Multi-specific antigen-binding molecule having alternative function to function of blood coagulation factor viii

Abstract

To provide a multi-specific antigen-binding molecule having an alternative function to the function of blood coagulation factor VIII.SOLUTION: The present invention provides a multi-specific antigen-binding molecule with high activity substituting for the function of blood coagulation factor VIII. The molecule is found out among various prepared bispecific antibodies each specifically binding to both the blood coagulation factor IX / activated blood coagulation factor IX and the blood coagulation factor X and having an alternative function to the cofactor function of the blood coagulation factor VIII, i.e., the function of promoting the activation of the blood coagulation factor X by the activated blood coagulation factor IX.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a multispecific antigen-binding molecule having a function of substituting for the function of blood coagulation factor VIII, which is a cofactor that enhances an enzymatic reaction, and a pharmaceutical composition containing the molecule as an active ingredient.

Background Art

[0002] Hemophilia A is a bleeding disorder due to congenital hypofunction or deficiency of blood coagulation factor VIII (F.VIII). For bleeding in patients with hemophilia A, an F.VIII preparation is usually administered (on-demand administration). In recent years, for the purpose of preventing bleeding events, an F.VIII preparation is prophylactically administered (prophylactic administration, Non-Patent Documents 1 and 2). The blood half-life of the F.VIII preparation is about 12 to 16 hours. Therefore, for continuous prevention, the F.VIII preparation is administered to patients three times a week (Non-Patent Documents 3 and 4). In on-demand administration, in order to prevent rebleeding, the F.VIII preparation is additionally administered at regular intervals as needed. In addition, the administration of the F.VIII preparation is carried out intravenously. Therefore, there has been a strong demand for a drug with a lower administration burden compared to the F.VIII preparation.

[0003] Occasionally, an antibody (inhibitor) against F.VIII occurs in hemophilia patients. The inhibitor nullifies the effect of the F.VIII preparation. For bleeding in patients in whom an inhibitor has developed (inhibitor patients), a bypass preparation is administered. Their mechanism of action is independent of the function of F.VIII, that is, the function of catalyzing the activation of blood coagulation factor X (F.X) by activated blood coagulation factor IX (F.IXa). Therefore, there are cases where the bypass preparation cannot sufficiently stop bleeding. Therefore, there has been a strong demand for a drug that is not affected by the presence of an inhibitor and substitutes for the function of F.VIII.

[0004] Recently, as a solution thereto, antibodies that substitute for the function of F.VIII and their use have been disclosed (Patent Documents 1, 2, and 3). Although these antibodies are considered to be effective also for acquired hemophilia having anti-F.VIII autoantibodies and von Willebrand disease caused by functional abnormalities or deficiencies of von Willebrand factor (vWF), the activity of substituting for the function of F.VIII has not necessarily been sufficient. Therefore, as a drug showing a higher hemostatic effect, an antibody having a higher activity of substituting for the function of F.VIII than the above antibody has been desired.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a multispecific antigen-binding molecule having a function of substituting for the function of F.VIII, which is a cofactor that enhances an enzymatic reaction.

Means for Solving the Problems

[0008] As a result of intensive research, the present inventors have found that among various bispecific antibodies that specifically bind to both F.IX / F.IXa and F.X and have a function (F.Xa production promoting function) that substitutes for the cofactor function of F.VIII, i.e., the function of promoting F.X activation by F.IXa, they have successfully found a bispecific antibody having a F.Xa production promoting activity superior to known ones.

[0009] Furthermore, they have successfully found the positions of the amino acid sequences of the antibody that are important for improving the F.Xa production promoting activity of the bispecific antibody having an activity substituting for the function of F.VIII, and by substituting the amino acids, they have successfully obtained a bispecific antibody in which the activity substituting for the F.VIII function is further increased. Also, they have successfully obtained a bispecific antibody that satisfies both properties of not only having a high activity substituting for the function of F.VIII but also having a low F.Xase inhibitory action, which is very difficult.

[0010] That is, the present invention relates to a multispecific antigen-binding molecule having a function substituting for the function of F.VIII, which is a cofactor that enhances an enzymatic reaction, and a pharmaceutical composition containing the molecule as an active ingredient, and more specifically relates to the following. 〔1〕 A multispecific antigen-binding molecule having a function substituting for the function of blood coagulation factor VIII, comprising a first antigen-binding site that recognizes blood coagulation factor IX and / or activated blood coagulation factor IX, and a second antigen-binding site that recognizes blood coagulation factor X, wherein the function substituting for the function of the blood coagulation factor VIII is a function resulting from a high activated blood coagulation factor X (F.Xa) production promoting activity as compared with a bispecific antibody (hA69-KQ / hB26-PF / hAL-AQ) having a heavy chain consisting of SEQ ID NOs: 165 and 166 and a common light chain consisting of SEQ ID NO: 167. 〔2〕 The multispecific antigen-binding molecule according to 〔1〕, comprising a first polypeptide comprising a first antigen-binding site that recognizes factor IX of blood coagulation and / or activated factor IX of blood coagulation, and a third polypeptide comprising a third antigen-binding site that recognizes factor IX of blood coagulation and / or activated factor IX of blood coagulation, and a second polypeptide comprising a second antigen-binding site that recognizes factor X of blood coagulation and a fourth polypeptide comprising a fourth antigen-binding site that recognizes factor X of blood coagulation. 〔3〕 The multispecific antigen-binding molecule according to 〔2〕, wherein the first polypeptide and the third polypeptide each comprise an antigen-binding site of the heavy chain or light chain of an antibody against factor IX of blood coagulation or activated factor IX of blood coagulation, and the second polypeptide and the fourth polypeptide each comprise an antigen-binding site of the heavy chain or light chain of an antibody against factor X of blood coagulation. 〔4〕 The multispecific antigen-binding molecule according to 〔3〕, wherein the antigen-binding site of the first polypeptide comprises an antigen-binding site comprising a heavy chain CDR consisting of any of the amino acid sequences selected from the following (a1) to (a11) or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide has an antigen-binding site comprising a heavy chain CDR consisting of any of the amino acid sequences selected from the following (b1) to (b11) or an antigen-binding site functionally equivalent thereto: (a1) An antigen-binding site having the amino acid sequences of heavy chain CDR1, 2, and 3 described in SEQ ID NOs: 75, 76, 77 (heavy chain CDRs of Q1). (a2) An antigen-binding site having the amino acid sequences of heavy chain CDR1, 2, and 3 described in SEQ ID NOs: 78, 79, 80 (heavy chain CDRs of Q31). (a3) An antigen-binding site having the amino acid sequences of heavy chain CDR1, 2, and 3 described in SEQ ID NOs: 81, 82, 83 (heavy chain CDRs of Q64). (a4) An antigen-binding site having the amino acid sequences of heavy chain CDR1, 2, and 3 described in SEQ ID NOs: 84, 85, 86 (heavy chain CDRs of Q85). (a5) An antigen-binding site having the amino acid sequences of heavy chain CDR1, 2, and 3 described in SEQ ID NOs: 87, 88, 89 (heavy chain CDRs of Q153). (a6) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 90, 91, 92 (H-chain CDRs of Q354), (a7) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 93, 94, 95 (H-chain CDRs of Q360), (a8) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 96, 97, 98 (H-chain CDRs of Q405), (a9) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 99, 100, 101 (H-chain CDRs of Q458), (a10) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 102, 103, 104 (H-chain CDRs of Q460), (a11) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 105, 106, 107 (H-chain CDRs of Q499), (b1) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 108, 109, 110 (H-chain CDRs of J232), (b2) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 111, 112, 113 (H-chain CDRs of J259), (b3) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 114, 115, 116 (H-chain CDRs of J268), (b4) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 117, 118, 119 (H-chain CDRs of J300), (b5) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 120, 121, 122 (H-chain CDRs of J321), (b6) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 123, 124, 125 (H-chain CDRs of J326), (b7) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 126, 127, 128 (H-chain CDRs of J327), (b8) An antigen-binding site having the amino acid sequences of H chain CDR1, 2, and 3 described in SEQ ID NOs: 129, 130, 131 (H chain CDRs of J339), (b9) An antigen-binding site having the amino acid sequences of H chain CDR1, 2, and 3 described in SEQ ID NOs: 132, 133, 134 (H chain CDRs of J344), (b10) An antigen-binding site having the amino acid sequences of H chain CDR1, 2, and 3 described in SEQ ID NOs: 135, 136, 137 (H chain CDRs of J346), (b11) An antigen-binding site having the amino acid sequences of H chain CDR1, 2, and 3 described in SEQ ID NOs: 174, 175, 176 (H chain CDRs of J142). [5] The antigen-binding site of the first polypeptide comprises an antigen-binding site comprising an H chain variable region consisting of any one of the amino acid sequences selected from the following (a1) to (a11) or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site comprising an H chain variable region consisting of any one of the amino acid sequences selected from the following (b1) to (b11) or an antigen-binding site functionally equivalent thereto. The multispecific antigen-binding molecule according to [3]: (a1) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 35 (H chain variable region of Q1), (a2) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 36 (H chain variable region of Q31), (a3) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 37 (H chain variable region of Q1), (a4) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 38 (H chain variable region of Q85), (a5) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 39 (H chain variable region of Q153), (a6) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 40 (H chain variable region of Q354), (a7) An antigen-binding site having the amino acid sequence of the H chain variable region described in SEQ ID NO: 41 (H chain variable region of Q360), (a8) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 42 (heavy chain variable region of Q405), (a9) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 43 (heavy chain variable region of Q458), (a10) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 44 (heavy chain variable region of Q460), (a11) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 45 (heavy chain variable region of Q499), (b1) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 46 (heavy chain variable region of J232), (b2) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 47 (heavy chain variable region of J259), (b3) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 48 (heavy chain variable region of J268), (b4) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 49 (heavy chain variable region of J300), (b5) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 50 (heavy chain variable region of J321), (b6) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 51 (heavy chain variable region of J326), (b7) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 52 (heavy chain variable region of J327), (b8) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 53 (heavy chain variable region of J339), (b9) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 54 (heavy chain variable region of J344), (b10) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 55 (heavy chain variable region of J346), (b11) An antigen-binding site having the amino acid sequence of the heavy chain variable region described in SEQ ID NO: 172 (heavy chain variable region of J142). 〔6〕The antigen-binding site contained in the third polypeptide and the fourth polypeptide comprises an antigen-binding site containing an L-chain CDR consisting of any amino acid sequence selected from the following (c1) to (c10) or an antigen-binding site functionally equivalent thereto, the multispecific antigen-binding molecule according to 〔3〕: (c1) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 138, 139, and 140 (L-chain CDRs of L2), (c2) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 141, 142, and 143 (L-chain CDRs of L45), (c3) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 144, 145, and 146 (L-chain CDRs of L248), (c4) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 147, 148, and 149 (L-chain CDRs of L324), (c5) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 150, 151, and 152 (L-chain CDRs of L334), (c6) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 153, 154, and 155 (L-chain CDRs of L377), (c7) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 156, 157, and 158 (L-chain CDRs of L404), (c8) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 159, 160, and 161 (L-chain CDRs of L406), (c9) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 137, 138, and 139 (L-chain CDRs of L408), (c10) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 177, 178, and 179 (L-chain CDRs of L180). 〔7〕The antigen-binding site contained in the third polypeptide and the fourth polypeptide is an antigen-binding site containing a light chain variable region consisting of any amino acid sequence selected from the following (c1) to (c10), or an antigen-binding site functionally equivalent thereto. The multispecific antigen-binding molecule according to 〔3〕: (c1) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 56 (light chain variable region of L2). (c2) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 57 (light chain variable region of L45). (c3) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 58 (light chain variable region of L248). (c4) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 59 (light chain variable region of L324). (c5) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 60 (light chain variable region of L334). (c6) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 61 (light chain variable region of L377). (c7) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 62 (light chain variable region of L404). (c8) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 63 (light chain variable region of L406). (c9) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 64 (light chain variable region of L408). (c10) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 173 (light chain variable region of L180). 〔8〕Furthermore, the first and second polypeptides contain the heavy chain constant region of an antibody, and the third and fourth polypeptides contain the light chain constant region of an antibody. The multispecific antigen-binding molecule according to 〔3〕. The multispecific antigen-binding molecule according to [3], wherein the first and second polypeptides comprise the heavy chain constant region of an antibody, and the third and fourth polypeptides comprise the light chain constant region of an antibody, and wherein the third polypeptide and the fourth polypeptide are a common light chain. 〔10〕 The multispecific antigen-binding molecule according to [8] or [9], wherein the first polypeptide comprises the heavy chain constant region of an antibody consisting of any amino acid sequence selected from the following groups (d1) to (d6) or any amino acid sequence selected from the following groups (d7) to (d9), and the second polypeptide comprises the heavy chain constant region of an antibody consisting of any amino acid sequence selected from a group different from the first polypeptide: (d1) The heavy chain constant region set forth in SEQ ID NO: 65 (G4k). (d2) The heavy chain constant region set forth in SEQ ID NO: 66 (z7). (d3) The heavy chain constant region set forth in SEQ ID NO: 67 (z55). (d4) The heavy chain constant region set forth in SEQ ID NO: 68 (z106). (d5) The heavy chain constant region set forth in SEQ ID NO: 69 (z118). (d6) The heavy chain constant region set forth in SEQ ID NO: 70 (z121). (d7) The heavy chain constant region set forth in SEQ ID NO: 71 (G4h). (d8) The heavy chain constant region set forth in SEQ ID NO: 72 (z107). (d9) The heavy chain constant region set forth in SEQ ID NO: 73 (z119). 〔11〕 The multispecific antigen-binding molecule according to [8] or [9], wherein the third and fourth polypeptides comprise the light chain constant region of an antibody consisting of the following amino acid sequence (e): (e) The light chain constant region set forth in SEQ ID NO: 74 (k). 〔12〕The multispecific antigen-binding molecule according to 〔8〕or 〔9〕, wherein the first polypeptide comprises any one of the antibody heavy chains selected from the following (a1) to (a14), the second polypeptide comprises any one of the antibody heavy chains selected from the following (b1) to (b12), and the third and fourth polypeptides comprise any one of the antibody light chains selected from the following (c1) to (c10): (a1) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 (Q1-G4k); (a2) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 (Q31-z7); (a3) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 (Q64-z55); (a4) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 10 (Q64-z7); (a5) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 (Q85-G4k); (a6) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 (Q153-G4k); (a7) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 13 (Q354-z106); (a8) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 (Q360-G4k); (a9) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 15 (Q360-z118); (a10) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 16 (Q405-G4k); (a11) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 17 (Q458-z106); (a12) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18 (Q460-z121); (a13) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 19 (Q499-z118); (a14) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 (Q499-z121); (b1) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4 (J268-G4h); (b2) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 (J321-G4h), (b3) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6 (J326-z107), (b4) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 (J344-z107), (b5) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 21 (J232-G4h), (b6) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 22 (J259-z107), (b7) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 23 (J300-z107), (b8) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 24 (J327-z107), (b9) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 25 (J327-z119), (b10) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 26 (J339-z119), (b11) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 27 (J346-z107), (b12) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 170 (J142-G4h), (c1) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 8 (L2-k), (c2) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 9 (L45-k), (c3) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 28 (L248-k), (c4) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 29 (L324-k), (c5) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 (L334-k), (c6) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 31 (L377-k), (c7) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 32 (L404-k), (c8) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 33 (L406-k), (c9) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 34 (L408-k), (c10) An antibody light chain consisting of the amino acid sequence set forth in SEQ ID NO: 171 (L180-k).

