, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 166, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 177; (m1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 156, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 165, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 176; (n1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 156, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 166, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 177; (o1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 157, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 178; (p1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 146, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 158, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 168, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 179; (q1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 159, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 167, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 178; (r1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 159, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 169, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 180; (s1) a bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 147, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 157, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 170, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 181; (t1) A bispecific antibody comprising a first antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 148, a first antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 160, a second antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 171, and a second antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 182.

[0026] Neutralization In the present invention, "neutralization" in a substance that neutralizes a substance having a functional substituting activity for F.VIII refers to, for example, completely or partially inhibiting the F.VIII substituting activity of the substance having the functional substituting activity for F.VIII. For example, when the substance having the functional substituting activity for F.VIII is an antibody, completely or partially inhibiting the F.VIII substituting activity can be achieved by, but is not limited to, completely or partially inhibiting the binding of the antibody to an antigen.

[0027] antibody Examples of antibodies that bind to substances having functional substitutable activity for F.VIII include, for example, when the substance having functional substitutable activity for F.VIII is a bispecific antibody that binds to F.IX and / or F.IXa and FX and / or F.Xa, the antibodies can be selected from the group consisting of antibodies that bind to Fabs containing an antigen-binding site that binds to F.IX, antibodies that bind to Fabs containing an antigen-binding site that binds to F.IXa, antibodies that bind to Fabs containing an antigen-binding site that binds to FX, antibodies that bind to Fabs containing an antigen-binding site that binds to F.Xa, and bispecific antibodies that bind to Fabs containing antigen-binding sites that bind to F.IX and / or F.IXa and Fabs containing antigen-binding sites that bind to FX and / or F.Xa. The above antibodies can be used alone or in combination, and can also be used as combined compositions or kits. For example, multiple antibodies that bind to Fabs containing antigen-binding sites that bind to a single antigen can be used, such as multiple antibodies that bind to Fabs containing antigen-binding sites that bind to F.IX. When the substance having functional substitutable activity for F.VIII is a bispecific antibody that binds to F.IX and / or F.IXa and FX and / or F.Xa, the following combinations can be used, for example: (a) An antibody that binds to a Fab containing an antigen-binding site that binds to F.IX and an antibody that binds to a Fab containing an antigen-binding site that binds to FX (b) an antibody that binds to a Fab containing an antigen-binding site that binds to F.IXa and an antibody that binds to a Fab containing an antigen-binding site that binds to FX; (c) an antibody that binds to a Fab containing an antigen-binding site that binds to F.IX and an antibody that binds to a Fab containing an antigen-binding site that binds to F.IXa (d) an antibody that binds to a Fab containing an antigen-binding site that binds to F.IX, an antibody that binds to a Fab containing an antigen-binding site that binds to FX, and an antibody that binds to a Fab containing an antigen-binding site that binds to F.IXa That is, the present invention relates to compositions or kits comprising the neutralizing antibody combinations described herein.

[0028] An example of an antibody that binds to Fab containing an antigen-binding site that binds to F.IX and / or F.IXa is antibody IDA0288. The nucleotide sequences of the variable regions and the predicted amino acid sequences were analyzed using GENETYX Ver. 9 (GENETYX CORPORATION).

[0029] The amino acid and nucleic acid sequences of the heavy chain variable region of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 1 Nucleic acid sequence: SEQ ID NO:5

[0030] The amino acid and nucleic acid sequences of the light chain variable region of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO:2 Nucleic acid sequence: SEQ ID NO:6

[0031] The amino acid and nucleic acid sequences of the heavy chain CDRs 1 to 3 of IDA0288 are shown in 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 nucleic acid sequence: SEQ ID NO: 15 CDR2 nucleic acid sequence: SEQ ID NO: 16 CDR3 nucleic acid sequence: SEQ ID NO: 17

[0032] The amino acid and nucleic acid sequences of the light chain CDRs 1 to 3 of IDA0288 are shown in 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 nucleic acid sequence: SEQ ID NO:21 CDR2 nucleic acid sequence: SEQ ID NO:22 CDR3 nucleic acid sequence: SEQ ID NO:23

[0033] The amino acid and nucleic acid sequences of the antibody heavy chain constant region of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 123 Nucleic acid sequence: SEQ ID NO:125

[0034] The amino acid and nucleic acid sequences of the antibody light chain constant region of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 124 Nucleic acid sequence: SEQ ID NO:126

[0035] The amino acid and nucleic acid sequences of the antibody heavy chain of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 131 Nucleic acid sequence: SEQ ID NO:133

[0036] The amino acid and nucleic acid sequences of the antibody light chain of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 132 Nucleic acid sequence: SEQ ID NO:134

[0037] An example of an antibody that binds to Fab containing an antigen-binding site that binds to FX and / or F.Xa is the IDA0339 antibody. The nucleotide sequences of the variable regions and the predicted amino acid sequences were analyzed using GENETYX Ver. 9 (GENETYX CORPORATION).