[13] The first polypeptide comprises an antigen-binding site that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of the antibody described in any one of (a1) to (a14) of

[12] and the light chain of the antibody described in any one of (c1) to (c10) binds, and the second polypeptide comprises an antigen-binding site that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of the antibody described in any one of (b1) to (b12) of

[12] and the light chain of the antibody described in any one of (c1) to (c10) binds. The multispecific antigen-binding molecule according to [1].

[14] The first polypeptide comprises the heavy chain of any antibody selected from the following (e1) to (e3), the second polypeptide comprises the heavy chain of any antibody selected from the following (f1) to (f3), and the third and fourth polypeptides comprise the light chain of any antibody selected from the following (g1) to (g4). The multispecific antigen-binding molecule according to [8] or [9]: (e1) The heavy chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of the antibody described in any one of (a1) to (a14) of

[12] and the light chain of the antibody described in any one of (c1) to (c10) binds, (e2) The heavy chain of an antibody in which at least one amino acid residue selected from the amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b, and 102 according to Kabat numbering in the heavy chain of any antibody selected from (e1) is substituted with another amino acid, The H chain of any antibody selected from (e3) and (e1), wherein the amino acid residue at position 34 according to Kabat numbering is isoleucine, the amino acid residue at position 35 is asparagine, glutamine or serine, the amino acid residue at position 49 is serine, the amino acid residue at position 61 is arginine, the amino acid residue at position 62 is glutamic acid, the amino acid residue at position 96 is serine or threonine, the amino acid residue at position 98 is lysine or arginine, the amino acid residue at position 100 is phenylalanine or tyrosine, the amino acid residue at position 100b is glycine, or the amino acid residue at position 102 is tyrosine. (f1) The H chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the H chain of any one of the antibodies (b1) to (b12) and the L chain of any one of the antibodies (c1) to (c10) in

[12] binds. (f2) The H chain of an antibody in which at least one amino acid residue selected from the amino acid residues at positions 35, 53, 73, 76, 96, 98, 100 and 100a according to Kabat numbering in the H chain of any one of the antibodies in (f1) is substituted with another amino acid. (f3) The H chain of an antibody in (f1), wherein the amino acid residue at position 35 according to Kabat numbering is aspartic acid, the amino acid residue at position 53 is arginine, the amino acid residue at position 73 is lysine, the amino acid residue at position 76 is glycine, the amino acid residue at position 96 is lysine or arginine, the amino acid residue at position 98 is tyrosine, the amino acid residue at position 100 is tyrosine, or the amino acid residue at position 100a is histidine. (g1) The L chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the H chain of any one of the antibodies (a1) to (a14) and the L chain of any one of the antibodies (c1) to (c10) in

[12] binds. (g2) The L chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the H chain of any one of the antibodies (b1) to (b12) and the L chain of any one of the antibodies (c1) to (c10) in

[12] binds. The light chain of an antibody of either (g3), (g1), or (g2), wherein at least one amino acid residue selected from the amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 according to Kabat numbering is substituted with another amino acid. (g4) The light chain of an antibody of either (g1) or (g2), wherein the amino acid residue at position 27 according to Kabat numbering is lysine or arginine, the amino acid residue at position 30 is glutamic acid, the amino acid residue at position 31 is arginine, the amino acid residue at position 32 is glutamine, the amino acid residue at position 50 is arginine or glutamine, the amino acid residue at position 52 is serine, the amino acid residue at position 53 is arginine, the amino acid residue at position 54 is lysine, the amino acid residue at position 55 is glutamic acid, the amino acid residue at position 92 is serine, the amino acid residue at position 93 is serine, the amino acid residue at position 94 is proline, or the amino acid residue at position 95 is proline.

[15] The multispecific antigen-binding molecule according to any one of [1] to

[14] , wherein the multispecific antigen-binding molecule is a multispecific antibody.

[16] The bispecific antibody according to any one of the following (a) to (u): (a) A bispecific antibody (Q1-G4k / J268-G4h / L45-k) in which the first polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are common light chains consisting of the amino acid sequence set forth in SEQ ID NO: 9. (b) A bispecific antibody (Q1-G4k / J321-G4h / L45-k) in which the first polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are common light chains consisting of the amino acid sequence set forth in SEQ ID NO: 9. (c) A bispecific antibody (Q31-z7 / J326-z107 / L2-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 8. (d) A bispecific antibody (Q64-z55 / J344-z107 / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 3, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9. (e) A bispecific antibody (Q64-z7 / J326-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (f) A bispecific antibody (Q64-z7 / J344-z107 / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (g) A bispecific antibody (Q85-G4k / J268-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (h) A bispecific antibody (Q85-G4k / J321-G4h / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (i) A bispecific antibody (Q153-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (j) A bispecific antibody (Q354-z106 / J259-z107 / L324-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 13, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 22, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 29. (k) A bispecific antibody (Q360-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (l) A bispecific antibody (Q360-z118 / J300-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 23, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (m) A bispecific antibody (Q405-G4k / J232-G4h / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (n) A bispecific antibody (Q458-z106 / J346-z107 / L408-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 34. (o) A bispecific antibody (Q460-z121 / J327-z119 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (p) A bispecific antibody (Q499-z118 / J327-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (q) A bispecific antibody (Q499-z118 / J327-z107 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (r) A bispecific antibody (Q499-z118 / J346-z107 / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (s) A bispecific antibody (Q499-z121 / J327-z119 / L404-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 32. (t) A bispecific antibody (Q499-z121 / J339-z119 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 26, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (u) A bispecific antibody (Q153-G4k / J142-G4h / L180-k) in which the first polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 170, and the third and fourth polypeptides are common light chains consisting of the amino acid sequence set forth in SEQ ID NO: 171. 〔17〕 A nucleic acid encoding the multispecific antigen-binding molecule according to any one of 〔1〕 to 〔15〕 or the bispecific antibody according to 〔16〕. 〔18〕 A vector into which the nucleic acid according to 〔17〕 has been inserted. 〔19〕 A cell containing the nucleic acid according to 〔17〕 or the vector according to 〔18〕. 〔20〕 A method for producing the multispecific antigen-binding molecule according to any one of 〔1〕 to 〔15〕 or the bispecific antibody according to 〔16〕 by culturing the cell according to 〔19〕. 〔21〕 A pharmaceutical composition comprising the multispecific antigen-binding molecule according to any one of 〔1〕 to 〔15〕 or the bispecific antibody according to 〔16〕, and a pharmaceutically acceptable carrier. 〔22〕 The composition according to 〔21〕, which is a pharmaceutical composition used for the prevention and / or treatment of bleeding, a disease accompanied by bleeding, or a disease caused by bleeding. 〔23〕 The composition according to 〔22〕, wherein the bleeding, the disease accompanied by bleeding, or the disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. 〔24〕 The composition according to 〔23〕, wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is hemophilia A. 〔25〕 The composition according to 〔23〕, wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is a disease in which an inhibitor against blood coagulation factor VIII and / or activated blood coagulation factor VIII has appeared. The composition according to

[23] , wherein the disease caused and / or developed by the decrease or deficiency of the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is acquired hemophilia. The composition according to

[23] , wherein the disease caused and / or developed by the decrease of the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII is von Willebrand disease. A method for preventing and / or treating bleeding, a disease accompanied by bleeding or a disease caused by bleeding, comprising the step of administering the multispecific antigen-binding molecule according to any one of [1] to

[15] or the bispecific antibody according to

[16] , or the composition according to any one of

[21] to

[27] . A kit for use in the method for preventing and / or treating according to

[28] , comprising at least the multispecific antigen-binding molecule according to any one of [1] to

[15] or the bispecific antibody according to

[16] , or the composition according to any one of

[21] to

[27] .

[0011] The present invention also relates to the following. Use of the multispecific antigen-binding molecule according to any one of [1] to

[15] or the bispecific antibody according to

[16] , or the composition according to any one of

[21] to

[27] in the manufacture of a prophylactic and / or therapeutic agent for bleeding, a disease accompanied by bleeding or a disease caused by bleeding. The multispecific antigen-binding molecule according to any one of [1] to

[15] or the bispecific antibody according to

[16] , or the composition according to any one of

[21] to

[27] for preventing and / or treating bleeding, a disease accompanied by bleeding or a disease caused by bleeding.

[0012] The present invention also relates to a bispecific antibody having a function of substituting for the function of F.VIII, which is a cofactor that enhances an enzymatic reaction, and a pharmaceutical composition containing the antibody as an active ingredient, and more specifically relates to the following. A bispecific antibody having a function to substitute for the function of blood coagulation factor VIII, comprising a first antigen-binding site that recognizes blood coagulation factor IX and / or activated blood coagulation factor IX, and a second antigen-binding site that recognizes blood coagulation factor X, wherein the bispecific antibody is any one of the following (a) to (u); (a) A bispecific antibody (Q1-G4k / J268-G4h / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9, (b) A bispecific antibody (Q1-G4k / J321-G4h / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9, (c) A bispecific antibody (Q31-z7 / J326-z107 / L2-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 8, (d) A bispecific antibody (Q64-z55 / J344-z107 / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 3, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9, (e) A bispecific antibody (Q64-z7 / J326-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30, (f) A bispecific antibody (Q64-z7 / J344-z107 / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (g) A bispecific antibody (Q85-G4k / J268-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (h) A bispecific antibody (Q85-G4k / J321-G4h / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (i) A bispecific antibody (Q153-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (j) A bispecific antibody (Q354-z106 / J259-z107 / L324-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 13, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 22, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 29. (k) A bispecific antibody (Q360-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (l) A bispecific antibody (Q360-z118 / J300-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 23, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (m) A bispecific antibody (Q405-G4k / J232-G4h / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (n) A bispecific antibody (Q458-z106 / J346-z107 / L408-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 34. (o) A bispecific antibody (Q460-z121 / J327-z119 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (p) A bispecific antibody (Q499-z118 / J327-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (q) A bispecific antibody (Q499-z118 / J327-z107 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (r) A bispecific antibody (Q499-z118 / J346-z107 / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (s) A bispecific antibody (Q499-z121 / J327-z119 / L404-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 32. (t) A bispecific antibody (Q499-z121 / J339-z119 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 26, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (u) A bispecific antibody (Q153-G4k / J142-G4h / L180-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 170, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 171.

[33] A nucleic acid encoding the bispecific antibody according to

[32] .

[34] A vector into which the nucleic acid according to

[33] has been inserted.

[35] A cell containing the nucleic acid according to

[33] or the vector according to

[34] .

[36] A method for producing the bispecific antibody according to

[32] by culturing the cell according to

[35] .

[37] A pharmaceutical composition comprising the bispecific antibody according to

[32] and a pharmaceutically acceptable carrier.

[38] The composition according to

[37] , which is a pharmaceutical composition used for the prevention and / or treatment of bleeding, a disease accompanied by bleeding, or a disease caused by bleeding. The composition according to

[38] , wherein bleeding, a disease accompanied by bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of factor VIII of blood coagulation and / or activated factor VIII of blood coagulation. The composition according to

[39] , wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of factor VIII of blood coagulation and / or activated factor VIII of blood coagulation is hemophilia A. The composition according to

[39] , wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of factor VIII of blood coagulation and / or activated factor VIII of blood coagulation is a disease in which an inhibitor against factor VIII of blood coagulation and / or activated factor VIII of blood coagulation appears. The composition according to

[39] , wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of factor VIII of blood coagulation and / or activated factor VIII of blood coagulation is acquired hemophilia. The composition according to

[39] , wherein the disease that develops and / or progresses due to a decrease in the activity of factor VIII of blood coagulation and / or activated factor VIII of blood coagulation is von Willebrand disease. A method for preventing and / or treating bleeding, a disease accompanied by bleeding, or a disease caused by bleeding, comprising the step of administering the bispecific antibody according to

[32] or the composition according to any one of

[37] to

[43] . A kit for use in the method for preventing and / or treating according to

[44] , comprising the bispecific antibody according to

[32] or the composition according to any one of

[37] to

[43] . Use of the bispecific antibody according to

[32] or the composition according to any one of

[37] to

[43] in the manufacture of a prophylactic and / or therapeutic agent for bleeding, a disease accompanied by bleeding, or a disease caused by bleeding. The bispecific antibody according to

[32] or the composition according to any one of

[37] to

[43] for preventing and / or treating bleeding, a disease accompanied by bleeding, or a disease caused by bleeding.

Advantages of the Invention

[0013] The present invention provides a multi - specific antigen - binding molecule that is an antibody recognizing both an enzyme and the substrate of the enzyme and has high activity to substitute for the function of F.VIII. Also provided is a multi - specific antigen - binding molecule that is an antibody recognizing both an enzyme and the substrate of the enzyme, has high activity to substitute for the function of F.VIII, and has low F.Xase inhibitory activity. Since humanized antibodies are generally considered to have high stability and low immunogenicity in blood, the multi - specific antibody of the present invention is considered to be extremely promising as a pharmaceutical.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Mode for Carrying Out the Invention

[0015] The multispecific antigen-binding molecules described in this specification include a first antigen-binding site and a second antigen-binding site that can specifically bind to at least two different antigens. The first antigen-binding site and the second antigen-binding site are not particularly limited as long as they each have binding activity to F.IX and / or F.IXa and F.X. Examples include sites necessary for binding to antigens such as antibodies, Scaffold molecules (antibody-like molecules), peptides, or fragments containing such sites. A Scaffold molecule is a molecule that exerts its function by binding to a target molecule, and any polypeptide can be used as long as it is a structurally stable polypeptide capable of binding to at least one target antigen. Examples of such polypeptides include, for example, antibody variable regions, fibronectin (WO2002 / 032925), Protein A domain (WO1995 / 001937), LDL receptor A domain (WO2004 / 044011, WO2005 / 040229), ankyrin (WO2002 / 020565), etc. In addition, molecules described by Nygren et al. (Current Opinion in Structural Biology, 7:463-469 (1997), Journal of Immunol Methods, 290:3-28 (2004)), Binz et al. (Nature Biotech 23:1257-1266 (2005)), Hosse et al. (Protein Science 15:14-27 (2006)) can be mentioned. Also, peptide molecules capable of binding to a target antigen as described in Curr Opin Mol Ther. 2010 Aug;12(4):487-95. and Drugs. 2008;68(7):901-12. can be used.

[0016] In the present invention, the multispecific antigen-binding molecule is not particularly limited as long as it can bind to at least two different antigens. Examples include polypeptides containing the above antigen-binding sites such as antibodies, Scaffold molecules, and fragments thereof, aptamers composed of nucleic acid molecules or peptides, etc. It may be a single molecule or a multimer thereof. Preferred multispecific antigen-binding molecules include multispecific antibodies that can specifically bind to at least two different antigens. Among the antibodies having an activity to substitute for the function of F.VIII of the present invention, particularly preferred antibodies include bispecific antibodies (BsAb) (sometimes referred to as bispecific antibodies) that can specifically bind to two different antigens.

[0017] In the present invention, the "common L chain" is an L chain that can associate with two or more different H chains and can exhibit binding ability to each antigen. Here, the "different H chains" preferably refer to the H chains of antibodies against different antigens, but are not limited thereto, and mean H chains with different amino acid sequences from each other. The common L chain can be obtained, for example, according to the method described in WO2006 / 109592.

[0018] The multispecific antigen-binding molecule (preferably a bispecific antibody) in the present invention is a molecule composed of an antibody or an antibody fragment having specificity for two or more different antigens. The antibody of the present invention is not particularly limited, but is preferably monoclonal. Monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified genetically recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0019] Also, the L chains of the antibodies that become the multispecific antigen-binding molecules of the present invention may be different, but preferably have a common L chain.

[0020] The multispecific antigen-binding molecule in the present invention is preferably a recombinant antibody produced using genetic recombination technology. (See, for example, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). A recombinant antibody can be obtained by cloning the DNA encoding it from antibody-producing cells such as hybridomas or immunized lymphocytes that produce antibodies, incorporating it into an appropriate vector, and introducing this into a host (host cell) for production.

[0021] Furthermore, the antibody in the present invention may include not only whole antibodies but also antibody fragments, miniaturized antibodies, and antibody modifications. For example, antibody fragments and miniaturized antibodies include diabody (Db), linear antibodies, single-chain antibody (hereinafter also referred to as scFv) molecules, and the like. Here, the "Fv" fragment is the smallest antibody fragment and contains a complete antigen recognition site and binding site.

[0022] The "Fv" fragment is a dimer (VH-VL dimer) in which one heavy-chain variable region (VH) and one light-chain variable region (VL) are strongly linked by non-covalent bonds. The three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the VH-VL dimer. The six CDRs confer an antigen-binding site on the antibody. However, even one variable region (or half of the Fv containing only the three CDRs specific for the antigen), although having a lower affinity than the full binding site, has the ability to recognize and bind the antigen.

[0023] In addition, the Fab fragment (also called F(ab)) further contains the constant region of the L chain and the constant region of the H chain (CH1). The Fab' fragment differs from the Fab fragment in that it additionally has several residues derived from the carboxy terminus of the H chain CH1 region containing one or more cysteines from the hinge region of the antibody. Fab'-SH refers to Fab' in which one or more cysteine residues in the constant region have free thiol groups. The F(ab') fragment is produced by cleavage of the disulfide bond at the cysteine in the hinge portion of the F(ab')2 pepsin digest. Other chemically linked antibody fragments are also known to those skilled in the art.

[0024] A diabody refers to a bivalent, small-sized antibody constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097, WO93 / 11161, etc.). A diabody is a dimer composed of two polypeptide chains, and each polypeptide chain has the L chain variable region (VL) and the H chain variable region (VH) in the same chain, which are linked by a short linker, for example, preferably 2 to 12 residues, more preferably 3 to 10 residues, and particularly about 5 residues, such that they cannot bind to each other. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment due to the short linker between them and form a dimer, the diabody has two antigen-binding sites.

[0025] Single-chain antibodies or scFv antibody fragments contain the VH and VL regions of the antibody, and these regions are present in a single polypeptide chain. Generally, the Fv polypeptide further contains a polypeptide linker between the VH and VL regions, whereby the scFv can form the structure necessary for antigen binding (for a review of scFv, see Pluckthun, "The Pharmacology of Monoclonal Antibodies," Vol. 113 (Rosenburg and Moore eds., Springer Verlag, New York, pp. 269-315, 1994)). The linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable regions linked to both ends thereof.

[0026] IgG-type bispecific antibodies can be secreted by a hybrid hybridoma (quadroma) generated by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al., Nature 1983, 305: 537-540). They can also be secreted by co-expressing the genes of the L chain and H chain that make up the two types of IgG of interest, a total of four genes, by introducing them into cells.

[0027] At this time, by performing appropriate amino acid substitutions in the CH3 region of the H chain, it is also possible to preferentially secrete a heterologous combination of IgG for the H chain (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.).

[0028] In addition, regarding the L chain, since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of binding to both H chains can be obtained, and the antibody of the present invention is characterized by having a common L chain. By introducing this common L chain and both H chain genes into cells to express IgG, efficient expression of bispecific IgG becomes possible.

[0029] Also, bispecific antibodies can be prepared by chemically cross-linking Fab'. For example, Fab' prepared from one antibody is maleimidated with o-PDM (ortho-phenylenedi-maleimide), and this is reacted with Fab' prepared from the other antibody to cross-link Fab' derived from different antibodies to produce bispecific F(ab')2 (Keler T et al. Cancer Research 1997, 57: 4008-4014). Also, methods of chemically binding antibody fragments such as Fab'-thionitrobenzoic acid (TNB) derivatives and Fab'-thiol (SH) are known (Brennan M et al. Science 1985, 229: 81-83).

[0030] Leucine zippers derived from Fos, Jun, etc. can also be used instead of chemical cross-linking. Fos and Jun form homodimers, but the fact that they preferentially form heterodimers is utilized. Fab' with an added Fos leucine zipper and the other Fab' with an added Jun are prepared by expression. Bispecific F(ab')2 can be formed by mixing and reacting the monomeric Fab'-Fos and Fab'-Jun reduced under mild conditions (Kostelny SA et al. J of Immunology, 1992, 148: 1547-53). This method is not limited to Fab' and can also be applied to scFv, Fv, etc.

[0031] In addition, bispecific antibodies such as IgG-scFv (Protein Eng Des Sel. 2010 Apr;23(4):221-8), sc(Fv)2 such as BiTE (Drug Discov Today. 2005 Sep 15;10(18):1237-44.), DVD-Ig (Nat Biotechnol. 2007 Nov;25(11):1290-7. Epub 2007 Oct 14., MAbs. 2009 Jul;1(4):339-47. Epub 2009 Jul 10.), etc. (IDrugs 2010, 13:698-700), two-in-one antibodies (Science. 2009 Mar 20;323(5921):1610-4., Immunotherapy. 2009 Sep;1(5):749-51.), bispecific antibodies such as Tri-Fab, tandem scFv, diabody, etc. (MAbs. 2009 November ; 1(6): 539-547.) are also known. Furthermore, even when using molecular forms such as scFv-Fc and scaffold-Fc, by preferentially secreting heterologous combinations of Fc (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.), bispecific antibodies can be efficiently produced.

[0032] Bispecific antibodies can also be produced in diabodies. Bispecific diabodies are heterodimers of two cross-over scFv fragments. That is, they can be formed by constructing a heterodimer using VH (A)-VL (B) and VH (B)-VL (A) prepared by connecting the VH and VL derived from two types of antibodies A and B with a relatively short linker of around 5 residues (Holliger P et al. Proc of the National Academy of Sciences of the USA 1993, 90: 6444-6448).

[0033] At this time, two kinds of scFvs are linked with a flexible and relatively long linker of about 15 residues (single-chain diabody: Kipriyanov SM et al. J of Molecular Biology. 1999, 293: 41-56), and the target configuration can also be promoted by performing appropriate amino acid substitutions (knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788, VH / VL interface engineering: Igawa T et al. Protein Eng Des Sel. 2010, 8:667-77.).

[0034] sc(Fv)2 that can be prepared by linking two kinds of scFvs with a flexible and relatively long linker of about 15 residues can also become a bispecific antibody (Mallender WD et al. J of Biological Chemistry, 1994, 269: 199-206).

[0035] Examples of antibody modifications include antibodies conjugated with various molecules such as polyethylene glycol (PEG). The antibodies of the present invention also include these antibody modifications. In the antibody modifications of the present invention, the substances to be conjugated are not limited. Such antibody modifications can be obtained by chemically modifying the obtained antibodies. These methods are already established in this field.

[0036] The antibodies of the present invention include human antibodies, mouse antibodies, rat antibodies, etc., and their origins are not limited. Also, genetically modified antibodies such as chimeric antibodies and humanized antibodies may be used.

[0037] Methods for obtaining human antibodies are already known. For example, a transgenic animal having all repertoires of human antibody genes can be immunized with a target antigen to obtain a target human antibody (see International Patent Application Publication Nos. WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, WO 96 / 33735).

[0038] Genetically modified antibodies can be produced using known methods. Specifically, for example, a chimeric antibody is an antibody consisting of the variable regions of the H chain and the L chain of an antibody of an immunized animal and the constant regions of the H chain and the L chain of a human antibody. A DNA encoding the variable region of an antibody derived from an immunized animal is ligated to a DNA encoding the constant region of a human antibody, and this is incorporated into an expression vector and introduced into a host for production, whereby a chimeric antibody can be obtained.