[0038] The amino acid and nucleic acid sequences of the heavy chain variable region of IDA0339 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO:3 Nucleic acid sequence: SEQ ID NO:7

[0039] The amino acid and nucleic acid sequences of the light chain variable region of IDA0339 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO:4 Nucleic acid sequence: SEQ ID NO:8

[0040] The amino acid and nucleic acid sequences of the heavy chain CDRs 1 to 3 of IDA0339 are shown in 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 nucleic acid sequence: SEQ ID NO:27 CDR2 nucleic acid sequence: SEQ ID NO:28 CDR3 nucleic acid sequence: SEQ ID NO:29

[0041] The amino acid and nucleic acid sequences of the light chain CDRs 1 to 3 of IDA0339 are shown in 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 nucleic acid sequence: SEQ ID NO:33 CDR2 nucleic acid sequence: SEQ ID NO:34 CDR3 nucleic acid sequence: SEQ ID NO:35

[0042] The amino acid and nucleic acid sequences of the antibody heavy chain constant region of IDA0339 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 127 Nucleic acid sequence: SEQ ID NO:129

[0043] The amino acid and nucleic acid sequences of the antibody light chain constant region of IDA0339 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 128 Nucleic acid sequence: SEQ ID NO:130

[0044] The amino acid and nucleic acid sequences of the antibody heavy chain of IDA0339 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 135 Nucleic acid sequence: SEQ ID NO:137

[0045] The amino acid and nucleic acid sequences of the antibody light chain of IDA0288 are shown in the following SEQ ID NOs: Amino acid sequence: SEQ ID NO: 136 Nucleic acid sequence: SEQ ID NO:138

[0046] The term "antibody" is used in the broadest sense and may be a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (e.g., bispecific antibody), antibody derivative, or modified antibody, as long as it exhibits the desired biological activity (Miller K et al. J Immunol. 2003, 170(9), 4854-61). Antibodies may be murine, human, humanized, chimeric, or derived from other species or may be artificially synthesized. The antibodies disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Immunoglobulins may be derived from any species (e.g., human, mouse, or rabbit). The terms "antibody," "immunoglobulin," and "immunoglobulin" are used interchangeably and broadly.

[0047] An "antibody derivative" includes a portion of an antibody, preferably the variable region of the antibody, or at least the antigen-binding region of the antibody. Antibody derivatives include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies (scFv), sc(Fv)2, Fab3, domain antibodies (dAb) (WO 2004 / 058821, WO 2003 / 002609), diabodies, triabodies, tetrabodies, minibodies, and multispecific antibodies formed from antibody derivatives. Here, "Fab" is composed of one light chain and the CH1 region and variable region of one heavy chain. "Fv" is the smallest antibody derivative and contains the complete antigen-recognition and antigen-binding regions. Antibody derivatives may also be fusions with, for example, the Fc of an IgG antibody. See, for example, U.S. Pat. No. 5,641,870, Example 2; 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; Wu et al. Nat Biotechnol. 2007, 25(11), 1290-7.

[0048] Modified antibodies include, for example, antibodies conjugated with various molecules such as polyethylene glycol (PEG). These modified antibodies are also encompassed by the antibodies of the present invention. There are no limitations on the substances conjugated to the modified antibodies of the present invention. Such modified antibodies can be obtained by chemically modifying the antibodies obtained. These methods have already been established in this field.

[0049] A "bispecific" antibody refers to an antibody that has variable regions that recognize different epitopes within the same antibody molecule. A bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen. Bispecific antibodies may include not only whole antibodies but also antibody derivatives. The antibodies of the present invention also include bispecific antibodies. In this specification, anti-F.IXa / FX bispecific antibody is used synonymously with a bispecific antibody that binds to both F.IXa and FX.

[0050] Methods for producing recombinant antibodies The antibody may be a recombinant antibody produced using genetic engineering technology. Recombinant antibodies can be obtained by cloning the DNA encoding the antibody from antibody-producing cells such as hybridomas or sensitized lymphocytes that produce antibodies, incorporating it into a vector, and introducing this into a host (host cell) for production.

[0051] The antibody may be of any origin, including human, mouse, and rat antibodies, and may also be a genetically modified antibody such as a chimeric antibody or a humanized antibody.