[0039] A humanized antibody is a modified antibody also referred to as a reshaped human antibody. A humanized antibody is constructed by transplanting the CDRs of an antibody derived from an immunized animal into the complementarity-determining regions of a human antibody. General genetic recombination techniques thereof 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).

[0040] The multispecific antigen-binding molecule of the present invention is a multispecific antigen-binding molecule that recognizes F.IX and / or F.IXa, and F.X and has a function of substituting for the function of cofactor F.VIII. Compared with hA69-KQ / hB26-PF / hAL-AQ (described in WO2006 / 109592), which is known as a bispecific antibody having a function of substituting for the function of F.VIII, it is characterized by having high F.Xa production promoting activity. Further, the antibody of the present invention usually has a structure including the variable region in an anti-F.IXa antibody and the variable region in an anti-F.X antibody.

[0041] That is, the present invention provides a multispecific antigen-binding molecule having a function that substitutes for the function of F.VIII, which comprises a first antigen-binding site that recognizes F.IX and / or F.IXa and a second antigen-binding site that recognizes F.X, wherein the function that substitutes for the function of F.VIII is a function resulting from a high F.Xa production promoting activity as compared with a bispecific antibody (hA69-KQ / hB26-PF / hAL-AQ) having an H chain consisting of SEQ ID NOs: 165 and 166 and a common L chain consisting of SEQ ID NO: 167.

[0042] The multispecific antigen-binding molecule in the present invention comprises a first polypeptide and a third polypeptide containing an antigen-binding site that recognizes F.IX and / or F.IXa, and a second polypeptide and a fourth polypeptide containing an antigen-binding site that recognizes F.X. The first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide contain an antigen-binding site of the H chain of an antibody and an antigen-binding site of the L chain of an antibody.

[0043] For example, in the multispecific antigen-binding molecule of the present invention, the first polypeptide and the third polypeptide each contain an antigen-binding site of the H chain or the L chain of an antibody against F.IX or F.IXa, and the second polypeptide and the fourth polypeptide each contain an antigen-binding site of the H chain or the L chain of an antibody against F.X. At this time, the antigen-binding sites of the L chains of the antibodies contained in the first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide may be a common L chain.

[0044] The polypeptide containing the antigen-binding site of the L chain of the antibody in the present invention preferably contains all or part of the sequence of the L chain of an antibody that binds to F.IX, F.IXa and / or F.X.

[0045] As a preferred embodiment of the antigen-binding site of the first polypeptide of the antibody of the present invention, specifically, as described in the examples below The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q1 (SEQ ID NOs: 75, 76, 77), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q31 (SEQ ID NOs: 78, 79, 80), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q64 (SEQ ID NOs: 81, 82, 83), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q85 (SEQ ID NOs: 84, 85, 86), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q153 (SEQ ID NOs: 87, 88, 89), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q354 (SEQ ID NOs: 90, 91, 92), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q360 (SEQ ID NOs: 93, 94, 95), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q405 (SEQ ID NOs: 96, 97, 98), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q458 (SEQ ID NOs: 99, 100, 101), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q460 (SEQ ID NOs: 102, 103, 104), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of Q499 (SEQ ID NOs: 105, 106, 107), An antigen-binding site consisting of the amino acid sequences thereof, or an antigen-binding site functionally equivalent thereto can be mentioned.

[0046] Furthermore, as a preferred embodiment, the antigen-binding site of the second polypeptide is specifically the one described in the following examples The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J232 (SEQ ID NOs: 108, 109, 110), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J259 (SEQ ID NOs: 111, 112, 113), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J268 (SEQ ID NOs: 114, 115, 116), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J300 (SEQ ID NOs: 117, 118, 119), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J321 (SEQ ID NOs: 120, 121, 122), The amino acid sequences of CDR1, 2, and 3 of the heavy chain of J326 (SEQ ID NOs: 123, 124, 125), The amino acid sequences of the H-chain CDR1, CDR2, and CDR3 of J327 (SEQ ID NOs: 126, 127, 128), The amino acid sequences of the H-chain CDR1, CDR2, and CDR3 of J339 (SEQ ID NOs: 129, 130, 131), The amino acid sequences of the H-chain CDR1, CDR2, and CDR3 of J344 (SEQ ID NOs: 132, 133, 134), The amino acid sequences of the H-chain CDR1, CDR2, and CDR3 of J346 (SEQ ID NOs: 135, 136, 137), The amino acid sequences of the H-chain CDR1, CDR2, and CDR3 of J142 (SEQ ID NOs: 174, 175, 176), and antigen-binding sites consisting of the amino acid sequences described above, or antigen-binding sites functionally equivalent thereto can be mentioned.

[0047] That is, the present invention provides a multispecific antigen-binding molecule in which the antigen-binding site of the first polypeptide comprises an antigen-binding site comprising an H-chain CDR consisting of any of the amino acid sequences selected from the following (a1) to (a11) or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site comprising an H-chain CDR consisting of any of the amino acid sequences selected from the following (b1) to (b11) or an antigen-binding site functionally equivalent thereto: (a1) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 75, 76, 77 (H-chain CDRs of Q1), (a2) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 78, 79, 80 (H-chain CDRs of Q31), (a3) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 81, 82, 83 (H-chain CDRs of Q64), (a4) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 84, 85, 86 (H-chain CDRs of Q85), (a5) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 87, 88, 89 (H-chain CDRs of Q153), (a6) An antigen-binding site having the amino acid sequences of the H-chain CDR1, CDR2, and CDR3 described in SEQ ID NOs: 90, 91, 92 (H-chain CDRs of Q354), (a7) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 93, 94, 95 (H-chain CDRs of Q360), (a8) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 96, 97, 98 (H-chain CDRs of Q405), (a9) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 99, 100, 101 (H-chain CDRs of Q458), (a10) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 102, 103, 104 (H-chain CDRs of Q460), (a11) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 105, 106, 107 (H-chain CDRs of Q499), (b1) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 108, 109, 110 (H-chain CDRs of J232), (b2) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 111, 112, 113 (H-chain CDRs of J259), (b3) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 114, 115, 116 (H-chain CDRs of J268), (b4) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 117, 118, 119 (H-chain CDRs of J300), (b5) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 120, 121, 122 (H-chain CDRs of J321), (b6) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 123, 124, 125 (H-chain CDRs of J326), (b7) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 126, 127, 128 (H-chain CDRs of J327), (b8) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 129, 130, 131 (H-chain CDRs of J339), (b9) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 132, 133, 134 (H-chain CDRs of J344), (b10) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 135, 136, 137 (H-chain CDRs of J346), (b11) An antigen-binding site having the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 174, 175, 176 (H-chain CDRs of J142).

[0048] Also, as preferred embodiments of the antigen-binding sites of the third and fourth polypeptides, specifically, the sequences of L-chain CDR1, 2, and 3 of L2 (SEQ ID NOs: 138, 139, 140) described in the examples below, sequences of L-chain CDR1, 2, and 3 of L45 (SEQ ID NOs: 141, 142, 143), sequences of L-chain CDR1, 2, and 3 of L248 (SEQ ID NOs: 144, 145, 146), sequences of L-chain CDR1, 2, and 3 of L324 (SEQ ID NOs: 147, 148, 149), sequences of L-chain CDR1, 2, and 3 of L334 (SEQ ID NOs: 150, 151, 152), sequences of L-chain CDR1, 2, and 3 of L377 (SEQ ID NOs: 153, 154, 155), sequences of L-chain CDR1, 2, and 3 of L404 (SEQ ID NOs: 156, 157, 158), sequences of L-chain CDR1, 2, and 3 of L406 (SEQ ID NOs: 159, 160, 161), sequences of L-chain CDR1, 2, and 3 of L408 (SEQ ID NOs: 162, 163, 164), sequences of L-chain CDR1, 2, and 3 of L180 (SEQ ID NOs: 177, 178, 179), antigen-binding sites consisting of the amino acid sequences described above, or antigen-binding sites functionally equivalent to these can be mentioned.

[0049] That is, the present invention provides a multispecific antigen-binding molecule in which the antigen-binding sites contained in the third polypeptide and the fourth polypeptide include an antigen-binding site comprising an L-chain CDR consisting of any amino acid sequence selected from the following (c1) to (c10), or an antigen-binding site functionally equivalent thereto: (c1) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 138, 139, and 140 (L-chain CDRs of L2), (c2) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 141, 142, and 143 (L-chain CDRs of L45), (c3) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 144, 145, and 146 (L-chain CDRs of L248), (c4) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 147, 148, and 149 (L-chain CDRs of L324), (c5) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 150, 151, and 152 (L-chain CDRs of L334), (c6) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 153, 154, and 155 (L-chain CDRs of L377), (c7) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 156, 157, and 158 (L-chain CDRs of L404), (c8) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 159, 160, and 161 (L-chain CDRs of L406), (c9) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 137, 138, and 139 (L-chain CDRs of L408), (c10) An antigen-binding site having the amino acid sequences of L-chain CDR1, 2, and 3 described in SEQ ID NOs: 177, 178, and 179 (L-chain CDRs of L180).

[0050] The amino acid sequences of the heavy chain variable regions of Q1, Q31, Q64, Q85, Q153, Q354, Q360, Q405, Q458, Q460, and Q499 described in the present invention are respectively represented by the following sequence numbers. Q1: Sequence number: 35 Q31: Sequence number: 36 Q64: Sequence number: 37 Q85: Sequence number: 38 Q153: Sequence number: 39 Q354: Sequence number: 40 Q360: Sequence number: 41 Q405: Sequence number: 42 Q458: Sequence number: 43 Q460: Sequence number: 44 Q499: Sequence number: 45

[0051] The amino acid sequences of the heavy chain variable regions of J232, J259, J268, J300, J321, J326, J327, J339, J344, J346, and J142 described in the present invention are respectively represented by the following sequence numbers. J232: Sequence number: 46 J259: Sequence number: 47 J268: Sequence number: 48 J300: Sequence number: 49 J321: Sequence number: 50 J326: Sequence number: 51 J327: Sequence number: 52 J339: Sequence number: 53 J344: Sequence number: 54 J346: Sequence number: 55 J142: Sequence number: 172

[0052] That is, the present invention provides a multispecific antigen-binding molecule in which the antigen-binding site of the first polypeptide comprises an antigen-binding site comprising a heavy-chain variable region consisting of any amino acid sequence selected from the following (a1) to (a11) or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site comprising a heavy-chain variable region consisting of any amino acid sequence selected from the following (b1) to (b11) or an antigen-binding site functionally equivalent thereto: (a1) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 35 (heavy-chain variable region of Q1), (a2) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 36 (heavy-chain variable region of Q31), (a3) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 37 (heavy-chain variable region of Q1), (a4) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 38 (heavy-chain variable region of Q85), (a5) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 39 (heavy-chain variable region of Q153), (a6) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 40 (heavy-chain variable region of Q354), (a7) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 41 (heavy-chain variable region of Q360), (a8) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 42 (heavy-chain variable region of Q405), (a9) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 43 (heavy-chain variable region of Q458), (a10) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 44 (heavy-chain variable region of Q460), (a11) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 45 (heavy-chain variable region of Q499), (b1) An antigen-binding site having the amino acid sequence of the heavy-chain variable region set forth in SEQ ID NO: 46 (heavy-chain variable region of J232), (b2) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 47 (heavy-chain variable region of J259), (b3) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 48 (heavy-chain variable region of J268), (b4) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 49 (heavy-chain variable region of J300), (b5) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 50 (heavy-chain variable region of J321), (b6) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 51 (heavy-chain variable region of J326), (b7) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 52 (heavy-chain variable region of J327), (b8) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 53 (heavy-chain variable region of J339), (b9) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 54 (heavy-chain variable region of J344), (b10) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 55 (heavy-chain variable region of J346), (b11) An antigen-binding site having the amino acid sequence of the heavy-chain variable region described in SEQ ID NO: 172 (heavy-chain variable region of J142).

[0053] Also, the amino acid sequences of the light-chain variable regions of L2, L45, L248, L324, L334, L377, L404, L406, L408, and L180 described in the present invention are respectively represented by the following SEQ ID NOs. L2: SEQ ID NO: 56 L45: SEQ ID NO: 57 L248: SEQ ID NO: 58 L324: SEQ ID NO: 59 L334: SEQ ID NO: 60 L377: SEQ ID NO: 61 L404: SEQ ID NO: 62 L406: SEQ ID NO: 63 L408: SEQ ID NO: 64 L180: Sequence number: 173

[0054] That is, the present invention provides a multispecific antigen-binding molecule in which the antigen-binding sites contained in the third polypeptide and the fourth polypeptide include an antigen-binding site comprising a light chain variable region consisting of any amino acid sequence selected from the following (c1) to (c10) or an antigen-binding site functionally equivalent thereto: (c1) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 56 (light chain variable region of L2), (c2) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 57 (light chain variable region of L45), (c3) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 58 (light chain variable region of L248), (c4) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 59 (light chain variable region of L324), (c5) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 60 (light chain variable region of L334), (c6) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 61 (light chain variable region of L377), (c7) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 62 (light chain variable region of L404), (c8) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 63 (light chain variable region of L406), (c9) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 64 (light chain variable region of L408), (c10) An antigen-binding site having the amino acid sequence of the light chain variable region described in SEQ ID NO: 173 (light chain variable region of L180).