[0052] Methods for obtaining human antibodies are already known. For example, a desired human antibody can be obtained by immunizing a transgenic animal having a full repertoire of human antibody genes with a desired antigen (see International Patent Application Publication Nos. WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735).

[0053] Genetically modified antibodies can be produced using known methods. Specifically, for example, chimeric antibodies are antibodies consisting of the heavy and light chain variable regions of an antibody from an immunized animal and the heavy and light chain constant regions of a human antibody. A chimeric antibody can be obtained by linking DNA encoding the variable region of an antibody from an immunized animal to DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host for production.

[0054] Humanized antibodies are modified antibodies, also known as reshaped human antibodies. Humanized antibodies are constructed by grafting the CDRs of an antibody derived from an immunized animal onto the complementarity-determining regions of a human antibody. General genetic recombination techniques for this purpose are also known (see European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96 / 02576, Sato K et al., Cancer Research 1993, 53: 851-856, International Patent Application Publication No. WO 99 / 51743).

[0055] A bispecific antibody is an antibody that has specificity for two different antigens.

[0056] Bispecific antibodies are not limited to IgG types, but for example, IgG type bispecific antibodies can be secreted by hybrid hybridomas (quadromas) generated by fusing two types of IgG antibody-producing hybridomas (Milstein C et al. Nature 1983, 305: 537-540). Alternatively, the genes for the light and heavy chains constituting the two types of IgG of interest, a total of four genes, can be introduced into cells and co-expressed to secrete the antibodies.

[0057] In this case, by making appropriate amino acid substitutions in the CH3 region of the heavy chain, it is possible to preferentially secrete IgGs with heterogeneous heavy chain combinations (Ridgway JB et al. Protein Engineering 1996, 9: 617-621; Merchant AM et al. Nature Biotechnology 1998, 16: 677-681; WO2006 / 106905; Davis JH et al. Protein Eng Des Sel. 2010, 4: 195-202.).

[0058] Furthermore, because there is less diversity in light chain variable regions than in heavy chain variable regions, it is expected that a common light chain capable of conferring binding ability to both heavy chains will be obtained, and the bispecific antibody of the present invention may be an antibody having a common light chain. Efficient expression of bispecific IgG is possible by expressing IgG by introducing this common light chain and both heavy chain genes into cells.

[0059] epitope In the present invention, an antibody, which is one embodiment of a substance that neutralizes a substance having functional substituting activity for F.VIII, also includes an antibody that binds to an epitope that overlaps with the epitope to which the antibody binds, and more preferably an antibody that binds to the same epitope.

[0060] Whether an antibody recognizes an overlapping or identical epitope with another antibody can be determined by examining the competition between the two epitopes. Antibody competition can be assessed by competitive binding assays, including enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer assay (FRET), and fluorescence microanalysis (FMAT®). The amount of antibody bound to an antigen is indirectly correlated with the binding ability of a candidate competing antibody (test antibody) that competes for binding to the overlapping or identical epitope. In other words, the greater the amount or affinity of the test antibody for the overlapping or identical epitope, the lower the amount of binding of the antibody to the antigen and the higher the amount of binding of the test antibody to the antigen. Specifically, an appropriately labeled antibody and the test antibody are simultaneously added to the antigen, and the bound antibody is detected using the label. The amount of antibody bound to the antigen can be easily measured by labeling the antibody in advance. The labeling method is not particularly limited, and a labeling method appropriate for the technique can be selected. Specific examples of labeling methods include fluorescent labeling, radiolabeling, and enzyme labeling.

[0061] Here, "antibodies that bind to overlapping epitopes" or "antibodies that bind to the same epitope" refers to the concentration (IC) of the labeled antibody that reduces the binding amount by 50% due to the binding of the unlabeled antibody. 50) to the IC of the unlabeled antibody to be tested. 50 The antibody is capable of reducing the amount of binding of the labeled antibody by at least 50% at a concentration that is usually 100-fold, preferably 80-fold, more preferably 50-fold, even more preferably 30-fold, and more preferably 10-fold higher than the labeled antibody. Analysis of the epitope recognized by the antibody can be performed by methods known to those skilled in the art, such as Western blotting.

[0062] In the present invention, antibodies that bind to an epitope that overlaps with or is identical to an epitope bound by a neutralizing antibody can be identified by measuring the binding activity of emicizumab to the anti-F.IX antibody (Q499-z121 / L404-k) and the anti-F.IX antibody QT15, in the case of neutralizing antibodies against antibodies having an antigen-binding site that binds to F.IX and / or F.IXa.