[0055] Note that the amino acid sequences of CDR1-3 and FR1-4 of each sequence are as described in FIGS. 3A-D.

[0056] When producing a full-length antibody using the variable region disclosed in the present invention, the constant region is not particularly limited, and a constant region known to those skilled in the art can be used. For example, the constant regions described in Sequences of proteins of immunological interest, (1991), U.S. Department of Health and Human Services. Public Health Service National Institutes of Health, An efficient route to human bispecific IgG, (1998). Nature Biotechnology vol. 16, 677-681, etc. can be used. Preferred examples of the antibody constant region of the present invention include the constant region of an IgG antibody. When using the constant region of an IgG antibody, its type is not limited, and the IgG constant regions of subclasses such as IgG1, IgG2, IgG3, IgG4 can be used. Further, amino acid mutations may be introduced into the constant region portion of the constant regions of these subclasses of IgG. Examples of the amino acid mutations to be introduced include those that increase or decrease the binding to the Fcγ receptor (Proc Natl Acad Sci U S A. 2006 Mar 14;103(11):4005-10., MAbs. 2009 Nov;1(6):572-9.), those that increase or decrease the binding to FcRn (J Biol Chem. 2001 Mar 2;276(9):6591-604, Int Immunol. 2006 Dec;18(12):1759-69., J Biol Chem. 2006 Aug 18;281(33):23514-24.), etc., but are not limited thereto. In order to produce a bispecific antibody, it is necessary to heteroassociate two types of H chains.To heterodimerize two types of H chains via the CH3 domain, it is possible to use techniques such as the knobs-into-hole technique (J Immunol Methods. 2001 Feb 1;248(1-2):7-15, J Biol Chem. 2010 Jul 2;285(27):20850-9.), the electrostatic repulsion technique (WO2006 / 106905), the SEEDbody technique (Protein Eng Des Sel. 2010 Apr;23(4):195-202.), etc. Furthermore, the antibodies of the present invention may have modified or deficient sugar chains. Examples of antibodies with modified sugar chains include, for example, antibodies with modified glycosylation (such as WO99 / 54342), antibodies deficient in fucose added to the sugar chain (WO00 / 61739, WO02 / 31140, WO2006 / 067847, WO2006 / 067913, etc.), antibodies having a sugar chain with bisecting GlcNAc (such as WO02 / 79255), etc. As methods for producing IgG antibodies deficient in sugar chains, methods of introducing mutations into asparagine at position 297 of the EU numbering (J Clin Pharmacol. 2010 May;50(5):494-506.), methods of producing IgG in Escherichia coli (J Immunol Methods. 2002 May 1;263(1-2):133-47., J Biol Chem. 2010 Jul 2;285(27):20850-9.), etc. are known. Also, it is possible to reduce heterogeneity associated with deletion of lysine at the C-terminus of IgG or mispairing of disulfide bonds in the hinge region of IgG2 by introducing amino acid deletions and substitutions (WO2009 / 041613).

[0057] For example, the present invention provides a multispecific antigen-binding molecule in which the first and second polypeptides comprise the heavy chain constant region of an antibody and the third and fourth polypeptides comprise the light chain constant region of an antibody.

[0058] The present invention also provides a multispecific antigen-binding molecule, wherein the first polypeptide comprises the heavy-chain constant region of an antibody consisting of any amino acid sequence selected from the following groups (d1) to (d6) or any amino acid sequence selected from the following groups (d7) to (d9), and the second polypeptide comprises the heavy-chain constant region of an antibody consisting of any amino acid sequence selected from a group different from the first polypeptide: (d1) The heavy-chain constant region set forth in SEQ ID NO: 65 (G4k); (d2) The heavy-chain constant region set forth in SEQ ID NO: 66 (z7); (d3) The heavy-chain constant region set forth in SEQ ID NO: 67 (z55); (d4) The heavy-chain constant region set forth in SEQ ID NO: 68 (z106); (d5) The heavy-chain constant region set forth in SEQ ID NO: 69 (z118); (d6) The heavy-chain constant region set forth in SEQ ID NO: 70 (z121); (d7) The heavy-chain constant region set forth in SEQ ID NO: 71 (G4h); (d8) The heavy-chain constant region set forth in SEQ ID NO: 72 (z107); (d9) The heavy-chain constant region set forth in SEQ ID NO: 73 (z119).

[0059] Furthermore, the present invention provides a multispecific antigen-binding molecule, wherein the third and fourth polypeptides comprise the light-chain constant region of an antibody consisting of the following amino acid sequence (e): (e) The light-chain constant region set forth in SEQ ID NO: 74 (k).

[0060] In the present invention, "substituting the function of F.VIII" means recognizing F.IX and / or F.IXa, and F.X, and promoting the activation of F.X (promoting the production of F.Xa).

[0061] The "FXa production promoting activity" in the present invention can be confirmed by evaluating the multispecific antigen-binding molecule of the present invention in a measurement system composed of, for example, FIXa (FIX activating enzyme), FIX, FX, F synthetic substrate S-2222 (synthetic substrate of FXa), and phospholipid. This measurement system shows a correlation with the 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 FVIII activity. Thromb Haemost 1985; 54: 811-23). That is, in this measurement system, a test substance showing higher FXa production promoting activity is expected to show a better hemostatic effect against bleeding symptoms in hemophilia A. Based on the results, as a multispecific antigen-binding molecule having an activity to substitute for the function of FVIII, any molecule having higher activity than hA69-KQ / hB26-PF / hAL-AQ can obtain excellent blood coagulation promoting activity and excellent effects as a pharmaceutical component for the prevention and / or treatment of bleeding, diseases accompanied by bleeding, or diseases caused by bleeding. In order to obtain excellent effects as the pharmaceutical component, for example, the FXa production promoting activity measured under the conditions described in [Example 2] is preferably not less than that of hA69-KQ / hB26-PF / hAL-AQ, and particularly preferably not less than or equal to that of Q153-G4k / J142-G4h / L180-k. Here, the FXa production promoting activity refers to the value obtained by subtracting the absorbance change value of the solvent for 20 minutes from the absorbance change value of the antibody solution for 20 minutes.

[0062] A preferred embodiment of the present invention is a multispecific antibody that recognizes FIX and / or FIXa, and FX and has a function to substitute for the function of FVIII.

[0063] The above-mentioned multispecific antibody of the present invention is preferably an antibody comprising the H-chain CDR of an anti-F.IX / F.IXa antibody or a CDR functionally equivalent thereto and the H-chain CDR of an anti-F.X antibody or a CDR functionally equivalent thereto.

[0064] Moreover, the antibody of the present invention is, in the anti-F.IX / IXa antibody, the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 75, 76, 77 (H-chain CDRs of Q1), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 78, 79, 80 (H-chain CDRs of Q31), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 81, 82, 83 (H-chain CDRs of Q64), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 84, 85, 86 (H-chain CDRs of Q85), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 87, 88, 89 (H-chain CDRs of Q153), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 90, 91, 92 (H-chain CDRs of Q354), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 93, 94, 95 (H-chain CDRs of Q360), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 96, 97, 98 (H-chain CDRs of Q405), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 99, 100, 101 (H-chain CDRs of Q458), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 102, 103, 104 (H-chain CDRs of Q460), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 105, 106, 107 (H-chain CDRs of Q499), an antigen-binding site consisting thereof or an antigen-binding site functionally equivalent thereto, and in the anti-F.X antibody the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 108, 109, 110 (H-chain CDRs of J232), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 111, 112, 113 (H-chain CDRs of J259), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 114, 115, 116 (H-chain CDRs of J268), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 117, 118, 119 (H-chain CDRs of J300), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 120, 121, 122 (H-chain CDRs of J321), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 123, 124, 125 (H-chain CDRs of J326), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 126, 127, 128 (H-chain CDRs of J327), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 129, 130, 131 (H-chain CDRs of J339), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 132, 133, 134 (H-chain CDRs of J344), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 135, 136, 137 (H-chain CDRs of J346), or the amino acid sequences of H-chain CDR1, 2, and 3 described in SEQ ID NOs: 174, 175, 176 (H-chain CDRs of J142), It is preferable that the antibody comprises an antigen-binding site consisting of the above or an antigen-binding site functionally equivalent thereto.

[0065] In the present invention, the antigen-binding sites being functionally equivalent means that the activities of substituting the functions of F.VIII possessed by the multispecific antigen-binding molecule containing the antigen-binding site are equivalent.

[0066] In the present invention, "equivalent" does not necessarily mean having the same level of activity, and the activity may be enhanced, or may have higher activity than hA69-KQ / hB26-PF / hAL-AQ in the above measurement system, or preferably, as long as it has equivalent or higher activity in promoting F.Xa production as measured under the conditions described in [Example 2] compared to Q153-G4k / J142-G4h / L180-k, the activity may be decreased.

[0067] As long as the above antibody has higher activity than hA69-KQ / hB26-PF / hAL-AQ in the measurement system of the above paragraph number

[0061] , preferably, the activity of promoting the production of F.Xa when measured under the conditions described in [Example 2] is equal to or higher than that of Q153-G4k / J142-G4h / L180-k, one or more amino acids in the amino acid sequence of the variable region (CDR sequence and / or FR sequence) may be substituted, deleted, added and / or inserted. In the amino acid sequence, as a method well known to those skilled in the art for substituting, deleting, adding and / or inserting one or more amino acids, a method for introducing mutations into proteins is known.For example, a person skilled in the art can prepare a mutant that is functionally equivalent to a multispecific polypeptide multimer having an activity that substitutes for the function of the target F.VIII by appropriately introducing mutations into the amino acid sequence using site-directed mutagenesis methods (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M.(1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500, Kramer,W, Drutsa,V, Jansen,HW, Kramer,B, Pflugfelder,M, and Fritz,HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ(1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. Enzymol. 154, 350-367, Kunkel,TA(1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc Natl Acad Sci U S A. 82, 488-492), etc.

[0068] Thus, in the variable region, one or more amino acids are mutated, and antibodies having an activity higher than that of hA69-KQ / hB26-PF / hAL-AQ in the measurement system of the above paragraph number

[0061] , preferably an activity promoting FXa production equal to or higher than that of Q153-G4k / J142-G4h / L180-k when measured under the conditions described in [Example 2] are also included in the antibodies of the present invention.

[0069] When modifying amino acid residues, it is desirable to mutate them to another amino acid in which the properties of the amino acid side chain are preserved. For example, as properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), amino acids having sulfur atom-containing side chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), and amino acids having aromatic-containing side chains (H, F, Y, W) can be mentioned (the parentheses represent the one-letter codes of the amino acids). Substitutions of amino acids within each of these groups are referred to as conservative substitutions. It is already known that a polypeptide having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids for a certain amino acid sequence maintains its biological activity (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81:5662-6; Zoller, M. J. and Smith, M., Nucleic Acids Res. (1982) 10:6487-500; Wang, A. et al., Science (1984) 224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79:6409-13). Such variants have at least 70%, more preferably at least 75%, more preferably at least 80%, still more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity with the amino acid sequence of the variable region (e.g., CDR sequence, FR sequence, entire variable region) of the present invention. In the present specification, sequence identity is defined as the ratio of residues identical to the residues of the amino acid sequence of the original heavy chain variable region or light chain variable region after aligning the sequences as necessary so that the sequence identity is maximized and introducing gaps as appropriate. The amino acid sequence identity can be determined by the method described below.

[0070] In addition, in the amino acid sequence of the variable region (CDR sequence and / or FR sequence), one or more amino acids are substituted, deleted, added, and / or inserted, and the activity is higher than that of hA69-KQ / hB26-PF / hAL-AQ in the measurement system of the above paragraph number

[0061] , preferably the amino acid sequence of the variable region having an activity of promoting F.Xa production equal to or higher than that of Q153-G4k / J142-G4h / L180-k when measured under the conditions described in [Example 2] can also be obtained from a nucleic acid that hybridizes to a nucleic acid consisting of a nucleotide sequence encoding the amino acid sequence of the variable region under stringent conditions. As stringent hybridization conditions for isolating a nucleic acid that hybridizes to a nucleic acid consisting of a nucleotide sequence encoding the amino acid sequence of the variable region under stringent conditions, hybridization conditions such as 6 M urea, 0.4% SDS, 0.5 x SSC, and 37 °C or hybridization conditions of equivalent stringency can be exemplified. If more stringent conditions, for example, conditions of 6 M urea, 0.4% SDS, 0.1 x SSC, and 42 °C are used, isolation of a nucleic acid with higher homology can be expected. The determination of the sequence of the isolated nucleic acid can be performed by known methods described below. The homology of the isolated nucleic acid has sequence identity of at least 50% or more, more preferably 70% or more, and even more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) over the entire nucleotide sequence.