[0063] In the present invention, antibodies that bind to an epitope that overlaps with or the same epitope as the epitope bound by a neutralizing antibody can be identified by measuring the binding activity of emicizumab with the anti-FX antibody (J327-z119 / L404-k) and the anti-FX antibody JT13 antibody in the case of neutralizing antibodies against antibodies having an antigen-binding site that binds to FX and / or F.Xa.

[0064] In compositions containing antibodies of the present invention, antibodies that bind to epitopes that overlap with or are identical to the epitopes bound by neutralizing antibodies of the present invention can be identified by measuring binding to a mixed composition of emicizumab and QT15 and JT13 antibodies.

[0065] Antibody production method The antibodies of the present invention can be produced by methods known to those skilled in the art. Specifically, DNA encoding the antibody of interest is incorporated into an expression vector. At this time, the DNA is incorporated into the expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In this case, an appropriate combination of host and expression vector can be used. The method of producing an antibody of the present invention can include a step of culturing the host cells. The host cells can be cultured by methods known to those skilled in the art.

[0066] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Furthermore, for the purpose of subcloning or excision of cDNA, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be used.

[0067] When vectors are used for the purpose of antibody production, expression vectors are particularly useful. For example, when the host is E. coli such as JM109, DH5α, HB101, or XL1-Blue, it is essential that the expression vector contain a promoter that enables efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter. In addition to the above vectors, other such vectors include pGEX-5X-1 (Pharmacia), the "QIAexpress system" (QIAGEN), pEGFP, and pET (in this case, the host is preferably BL21, which expresses T7 RNA polymerase).

[0068] The vector may also contain a signal sequence for polypeptide secretion. Signal sequences for polypeptide secretion known to those skilled in the art can be used. For example, when producing a polypeptide in the periplasm of E. coli, the pelB signal sequence (Lei, SP et al. J. Bacteriol. (1987) 169, 4397) can be used. Introduction of the vector into host cells can be carried out using, for example, the calcium chloride method or electroporation.

[0069] In addition to E. coli expression vectors, vectors for producing antibodies of the present invention include, for example, mammalian-derived expression vectors (e.g., pcDNA3 (Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, and pCDM8), insect cell-derived expression vectors (e.g., the "Bac-to-BAC baculovirus expression system" (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., the "Pichia Expression Kit" (Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis-derived expression vectors (e.g., pPL608 and pKTH50).

[0070] When the aim is to express the vector in animal cells such as CHO cells, COS cells, or NIH3T3 cells, it is essential to have a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), the MMTV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CAG promoter (Gene. (1991) 108, 193), or the CMV promoter. It is even more preferable if the vector also has a gene for selecting transformed cells. Examples of genes for selecting transformed cells include drug resistance genes that can be distinguished by drugs (neomycin, G418, etc.). Examples of vectors with such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0071] Furthermore, to achieve stable gene expression and increase the intracellular copy number of a gene, one method involves introducing a vector (e.g., pCHOI) containing a complementary DHFR gene into CHO cells deficient in the nucleic acid synthesis pathway and amplifying the gene with methotrexate (MTX). Another method involves transforming COS cells carrying a gene expressing SV40 T antigen on their chromosomes with a vector (e.g., pcD) containing an SV40 replication origin. Replication origins derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, to increase the gene copy number in a host cell system, the expression vector can contain a selection marker such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene.

[0072] The present invention also provides vectors into which nucleic acids encoding the antibodies described herein have been inserted. As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked thereto, and includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which they have been introduced. Some vectors are capable of mediating expression of nucleic acids operably linked thereto. Such vectors are also referred to herein as "expression vectors."

[0073] The present invention also provides isolated nucleic acids encoding antibodies of the present invention. Such nucleic acids may encode an amino acid sequence comprising a light chain variable region and / or an amino acid sequence comprising a heavy chain variable region of an antibody (e.g., the light chain and / or the heavy chain of an antibody). The isolated nucleic acids encoding the antibodies may be inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleic acid probes that specifically bind to genes encoding the heavy and / or light chains of the antibody).

[0074] The present invention also relates to cells containing nucleic acids encoding the antibodies described herein or vectors containing such nucleic acids. As used herein, a cell refers to a cell into which exogenous nucleic acid has been introduced (including the progeny of such cells). Cells of the present invention include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as the original transformed cell screened or selected for are also included. As used herein, a cell contains (1) a vector containing a nucleic acid encoding an amino acid sequence comprising an antibody light chain variable region and / or an amino acid sequence comprising an antibody heavy chain variable region, or (2) a first vector containing a nucleic acid encoding an amino acid sequence comprising an antibody light chain variable region and a second vector containing a nucleic acid encoding an amino acid sequence comprising an antibody heavy chain variable region (e.g., the cell has been transformed with these vectors). In the present invention, the host can be a eukaryote (for example, a CHO cell or a lymphoid cell (for example, a Y0, NS0, or SP20 cell)).

[0075] The antibodies of the present invention obtained by the methods described herein can be isolated from inside or outside host cells (e.g., from the culture medium) and purified as substantially pure, homogeneous antibodies. Antibody separation and purification can be performed using any separation and purification method commonly used for antibody purification, and are not limited in any way. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0076] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-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). These chromatographies can be performed using liquid-phase chromatography, such as HPLC and FPLC. Examples of columns used in affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A include Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). The present invention also encompasses antibodies highly purified using these purification methods.

[0077] The obtained antibodies can be purified to homogeneity. Antibodies can be separated and purified using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but these methods are not limited thereto. Columns used for affinity chromatography include Protein A columns and Protein G columns.

[0078] As described above, the neutralizing antibodies of the present invention can be used in methods for measuring F.VIII reactivity in the presence of a substance having F.VIII functional substituting activity, for example, in methods for measuring F.VIII activity or F.VIII inhibitor titer. The method for measuring F.VIII activity of the present invention comprises the step of contacting the following (1) and (2). The rest of the steps can be performed according to a commonly used method for measuring F.VIII activity. (1) A blood-derived sample containing a substance with F.VIII functional substitution activity (2) Substances that neutralize substances with functional substituting activity for F.VIII In the present invention, the substance that neutralizes a substance having a functional substituting activity for F.VIII is preferably an antibody that neutralizes a substance having a functional substituting activity for F.VIII, and more preferably the neutralizing antibodies IDA0288, IDA0339, etc. described herein. In the method of measuring F.VIII activity of the present invention, such substances may be used alone or in combination of two or more. Furthermore, in the present invention, the substance that neutralizes a substance having a functional substituting activity for F.VIII may be in the form of a composition. The composition may contain a buffer, a substance commonly used for measuring the reactivity of F.VIII, etc.

[0079] F.VIII activity measurement method Commonly used methods for measuring F.VIII activity include those known to those skilled in the art. For example, a one-stage clotting assay (Casillas et al., (1971) Coagulation 4: 107-11) based on clotting time (aPTT measurement) using factor VIII-deficient plasma (Sysmex, Kobe, Japan) can be used. The one-stage clotting assay is performed, for example, as follows: 50 μL of 10-fold diluted test plasma, 50 μL of F.VIII-deficient plasma, and 50 μL of APTT reagent are mixed and incubated at 37°C for 5 minutes. The clotting reaction is initiated by adding 50 μL of calcium solution, and the time to clotting is measured. Instead of the test plasma, a normal plasma dilution series (F.VIII activity in 10-fold diluted normal plasma is defined as 100%) is measured, and a calibration curve is created by plotting F.VIII activity on the horizontal axis and clotting time on the vertical axis. The coagulation time of the test plasma is converted into F.VIII activity from the calibration curve, and the F.VIII activity in the test plasma is calculated. In this specification, unless otherwise specified, the term "measurement of F.VIII activity" may also include "measurement of the activity of activated coagulation factor VIII (F.VIIIa)."

[0080] In addition to the one-stage clotting assay, methods for measuring F.VIII activity include thrombin generation assay (TGA), rotational thromboelastometry, F.VIII chromogenic assay, clot waveform analysis, and thrombin and activated factor X generation tests. The method for measuring the potency of an F.VIII inhibitor of the present invention comprises the steps of contacting (1) and (2) below. The rest of the procedure can be performed according to a commonly used method for measuring F.VIII inhibitor potency. (1) A blood-derived sample containing a substance with F.VIII functional substitution activity (2) Substances that neutralize substances with functional substituting activity for F.VIII

[0081] In the present invention, the neutralizing antibodies of the present invention can be used at a concentration that can neutralize substances that have functional substituting activity for F.VIII. Such concentrations can be appropriately adjusted by those skilled in the art. Specific examples include, but are not limited to, 1 to 100 μg / ml, preferably 5 to 80 μg / ml, more preferably 10 to 60 μg / ml, and particularly preferably 20 to 40 μg / ml.