[0071] Instead of the method using the above hybridization technique, it is also possible to isolate a nucleic acid that hybridizes to a nucleic acid consisting of a nucleotide sequence encoding the amino acid sequence of the variable region under stringent conditions by using a gene amplification method using a primer synthesized based on the nucleotide sequence information encoding the amino acid sequence of the variable region, for example, the polymerase chain reaction (PCR) method.

[0072] The identity of nucleotide sequences and amino acid sequences can be determined by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al., J. Mol. Biol. (1990) 215: 403-10). When analyzing nucleotide sequences by BLASTN based on BLAST, the parameters are, for example, score = 100 and wordlength = 12. When analyzing amino acid sequences by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and the Gapped BLAST program, the default parameters of each program are used. The specific methods of these analysis methods are known (see the website of BLAST (Basic Local Alignment Search Tool) of NCBI (National Center for Biotechnology Information); http: / / www.ncbi.nlm.nih.gov).

[0073] The present invention also provides an antibody that binds to an epitope overlapping with the epitope to which the above antibody binds.

[0074] Whether or not a certain antibody recognizes an epitope overlapping with that of another antibody can be confirmed by competition between the epitopes of the two. Competition between antibodies can be evaluated by a competitive binding assay, and examples of such means include enzyme-linked immunosorbent assay (ELISA), fluorescence energy transfer measurement (FRET), and fluorescence microplate assay technology (FMAT (registered trademark)). The amount of the antibody bound to the antigen is indirectly correlated with the binding ability of a candidate competing antibody (test antibody) that competes with respect to binding to the overlapping epitope. That is, the greater the amount and affinity of the test antibody for the overlapping epitope, the lower the amount of binding of the antibody to the antigen, and the greater the amount of binding of the test antibody to the antigen. Specifically, the antibody labeled with an appropriate label and the antibody to be evaluated are simultaneously added to the antigen, and the labeled antibody bound is detected using the label. The amount of the antibody bound to the antigen can be easily measured by previously labeling the antibody. This label is not particularly limited, but a labeling method corresponding to the technique is selected. Specific examples of the labeling method include fluorescence labeling, radioisotope labeling, and enzyme labeling.

[0075] For example, the antibody labeled with fluorescence and the unlabeled antibody or test antibody are simultaneously added to beads immobilized with F.IX, F.IXa, or F.X, and the labeled antibody is detected by fluorescence microplate assay technology.

[0076] The "antibody that binds to an overlapping epitope" as used herein refers to a concentration (IC 50 ) at which the binding amount of the labeled antibody is reduced by 50% due to the binding of the unlabeled antibody thereto. With respect to this concentration, the test antibody can reduce the binding amount of the labeled antibody by at least 50% at a concentration that is usually 100-fold, preferably 80-fold, more preferably 50-fold, still more preferably 30-fold, and even more preferably 10-fold higher than the IC 50 of the unlabeled antibody.

[0077] A multispecific antigen-binding molecule having an antigen-binding site of an antibody that binds to an epitope overlapping with the epitope to which the above antibody binds can obtain an activity to substitute for excellent F.VIII function. Further, the antigen-binding site of an antibody that binds to an epitope overlapping with the epitope to which the above antibody binds can be modified with one or more amino acids in order to obtain an activity to substitute for more excellent F.VIII function. After modifying the amino acids of the antigen-binding site, a multispecific antigen-binding molecule having an activity higher than that of hA69-KQ / hB26-PF / hAL-AQ, preferably an activity equivalent to or higher than that of Q153-G4k / J142-G4h / L180-k in terms of the F.Xa production promoting activity measured under the conditions described in [Example 2] is selected, whereby an activity to substitute for more excellent F.VIII function can be obtained. In order to obtain the activity to substitute for the excellent F.VIII function of the present invention, in particular, the following amino acid modifications are preferred. (1) At least one amino acid residue selected from the amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b, and 102 according to Kabat numbering in the H chain of an antibody that recognizes F.IX and / or F.IXa is substituted with another amino acid. (2) At least one amino acid residue selected from the amino acid residues at positions 35, 53, 73, 76, 96, 98, 100, and 100a according to Kabat numbering in the H chain of an antibody that recognizes F.X is substituted with another amino acid. (3) At least one amino acid residue selected from the amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 according to Kabat numbering in the L chain of the antibody is substituted with another amino acid.

[0078] In the present invention, the amino acids of the preferred antibody for obtaining the activity to substitute for more excellent F.VIII function can include the following (4) to (6). These amino acids may be those that the antibody H chain already has such amino acids in advance, or the antibody H chain having such a sequence by modifying the amino acids. (4) The antibody H chain that recognizes F.IX and / or F.IXa has an isoleucine as the amino acid residue at position 34, an asparagine, glutamine or serine as the amino acid residue at position 35, a serine as the amino acid residue at position 49, an arginine as the amino acid residue at position 61, a glutamic acid as the amino acid residue at position 62, a serine or threonine as the amino acid residue at position 96, a lysine or arginine as the amino acid residue at position 98, a phenylalanine or tyrosine as the amino acid residue at position 100, a glycine as the amino acid residue at position 100b, or a tyrosine as the amino acid residue at position 102, (5) The antibody H chain that recognizes F.X has an aspartic acid as the amino acid residue at position 35, an arginine as the amino acid residue at position 53, a lysine as the amino acid residue at position 73, a glycine as the amino acid residue at position 76, a lysine or arginine as the amino acid residue at position 96, a tyrosine as the amino acid residue at position 98, a tyrosine as the amino acid residue at position 100, or a histidine as the amino acid residue at position 100a, (6) The antibody L chain has a lysine or arginine as the amino acid residue at position 27, a glutamic acid as the amino acid residue at position 30, an arginine as the amino acid residue at position 31, a glutamine as the amino acid residue at position 32, an arginine or glutamine as the amino acid residue at position 50, a serine as the amino acid residue at position 52, an arginine as the amino acid residue at position 53, a lysine as the amino acid residue at position 54, a glutamic acid as the amino acid residue at position 55, a serine as the amino acid residue at position 92, a serine as the amino acid residue at position 93, a proline as the amino acid residue at position 94, or a proline as the amino acid residue at position 95 according to Kabat numbering.

[0079] Among the amino acid residues of the antibodies (1) to (6) described above, the amino acid residues at preferred positions for obtaining particularly excellent F.VIII-like activity can include the following (1) to (3). (1) Amino acid residues at positions 34, 35, 61, 98, 100, and 100b according to Kabat numbering in the H chain of an antibody that recognizes F.IX and / or F.IXa, particularly the amino acid residues at positions 61 and 100 (2) Amino acid residues at positions 35, 53, 73, 96, 98, 100, and 100a according to Kabat numbering in the H chain of an antibody that recognizes F.X (3) Amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 93, 94, and 95 according to Kabat numbering in the L chain of the antibody, particularly the amino acid residues at positions 27, 30, 31, 50, 53, 94, and 95

[0080] That is, the present invention provides a multispecific antigen-binding molecule in which the first polypeptide is composed of any antibody H chain selected from the following (a1) to (a14) and any antibody L chain selected from the following (c1) to (c10), and the second polypeptide is composed of any antibody H chain selected from the following (b1) to (b12) and any antibody L chain selected from the following (c1) to (c10): (a1) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 1 (Q1-G4k) (a2) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 2 (Q31-z7) (a3) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 3 (Q64-z55) (a4) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10 (Q64-z7) (a5) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11 (Q85-G4k) (a6) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12 (Q153-G4k) (a7) An antibody H chain consisting of the amino acid sequence set forth in SEQ ID NO: 13 (Q354-z106) (a8) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 14 (Q360-G4k), (a9) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 15 (Q360-z118), (a10) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 16 (Q405-G4k), (a11) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 17 (Q458-z106), (a12) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 18 (Q460-z121), (a13) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 19 (Q499-z118), (a14) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 20 (Q499-z121), (b1) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 4 (J268-G4h), (b2) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 5 (J321-G4h), (b3) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 6 (J326-z107), (b4) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 7 (J344-z107), (b5) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 21 (J232-G4h), (b6) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 22 (J259-z107), (b7) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 23 (J300-z107), (b8) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 24 (J327-z107), (b9) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 25 (J327-z119), (b10) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 26 (J339-z119), (b11) An antibody heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 27 (J346-z107), (b12) The antibody heavy chain consisting of the amino acid sequence described in Accession No. 170 (J142 - G4h), (c1) The antibody light chain consisting of the amino acid sequence described in Accession No. 8 (L2 - k), (c2) The antibody light chain consisting of the amino acid sequence described in Accession No. 9 (L45 - k), (c3) The antibody light chain consisting of the amino acid sequence described in Accession No. 28 (L248 - k), (c4) The antibody light chain consisting of the amino acid sequence described in Accession No. 29 (L324 - k), (c5) The antibody light chain consisting of the amino acid sequence described in Accession No. 30 (L334 - k), (c6) The antibody light chain consisting of the amino acid sequence described in Accession No. 31 (L377 - k), (c7) The antibody light chain consisting of the amino acid sequence described in Accession No. 32 (L404 - k), (c8) The antibody light chain consisting of the amino acid sequence described in Accession No. 33 (L406 - k), (c9) The antibody light chain consisting of the amino acid sequence described in Accession No. 34 (L408 - k), (c10) The antibody light chain consisting of the amino acid sequence described in Accession No. 171 (L180 - k).

[0081] Furthermore, the present invention provides a multispecific antigen - binding molecule, wherein the first polypeptide comprises an antigen - binding site that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of the antibody described in any one of (a1) - (a14) above and the light chain of the antibody described in any one of (c1) - (c10) above binds, and the second polypeptide comprises an antigen - binding site that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of the antibody described in any one of (b1) - (b12) above and the light chain of the antibody described in any one of (c1) - (c10) above binds.

[0082] Furthermore, the present invention provides a multispecific antigen-binding molecule, wherein the first polypeptide comprises the heavy chain of an antibody selected from any of the following (e1) to (e3), the second polypeptide comprises the heavy chain of an antibody selected from any of the following (f1) to (f3), and the third and fourth polypeptides comprise the light chain of an antibody selected from any of the following (g1) to (g4): (e1) The heavy chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of an antibody selected from any of (a1) to (a14) above and the light chain of an antibody described in any of (c1) to (c10) above binds, (e2) The heavy chain of an antibody in which at least one amino acid residue selected from the amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b, and 102 according to Kabat numbering in the heavy chain of an antibody selected from any of (e1) above is substituted with another amino acid, (e3) The amino acid residue at position 34 according to Kabat numbering in the heavy chain of an antibody selected from any of (e1) above is isoleucine, the amino acid residue at position 35 is asparagine, glutamine, or serine, the amino acid residue at position 49 is serine, the amino acid residue at position 61 is arginine, the amino acid residue at position 62 is glutamic acid, the amino acid residue at position 96 is serine or threonine, the amino acid residue at position 98 is lysine or arginine, the amino acid residue at position 100 is phenylalanine or tyrosine, the amino acid residue at position 100b is glycine, or the amino acid residue at position 102 is tyrosine in the heavy chain of the antibody, (f1) The heavy chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of the heavy chain of an antibody selected from any of (b1) to (b12) above and the light chain of an antibody selected from any of (c1) to (c10) above binds, (f2) The heavy chain of an antibody in which at least one amino acid residue selected from the amino acid residues at positions 35, 53, 73, 76, 96, 98, 100, and 100a according to Kabat numbering in the heavy chain of an antibody selected from any of (f1) above is substituted with another amino acid, (f3) An H chain of any of the antibodies in (f1) above, wherein the amino acid residue at position 35 according to Kabat numbering is aspartic acid, the amino acid residue at position 53 is arginine, the amino acid residue at position 73 is lysine, the amino acid residue at position 76 is glycine, the amino acid residue at position 96 is lysine or arginine, the amino acid residue at position 98 is tyrosine, the amino acid residue at position 100 is tyrosine, or the amino acid residue at position 100a is histidine, (g1) An L chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of an H chain of any of the antibodies in (a1) to (a14) above and an L chain of any of the antibodies in (c1) to (c10) above binds, (g2) An L chain of an antibody that binds to an epitope overlapping with the epitope to which an antibody consisting of an H chain of any of the antibodies in (b1) to (b12) above and an L chain of any of the antibodies in (c1) to (c10) above binds, (g3) An L chain of an antibody in either (g1) or (g2) above, wherein at least one amino acid residue selected from the amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 according to Kabat numbering is substituted with another amino acid, (g4) An L chain of an antibody in either (g1) or (g2) above, wherein the amino acid residue at position 27 according to Kabat numbering is lysine or arginine, the amino acid residue at position 30 is glutamic acid, the amino acid residue at position 31 is arginine, the amino acid residue at position 32 is glutamine, the amino acid residue at position 50 is arginine or glutamine, the amino acid residue at position 52 is serine, the amino acid residue at position 53 is arginine, the amino acid residue at position 54 is lysine, the amino acid residue at position 55 is glutamic acid, the amino acid residue at position 92 is serine, the amino acid residue at position 93 is serine, the amino acid residue at position 94 is proline, or the amino acid residue at position 95 is proline.