[0082] F.VIII inhibitor titration assay Commonly used methods for measuring F.VIII inhibitor titers include those known to those skilled in the art, such as the Bethesda assay (Kasper et al., (1975) Thrombos Diath Haemorrh 34:869-872), ELISA, and the Nijmegen Bethesda assay (Nijmegen modification assay) (Verbruggen et al., (1995) Thromb Haemost 73:247-251). The Bethesda assay is performed, for example, as follows: Equal volumes of normal plasma and test plasma are mixed and incubated at 37°C for 2 hours, after which the residual factor VIII activity in the normal plasma is measured by a one-stage clotting assay based on the activated partial thromboplastin time (APTT). The effect of inhibiting 50% of factor VIII activity in normal plasma is defined as 1 Bethesda (1 BU), and therefore F.VIII inhibitor titers are calculated in Bethesda units. If the F.VIII inhibitor titer in the test plasma is high and the residual F.VIII activity does not fall within the range of 25-75%, the test plasma is appropriately diluted with buffer solution, the Bethesda units are calculated again, and then the F.VIII inhibitor titer in the test plasma is calculated by multiplying by the dilution factor.

[0083] F.VIII inhibitor F.VIII inhibitors are alloantibodies against exogenous F.VIII that occur in 20-30% of hemophilia A patients. Even previously normal individuals may develop autoantibodies against F.VIII. Generally, most F.VIII inhibitor alloantibodies and autoantibodies function as anti-F.VIII neutralizing antibodies, reducing or eliminating F.VIII activity.

[0084] How to obtain samples In the present invention, the blood-derived sample is preferably a blood-derived sample collected from a subject. Such a blood-derived sample can be obtained from a subject who has been administered a substance having F.VIII replacement activity. Examples of subjects include patients with bleeding symptoms anywhere in the body (patients with bleeding disorders). Main bleeding sites include, but are not limited to, intra-articular, intramuscular, subcutaneous, oral, intracranial, gastrointestinal, and nasal cavities. Patients with bleeding disorders preferably include patients with bleeding disorders caused by reduced or deficient activity of F.VIII and / or F.VIIIa. Examples of patients with bleeding disorders caused by reduced or deficient activity of F.VIII and / or F.VIIIa include patients with bleeding symptoms who have congenital or acquired reduced or deficient activity of either or both of F.VIII and F.VIIIa. Decreased activity of F.VIII and F.VIIIa includes, but is not limited to, patients whose activity is preferably less than 40% (e.g., less than 40%, less than 30%, less than 20%, less than 10%), more preferably less than 10% (e.g., less than 10%, less than 9%, less than 8%, less than 7%, less than 6%), even more preferably less than 5% (e.g., less than 5%, less than 4%, less than 3%, less than 2%), and particularly preferably less than 1% compared to healthy individuals.

[0085] More specifically, examples of such diseases include, but are not limited to, diseases selected from hemophilia (hemophilia A, hemophilia B), acquired hemophilia, and von Willebrand disease caused by a dysfunction or deficiency of von Willebrand factor (vWF). Blood-derived samples include serum, plasma, and whole blood. In the present invention, it is preferable to use a plasma sample. Methods for obtaining blood-derived samples from subjects are well known to those skilled in the art.

[0086] Compositions and Kits The neutralizing antibodies of the present invention can be used to form compositions containing buffers necessary for the method of measuring F.VIII reactivity and substances commonly used for measuring F.VIII reactivity. Furthermore, the neutralizing antibodies of the present invention can be pre-packaged with various reagents, such as buffers necessary for the method of measuring F.VIII reactivity and substances commonly used for measuring F.VIII reactivity, and supplied as kits. Preferred examples of neutralizing antibodies constituting the compositions and kits of the present invention include the neutralizing antibodies IDA0288 and IDA0339 described herein. The compositions and kits of the present invention can contain either one of these neutralizing antibodies, or preferably both. The compositions and kits of the present invention can also contain, in addition to buffers, substances commonly used for measuring F.VIII reactivity, such as plasma samples isolated from humans with normal blood F.VIII activity and F.IX activity, substances with F.VIII replacement activity, substances usable for measuring F.VIII activity, and substances usable for measuring F.VIII inhibitor titers. The various reagents included in the kits can be in powder or liquid form depending on the intended use. These can be stored in appropriate containers and used as needed.

[0087] For example, a method using the antibody of the present invention can be used to diagnose the severity of a patient who has been administered a substance having a functional substituting activity for F.VIII. The reactivity of F.VIII can be measured using a method using the antibody of the present invention, and the severity and / or inhibitor titer of the patient can be diagnosed or assessed based on the measurement results. The diagnosis and assessment method can be performed by a method known to those skilled in the art. Those skilled in the art can determine a treatment plan for the patient based on the diagnosis and assessment. The present invention may include a step of determining the dosage of the substance having a functional substituting activity for F.VIII in accordance with the determined treatment plan, and a step of administering the substance having a functional substituting activity for F.VIII.

[0088] For example, the pharmacological activity of a substance having a functional substituting activity for F.VIII and / or an F.VIII preparation can be monitored in a patient administered with the substance and / or an F.VIII preparation using the antibody of the present invention. Monitoring can be performed by methods known to those skilled in the art.

[0089] Kits containing the antibodies of the present invention can be used, for example, as kits for diagnosing the severity of disease in patients administered with a substance that has functionally substituting activity for F.VIII. F.VIII reactivity can be measured using kits containing the antibodies of the present invention, and the severity and / or inhibitor titer of the patient can be diagnosed or assessed based on the measurement results. The diagnosis and assessment methods can be performed by methods known to those skilled in the art.

[0090] Kits containing the antibodies of the present invention can be used, for example, as kits for monitoring the pharmacological activity of F.VIII preparations in patients administered with substances having functional substituting activity for F.VIII and F.VIII preparations. Monitoring can be performed by methods known to those skilled in the art.

[0091] Patients who are candidates for methods using the antibodies of the present invention or kits containing the antibodies of the present invention include, for example, patients with hemophilia A, acquired hemophilia A, von Willebrand disease, and patients with hemophilia A who have developed inhibitors to F.VIII and / or F.VIIIa.

[0092] As used in this specification, an embodiment expressed by the expression "comprising..." encompasses an embodiment expressed by the expression "essentially consisting of..." as well as an embodiment expressed by the expression "consisting of...". The contents of all patents and references expressly cited herein are hereby incorporated by reference in their entirety. The present invention is further illustrated by, but not limited to, the following examples. [Example]

[0093] Example 1: Production of antibodies against emicizumab and anti-F.IXa / FX bispecific antibodies, determination of variable region sequences, and construction of expression vectors QT15 F(ab')2 or JT13 F(ab')2 was prepared by methods known to those skilled in the art and immunized four times into three New Zealand White rabbits (Kitayama Labes). Details of the anti-F.IX antibody QT15 and the anti-FX antibody JT13 are shown in Table 1.

[0094] [Table 1]

[0095] Peripheral blood mononuclear cells and splenocytes were collected one week after the final immunization. Cells that bound to QT15 whole antibody or JT13 whole antibody were enriched using MACS. Subsequently, cells were sorted using a cell sorter (FACS aria III, BD) with PE-labeled anti-rabbit IgG antibody (Southern Biotech) and QT15 whole antibody or JT13 whole antibody, and then cultured. Using the secreted antibodies in the B cell culture supernatant, B cells secreting antibodies that bound to the variable regions of either QT15 whole antibody or JT13 whole antibody and the variable region of emicizumab alone, but not to the constant region, were selected. The variable region sequences obtained from the selected B cells by RT-PCR were inserted into an expression vector containing the known rabbit IgG constant region sequences (heavy chain: IgG / SEQ ID NO: 123, light chain: Igκ / SEQ ID NO: 124). Using the resulting plasmids, we prepared two antibodies (designated IDA0288) that bind to the variable regions of QT15 whole and emicizumab but not to the constant region or whole JT13, and two antibodies (designated IDA0339) that bind to the variable regions of JT13 whole and emicizumab but not to the constant region or whole QT15. The nucleotide sequences of the regions encoding IDA0288 and IDA0339 were identified by DNA sequencing.

[0096] heavy chain variable region of IDA0288; Amino acid sequence: SEQ ID NO: 1 Nucleic acid sequence: SEQ ID NO:5 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 nucleic acid sequence: SEQ ID NO: 15 CDR2 nucleic acid sequence: SEQ ID NO: 16 CDR3 nucleic acid sequence: SEQ ID NO: 17

[0097] the light chain variable region of IDA0288; Amino acid sequence: SEQ ID NO:2 Nucleic acid sequence: SEQ ID NO:6 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 nucleic acid sequence: SEQ ID NO:21 CDR2 nucleic acid sequence: SEQ ID NO:22 CDR3 nucleic acid sequence: SEQ ID NO:23

[0098] heavy chain variable region of IDA0339; Amino acid sequence: SEQ ID NO:3 Nucleic acid sequence: SEQ ID NO:7 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 nucleic acid sequence: SEQ ID NO:27 CDR2 nucleic acid sequence: SEQ ID NO:28 CDR3 nucleic acid sequence: SEQ ID NO:29

[0099] the light chain variable region of IDA0339; Amino acid sequence: SEQ ID NO:4 Nucleic acid sequence: SEQ ID NO:8 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 nucleic acid sequence: SEQ ID NO:33 CDR2 nucleic acid sequence: SEQ ID NO:34 CDR3 nucleic acid sequence: SEQ ID NO:35