[0083] Also, for the antibody (clone) of the present invention, amino acid residues may be substituted for the purpose of avoiding deamidation, methionine oxidation, etc. or stabilizing the structure of the antibody.

[0084] The method for obtaining the multispecific antigen-binding molecule of the present invention is not particularly limited, and it may be obtained by any method. For example, as an example of a method for producing a bispecific antibody, after producing a bispecific antibody according to the methods described in WO2006 / 109592, WO2005 / 035756, WO2006 / 106905, WO2007 / 114325, an antibody having the target cofactor function substitution activity can be selected and obtained.

[0085] For example, in the present invention, a bispecific antibody described in any of the following (a) to (u) is provided: (a) A bispecific antibody (Q1-G4k / J268-G4h / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9; (b) A bispecific antibody (Q1-G4k / J321-G4h / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9; (c) A bispecific antibody (Q31-z7 / J326-z107 / L2-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 8; (d) A bispecific antibody (Q64-z55 / J344-z107 / L45-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 3, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 9; (e) A bispecific antibody (Q64-z7 / J326-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 6, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (f) A bispecific antibody (Q64-z7 / J344-z107 / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 7, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (g) A bispecific antibody (Q85-G4k / J268-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (h) A bispecific antibody (Q85-G4k / J321-G4h / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 5, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (i) A bispecific antibody (Q153-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. (j) A bispecific antibody (Q354-z106 / J259-z107 / L324-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 13, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 22, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 29. (k) A bispecific antibody (Q360-G4k / J232-G4h / L406-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 33, (l) A bispecific antibody (Q360-z118 / J300-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 23, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30, (m) A bispecific antibody (Q405-G4k / J232-G4h / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 21, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28, (n) A bispecific antibody (Q458-z106 / J346-z107 / L408-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 34, (o) A bispecific antibody (Q460-z121 / J327-z119 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30, (p) A bispecific antibody (Q499-z118 / J327-z107 / L334-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 30, (q) A bispecific antibody (Q499-z118 / J327-z107 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 24, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (r) A bispecific antibody (Q499-z118 / J346-z107 / L248-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 27, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 28. (s) A bispecific antibody (Q499-z121 / J327-z119 / L404-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 25, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 32. (t) A bispecific antibody (Q499-z121 / J339-z119 / L377-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 26, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 31. (u) A bispecific antibody (Q153-G4k / J142-G4h / L180-k) in which the first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 170, and the third and fourth polypeptides are a common L chain consisting of the amino acid sequence set forth in SEQ ID NO: 171.

[0086] The antibodies of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, or the presence, form, etc. of sugar chains depending on the cells, hosts, or purification methods that produce the antibodies described below. However, as long as the obtained antibodies have functions equivalent to those of the antibodies of the present invention, they are included in the present invention. For example, when the antibody of the present invention is expressed in prokaryotic cells, such as Escherichia coli, a methionine residue is added to the N-terminus of the amino acid sequence of the original antibody. The antibodies of the present invention also include such antibodies.

[0087] The bispecific antibodies of the present invention can be produced by methods known to those skilled in the art. For example, based on the sequences of the obtained anti-F.IX / F.IXa antibody or anti-F.X antibody, it is possible to produce antibodies using gene recombination techniques known to those skilled in the art. Specifically, a polynucleotide encoding the antibody is constructed based on the sequence of the anti-F.IX / F.IXa antibody or anti-F.X antibody, introduced into an expression vector, and then expressed in an appropriate host cell (for example, see Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137).

[0088] Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. Also, when the purpose is cDNA subcloning or excision, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7, etc. can be mentioned. When using a vector for the purpose of producing the antibody of the present invention, in particular, an expression vector is useful. As an expression vector, for example, when the purpose is expression in E. coli, in addition to having a feature that the vector can be amplified in E. coli, when the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, etc., a promoter that can be efficiently expressed in E. coli, for example, 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, etc. is essential. Such vectors include, in addition to the above vectors, pGEX-5X-1 (manufactured by Pharmacia), "QIAexpress system" (manufactured by Qiagen), pEGFP, or pET (in this case, the host is preferably BL21 expressing T7 RNA polymerase), etc.

[0089] Furthermore, the vector of the expression plasmid may contain a signal sequence for antibody secretion. As a signal sequence for antibody secretion, when producing it in the periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) can be used. Introduction of the vector into the host cell can be carried out, for example, using the calcium chloride method or the electroporation method.

[0090] In addition to Escherichia coli, for example, as vectors for producing the antibodies of the present invention, expression vectors derived from mammals (for example, pcDNA3 (manufactured by Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990, 18(17), p5322), pEF, pCDM8), expression vectors derived from insect cells (for example, "Bac-to-BAC baculovairus expression system" (manufactured by Gibco BRL), pBacPAK8), expression vectors derived from plants (for example, pMH1, pMH2), expression vectors derived from animal viruses (for example, pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (for example, pZIPneo), expression vectors derived from yeast (for example, "Pichia Expression Kit" (manufactured by Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (for example, pPL608, pKTH50) can be mentioned.

[0091] When aiming for expression in animal cells such as CHO cells, COS cells, and NIH3T3 cells, it is essential that the vector of the expression plasmid has a promoter necessary for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc. It is more preferable to have a gene for selecting transformation into cells (for example, a drug resistance gene that can be discriminated by a drug (such as neomycin, G418)). Examples of vectors having such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.

[0092] Furthermore, when aiming to stably express a gene and amplify the copy number of the gene in cells, a method can be used where a vector having a DHFR gene that complements a CHO cell lacking a nucleic acid synthesis pathway (for example, pSV2-dhfr (「Molecular Cloning 2nd edition」 Cold Spring Harbor Laboratory Press, (1989)), etc.) is introduced and amplified with methotrexate (MTX). Also, when aiming for transient expression of a gene, a method can be used where COS cells having a gene expressing SV40 T antigen on the chromosome are transformed with a vector having an SV40 origin of replication (such as pcD). As the origin of replication, those derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), etc. can also be used. Furthermore, for gene copy number amplification in the host cell line, the expression vector can include, as a selectable marker, an aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc.

[0093] The antibody of the present invention thus obtained can be isolated from inside or outside the host cell (such as the culture medium) and purified as a substantially pure and homogeneous antibody. For the separation and purification of the antibody, the separation and purification methods usually used for the purification of antibodies can be used, and there is no particular limitation. For example, by appropriately selecting and combining a chromatography column, a filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, dialysis, recrystallization, etc., the antibody can be separated and purified.

[0094] Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (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 carried out using liquid chromatography, such as HPLC, FPLC and other liquid chromatographies. Examples of columns used for affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A include Hyper D, POROS, Sepharose FF (GE Amersham Biosciences), etc. The present invention also includes highly purified antibodies using these purification methods.

[0095] The obtained antibody can be purified to homogeneity. For the separation and purification of the antibody, the separation and purification methods commonly used for ordinary proteins can be used. For example, chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, etc. can be appropriately selected and combined to separate and purify the antibody (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but it is not limited thereto. Examples of columns used for affinity chromatography include Protein A columns, Protein G columns, etc.

[0096] As one aspect of the antibody in the present invention, since it has a function of substituting for cofactor F.VIII, the antibody of the present invention is expected to be an effective drug for diseases caused by a decrease in the activity (function) of the cofactor. Examples of the above diseases include bleeding, diseases accompanied by bleeding, or diseases caused by bleeding. In particular, an excellent therapeutic effect on hemophilia, which causes bleeding disorders due to decreased function or deficiency of F.VIII / F.VIIIa, is considered. Among them, it is expected to be an excellent therapeutic agent for hemophilia A, which causes bleeding disorders due to congenital decreased function or deficiency of F.VIII / F.VIIIa.

[0097] In the present invention, a (pharmaceutical) composition containing the antibody described in the present invention and a pharmaceutically acceptable carrier is provided. For example, an antibody that recognizes both F.IX or F.IXa and F.X and substitutes for the function of F.VIII is expected to be a pharmaceutical (pharmaceutical composition) or drug for the prevention and / or treatment of, for example, bleeding, diseases accompanied by bleeding, or diseases caused by bleeding.

[0098] In the present invention, bleeding, diseases accompanied by bleeding, or diseases caused by bleeding are preferably diseases that develop and / or progress due to a decrease or deficiency in the activity of F.VIII and / or activated blood coagulation factor VIII (F.VIIIa). Examples of such diseases include the above-mentioned hemophilia A, diseases in which inhibitors against F.VIII / F.VIIIa appear, acquired hemophilia, von Willebrand disease, etc., but are not particularly limited to these diseases.

[0099] The pharmaceutical composition containing the antibody of the present invention as an active ingredient for therapeutic or prophylactic purposes can be formulated by mixing it, if necessary, with a suitable pharmaceutically acceptable carrier, medium, etc. that are inert to them. For example, sterilized water, physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphoric acid, citric acid, histidine, other organic acids, etc.), preservatives, surfactants (such as PEG, Tween, etc.), chelating agents (such as EDTA, etc.), binders, etc. can be mentioned. In addition, it may contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine, saccharides and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol. When making an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose, and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, can be mentioned, and it may be used in combination with a suitable solubilizing agent, such as alcohol (such as ethanol), polyalcohol (such as propylene glycol, PEG, etc.), nonionic surfactant (such as polysorbate 80, polysorbate 20, poloxamer 188, HCO-50, etc.). Also, by mixing hyaluronidase in the formulation, it is possible to administer a larger volume of liquid subcutaneously (Expert Opin Drug Deliv. 2007 Jul;4(4):427-40.).

[0100] Alternatively, the antibody of the present invention can be encapsulated in microcapsules (such as microcapsules of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.), or can be made into a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (see "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.). Further, methods for making the drug into a sustained-release drug are also known and can be applied to the antibody of the present invention (Langer et al., J.Biomed.Mater.Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105 (1982); U.S. Patent No. 3,773,919; European Patent Application Publication (EP) No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP No. 133,988).

[0101] The dosage of the pharmaceutical composition of the present invention is ultimately determined appropriately by a doctor's judgment in consideration of the type of dosage form, administration method, patient's age and weight, patient's symptoms, type and progression degree of the disease, etc. Generally, for adults, 0.1 to 2000 mg can be administered in divided doses of 1 to several times per day. More preferably, it is 0.2 to 1000 mg / day, even more preferably 0.5 to 500 mg / day, even more preferably 1 to 300 mg / day, even more preferably 3 to 100 mg / day, and most preferably 5 to 50 mg / day. These dosages vary depending on the patient's weight, age, administration method, etc., but those skilled in the art can appropriately select an appropriate dosage. The administration period is also preferably determined appropriately according to the patient's healing process, etc.

[0102] The present invention also provides a gene or nucleic acid encoding the antibody of the present invention. It is also conceivable to incorporate the gene or nucleic acid encoding the antibody of the present invention into a gene therapy vector for gene therapy. As the administration method, in addition to direct administration with naked plasmid, it can be packaged in liposomes or the like, or formed as various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated vectors, HVJ vectors (see Adolph, "Viral Genome Method", CRC Press, Florid (1996)), or coated on bead carriers such as colloidal gold particles (see WO93 / 17706, etc.) for administration. However, as long as the antibody can be expressed in vivo and exert its action, it may be administered by any method. Preferably, a sufficient amount is administered by an appropriate parenteral route (injection, infusion, or gas-induced particle bombardment method (by an electron gun, etc.), or a method via a mucosal route such as nasal drops, etc. through intravenous, intraperitoneal, subcutaneous, intradermal, intra-adipose tissue, intra-mammary tissue, inhalation, or intramuscular routes). The gene encoding the antibody of the present invention may be administered ex vivo by liposome transfection, particle bombardment method (U.S. Patent No. 4,945,050), or viral infection to blood cells and bone marrow-derived cells, etc., and then reintroducing the cells into the patient. In gene therapy, any gene encoding the antibody of the present invention can be used. For example, genes containing the nucleotide sequences encoding the CDRs of Q1, Q31, Q64, Q85, Q153, Q354, Q360, Q405, Q458, Q460, Q499, J232, J259, J268, J300, J321, J326, J327, J339, J344, J346, J142, L2, L45, L248, L324, L334, L377, L404, L406, L408, L180 mentioned above can be mentioned.

[0103] The present invention also provides a method for preventing and / or treating bleeding, a disease accompanied by bleeding, or a disease caused by bleeding, which includes the step of administering the antibody or composition of the present invention. The administration of the antibody or composition can be carried out, for example, by the methods described above.

[0104] Furthermore, the present invention provides a kit for use in the above method, which comprises at least the antibody or composition of the present invention. In addition, a syringe, a needle, a pharmaceutically acceptable medium, an alcohol swab, a band-aid, or an instruction manual describing the method of use can also be packaged in the kit. The present invention also relates to the use of the multispecific antigen-binding molecule or bispecific antibody, or composition of the present invention, in the manufacture of a prophylactic and / or therapeutic agent for bleeding, a disease accompanied by bleeding, or a disease caused by bleeding. The present invention also relates to the multispecific antigen-binding molecule or bispecific antibody, or composition of the present invention, for the prevention and / or treatment of bleeding, a disease accompanied by bleeding, or a disease caused by bleeding.