[0100] [Example 2] One-stage clotting assay using IDA0288 and IDA0339 or rAQ8 and rAJ540 with anti-F.IXa / FX bispecific antibodies for neutralization The expression clone plasmid prepared in Example 1 was introduced into Expi293 cells, followed by mass culture and purification using ProA to produce IDA0288 and IDA0339 antibodies. IDA0288, IDA0339, rAQ8 (heavy chain variable region: SEQ ID NO: 79, light chain variable region: SEQ ID NO: 80) and rAJ540 (heavy chain variable region: SEQ ID NO: 81, light chain variable region: SEQ ID NO: 82) described in the Examples of WO2016 / 047656 were each diluted with TBS to 300 μg / mL, and then equal amounts of IDA0288 and IDA0339 or rAQ8 and rAJ540 were mixed to prepare an anti-antibody solution.

[0101] Anti-F.IXa / FX bispecific antibodies (emicizumab, #1 to #6) were also prepared by standard methods. The amino acid sequences of these antibodies are summarized in Tables 2-1 and 2-2. Anti-F.IXa / FX bispecific antibodies (emicizumab, #1 to #6) were serially diluted with F.VIII-deficient plasma (Siemens) to 12.5 μg / mL, respectively, to prepare antibody solutions. 120 μL of the anti-antibody solution and 30 μL of the antibody solution were mixed in the combinations shown in Table 3 to prepare the measurement sample solution, which was then allowed to stand at room temperature for 5 minutes. 50 μL of the measurement sample solution and 50 μL of Thrombocheck APTT-SLA (Sysmex) were mixed and incubated at 37°C for 190 seconds. After incubation, 50 μL of 0.02 M calcium chloride solution (Sysmex) was added to initiate coagulation, and the coagulation time was measured using a CS-2000i (Sysmex). Details of the anti-F.IXa / FX bispecific antibody and measurement sample combinations are shown in Table 3 below.

[0102] [Table 2-1] [Table 2-2]

[0103] [Table 2-3]

[0104] [Table 3]

[0105] result The average clotting time was calculated from duplicate measurements and is shown in Figure 1. In the group without anti-antibody solution, the clotting time was shortened by each anti-F.IXa / FX bispecific antibody. In the group with anti-antibody solution IDA0288 / IDA0339, the clotting time was partially shortened in #1 and #2, but the clotting time with emicizumab and other anti-F.IXa / FX bispecific antibodies was similar to that in the group without anti-F.IXa / FX bispecific antibodies, indicating complete neutralization of the activity of emicizumab and other anti-F.IXa / FX bispecific antibodies. In the group with anti-antibody solution rAQ8 / rAJ540, the clotting time with emicizumab was similar to that in the group without anti-F.IXa / FX bispecific antibodies, but the clotting time with the addition of other anti-F.IXa / FX bispecific antibodies was partially shortened, and the activity of the other anti-F.IXa / FX bispecific antibodies was not completely neutralized. [Industrial Applicability]

[0106] The present invention provides an antibody for use in a method for measuring F.VIII reactivity in the presence of a bispecific antibody having F.VIII functional substituting activity, for example, a method for measuring F.VIII activity or F.VIII inhibitor titer. The method using the antibody of the present invention enables accurate measurement of F.VIII reactivity in patients undergoing treatment with a bleeding disorder such as hemophilia using the bispecific antibody.

Claims

1. An antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

2.

2. An antibody comprising an antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 131 and an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO:

132.

3. An antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

4.

4. An antibody comprising an antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 135 and an antibody light chain comprising the amino acid sequence set forth in SEQ ID NO:

136.

5. A nucleic acid encoding the antibody according to any one of claims 1 to 4.

6. A vector into which the nucleic acid according to claim 5 has been inserted.

7. A cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. A method for producing an antibody, comprising culturing the cell of claim 7.

9. A composition comprising the antibody of claim 1 and / or the antibody of claim 3.

10. A composition comprising the antibody of claim 2 and / or the antibody of claim 4.

11. A method for measuring the reactivity of coagulation factor VIII, comprising the step of contacting the following (1) and (2): (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated coagulation factor X; (2) An antibody according to claim 1 or 2 and / or an antibody according to claim 3 or 4.

12. A method for measuring the reactivity of coagulation factor VIII, comprising the step of contacting the following (1) and (2): (1) a blood-derived sample containing a bispecific antibody that binds to coagulation factor IX and / or activated coagulation factor IX and coagulation factor X and / or activated coagulation factor X; (2) The composition according to claim 9 or 10.

13. 13. A kit for use in the method of claim 12, comprising the composition of claim 9 or claim 10.

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