[0105] All prior art documents cited in this specification are hereby incorporated by reference into this specification.

Examples

[0106] Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

[0107] 〔Example 1〕Preparation of a bispecific antibody having F.Xa production promoting activity In WO 2006 / 109592, hA69-KQ / hB26-PF / hAL-AQ was obtained as a bispecific antibody having an activity to substitute for the function of F.VIII. However, it was considered possible that this antibody had an effect of inhibiting the reaction in which F.IXa activates F.X using F.VIIIa as a cofactor. As shown in FIG. 1, antibodies that bind to FIX / FIXa or FX may inhibit the formation of the complex of FIXa and FVIIIa (Factor Xase (FXase)), or may inhibit FXase activity (activation of FX). Hereinafter, the inhibition of FXase formation and / or the inhibition of FXase activity will be referred to as FXase inhibitory action. The FXase inhibitory action is an inhibition of the coagulation reaction using FVIIIa as a cofactor, and may suppress the function of FVIII remaining in the patient or the function of the administered FVIII preparation. Therefore, it is desirable that the double - specific antibody has high FXa - producing promoting activity, and further, it is desirable that the FXase inhibitory action is low. In particular, in the case of patients maintaining the function of FVIII or patients receiving treatment with an FVIII preparation, it is more desirable that the FXa - producing promoting activity and the FXase inhibitory action are separated as much as possible.

[0108] However, the FXase inhibitory action is due to the basic property of the antibody to bind to the antigen (FIXa and / or FX). On the other hand, in a double - specific antibody having FXa - producing promoting activity (substituting the function of FVIII), it is also necessary to bind to the antigen (FIXa and FX). Therefore, it is expected to be very difficult to obtain a double - specific antibody having FXa - producing promoting activity (substituting the function of FVIII) without the FXase inhibitory action. Similarly, it is expected to be very difficult to increase the desired FXa - producing promoting activity while reducing the FXase inhibitory action by introducing amino acid substitutions into the double - specific antibody.

[0109] The inventors of the present invention obtained antibody genes from antibody - producing cells of animals immunized with antigens (human FIXa or human FX), which are methods known to those skilled in the art, and introduced amino acid substitutions as necessary, thereby preparing about 200 types of antibody genes against human FIXa and human FX each. Each antibody gene was incorporated into an animal cell expression vector.

[0110] By simultaneously transfecting a mammalian cell such as HEK293H cells with an anti-human F.IXa antibody heavy chain expression vector, an anti-human F.X antibody heavy chain expression vector, and a common antibody light chain expression vector, more than 40,000 bispecific antibodies were transiently expressed as a combination of an anti-F.IXa antibody and an anti-F.X antibody. As a comparative control, hA69-KQ / hB26-PF / hAL-AQ (SEQ ID NO: 165 / 166 / 167), a bispecific antibody described in WO 2006 / 109592, was prepared. Since the mutations described in WO 2006 / 106905 or WO 1996 / 027011 were introduced into the CH3 domain of each heavy chain, it was considered that the bispecific antibody was mainly expressed. The antibody in the cell culture supernatant was purified by a method known to those skilled in the art using Protein A. The present inventors measured the F.Xa production promoting activity of these antibodies by the following method. All reactions were carried out at room temperature.

[0111] 5 μL of an antibody solution diluted with Tris-buffered saline containing 0.1% bovine serum albumin (hereinafter referred to as TBSB) was mixed with 2.5 μL of 27 ng / mL Human Factor IXa beta (Enzyme Research Laboratories) and 2.5 μL of 6 IU / mL Novact® M (Chemical and Serum Therapy Research Institute), and then incubated at room temperature for 30 minutes in a 384-well plate.

[0112] The enzyme reaction in this mixture was initiated by adding 5 μL of 24.7 μg / mL Human Factor X (Enzyme Research Laboratories), and after 10 minutes, the reaction was stopped by adding 5 μL of 0.5 M EDTA. The chromogenic reaction was initiated by adding 5 μL of a chromogenic substrate solution. After a 50-minute chromogenic reaction, the change in absorbance at 405 nm was measured using SpectraMax 340PC 384 (Molecular Devices). The F.Xa production promoting activity was shown as the value obtained by subtracting the absorbance of the antibody-free reaction solution from the absorbance of the antibody-added reaction solution. The solvents for Human Factor IXa, Novact (registered trademark) M, and Human Factor X were TBCP (93.75 μM phospholipid solution (SYSMEX CO.), TBSB containing 7.5 mM CaCl2 and 1.5 mM MgCl2). The chromogenic substrate solution, S-2222 TM (CHROMOGENIX), was dissolved in purified water to 1.47 mg / mL and used in this assay.

[0113] In order to evaluate the F.Xase inhibitory effect of the antibody, the present inventors measured the effect on F.X activation by F.IXa in the presence of F.VIIIa by the following method. All reactions were carried out at room temperature. 5 μL of the antibody solution diluted with Tris-buffered saline containing 0.1% bovine serum albumin (hereinafter referred to as TBSB) was mixed with 2.5 μL of 80.9 ng / mL Human Factor IXa beta (Enzyme Research Laboratories) and then incubated at room temperature for 30 minutes in a 384-well plate. Furthermore, 2.5 μL of 1.8 IU / mL F.VIIIa (the preparation method will be described later) was added. After 30 seconds, the enzymatic reaction in this mixture was initiated by adding 5 μL of 24.7 μg / mL Human Factor X (Enzyme Research Laboratories). After 6 minutes, 5 μL of 0.5 M EDTA was added to stop the reaction. The chromogenic reaction was initiated by adding 5 μL of the chromogenic substrate solution. After a 14-minute chromogenic reaction, the change in absorbance at 405 nm was measured using a SpectraMax 340PC 384 (Molecular Devices). The F.Xase inhibitory effect of the antibody was indicated by the value obtained by subtracting the absorbance of the reaction solution without antibody from the absorbance of the reaction solution with antibody added.

[0114] Note that F.VIIIa was prepared by mixing 5.4 IU / mL of Coagulate® FS (Bayer Yakuhin, Ltd.) and 1.11 μg / mL of Human alpha Thrombin (Enzyme Research Laboratories) at a volume ratio of 1:1, incubating at room temperature for 1 minute, and then adding 7.5 U / mL of Hirudin (Merck KgaA) in an amount equal to half the volume of the mixture. The prepared solution was used as 1.8 IU / mL of FVIIIa and assayed 1 minute after the addition of Hirudin. The solvents for Human Factor IXa, Human Factor X, Coagulate® FS, Human alpha Thrombin, and Hirudin were TBCP (TBSB containing 93.75 μM phospholipid solution (SYSMEX CO.), 7.5 mM CaCl2, and 1.5 mM MgCl2). The chromogenic substrate solution S-2222 TM (CHROMOGENIX) was dissolved in purified water to 1.47 mg / mL and used in this assay.

[0115] The F.Xa production promoting activities of each bispecific antibody are shown in FIGS. 3 and 4, and the F.Xase inhibitory effects of each bispecific antibody are shown in FIG. 5. Although various amino acid substitutions that increase the F.Xa production promoting activity were found, as expected, most of the amino acid substitutions that increase the F.Xa production promoting activity also increased the F.Xase inhibitory effect, and it was very difficult to suppress the F.Xase inhibitory effect and increase the F.Xa production promoting activity at the same time.

[0116] Under such circumstances, the inventors of the present application obtained Q1-G4k / J268-G4h / L45-k, Q1-G4k / J321-G4h / L45-k, Q31-z7 / J326-z107 / L2-k, and Q64-z55 / J344-z107 / L45-k as bispecific antibodies with high F.Xa production-promoting activity and low F.Xase inhibitory activity. Incidentally, as the anti-human F.IXa antibody H chain, Q1-G4k (SEQ ID NO: 1), Q31-z7 (SEQ ID NO: 2), Q64-z55 (SEQ ID NO: 3), and as the prototype anti-human F.X antibody H chain, J268-G4h (SEQ ID NO: 4), J321-G4h (SEQ ID NO: 5), J326-z107 (SEQ ID NO: 6), J344-z107 (SEQ ID NO: 7), and as the prototype common antibody L chain, L2-k (SEQ ID NO: 8), L45-k (SEQ ID NO: 9) were obtained. Before the hyphen in the sequence name indicates the variable region, and after the hyphen indicates the constant region. Each bispecific antibody name is shown by arranging the sequence names of each chain to be transfected.

[0117] Most of the bispecific antibodies having F.Xa production-promoting activity similar to hA69-KQ / hB26-PF / hAL-AQ had a high F.Xase inhibitory activity as expected, but these bispecific antibodies (Q1-G4k / J268-G4h / L45-k, Q1-G4k / J321-G4h / L45-k, Q31-z7 / J326-z107 / L2-k, Q64-z55 / J344-z107 / L45-k) were found to have higher F.Xa production-promoting activity and lower F.Xase inhibitory activity than hA69-KQ / hB26-PF / hAL-AQ described in WO 2006 / 109592. The inventors conducted studies to further increase the F.Xa production-promoting activity and attenuate the F.Xase inhibitory activity using these four antibodies as prototype antibodies. The screening of bispecific antibodies for increasing the F.Xa production-promoting activity and attenuating the F.Xase inhibitory activity is shown in Figure 2.

[0118] Example 2: Preparation of Modified Antibodies The inventors introduced various amino acid mutations that affect the F.Xa production-promoting activity and F.Xase inhibitory action found in Example 1 for each chain of the prototype antibody by methods known to those skilled in the art, such as PCR for mutagenesis, and combined them. By evaluating large-scale combinations of the modified chains, screening for amino acid substitutions that further increase the F.Xa production-promoting activity of the four prototype antibodies and attenuate the F.Xase inhibitory action was performed. Each modified bispecific antibody into which an amino acid substitution was introduced was transiently expressed and purified in the same manner as the prototype antibody. The F.Xa production-promoting activity of these antibodies was measured by the following method. All reactions were carried out at room temperature.

[0119] 5 μL of the antibody solution diluted with Tris-buffered saline containing 0.1% bovine serum albumin (hereinafter referred to as TBSB) was mixed with 2.5 μL of 27 ng / mL Human Factor IXa beta (Enzyme Research Laboratories) and 2.5 μL of 6 IU / mL Novact® M (Institute of Chemistry and Serum Therapy), and then incubated at room temperature for 30 minutes in a 384-well plate.

[0120] The enzymatic reaction in this mixture was initiated by adding 5 μL of 24.7 μg / mL Human Factor X (Enzyme Research Laboratories), and after 2 minutes, the reaction was stopped by adding 5 μL of 0.5 M EDTA. The color development reaction was initiated by adding 5 μL of the chromogenic substrate solution. After a 20-minute color development reaction, the change in absorbance at 405 nm was measured using SpectraMax 340PC 384 (Molecular Devices). The F.Xa production-promoting activity was indicated by the value obtained by subtracting the absorbance of the antibody-free reaction solution from the absorbance of the antibody-added reaction solution.

[0121] The solvents for Human Factor IXa, Novact (registered trademark) M, and Human Factor X were TBCP (93.75 μM lipid solution (SYSMEX CO.)), TBSB containing 7.5 mM CaCl2 and 1.5 mM MgCl2. The chromogenic substrate solution S-2222 TM (CHROMOGENIX) was dissolved in purified water to 1.47 mg / mL and used. The F.Xase inhibitory effect of the antibody was also evaluated by the method described above.

[0122] The F.Xa production promoting activity of each modified bispecific antibody is shown in Fig. 4, and the F.Xase inhibitory effect of each bispecific antibody is shown in Fig. 5. The inventors of the present application obtained Q85-G4k / J268-G4h / L406-k, Q85-G4k / J321-G4h / L334-k, Q64-z7 / J344-z107 / L406-k, and Q64-z7 / J326-z107 / L334-k as bispecific antibodies with high F.Xa production promoting activity and low F.Xase inhibitory effect. As the anti-human F.IXa antibody H chain, Q64-z7 (SEQ ID NO: 10), Q85-G4k (SEQ ID NO: 11), and as the common antibody L chain with increased F.Xa production promoting activity, L334-k (SEQ ID NO: 30), L406-k (SEQ ID NO: 33) were found. Although the F.Xase inhibitory effect of Q85-G4k / J268-G4h / L406-k, Q85-G4k / J321-G4h / L334-k, Q64-z7 / J344-z107 / L406-k, and Q64-z7 / J326-z107 / L334-k increased slightly, the F.Xa production promoting activity increased significantly. Since the F.Xa production promoting activity of these modified antibodies is much greater than the increase in the F.Xase inhibitory effect, the F.Xa production promoting activity and the F.Xase inhibitory effect could be further separated from the prototype antibody. Thus, a combination that suppresses the F.Xase inhibitory effect and increases the F.Xa production promoting activity was found.

[0123] In order to replace the function of F.VIII with a bispecific antibody, it is preferable that the F.Xa production promoting activity of the found prototype antibody is higher. On the other hand, for patients maintaining the function of F.VIII or patients receiving treatment with F.VIII preparations, it was considered more preferable that the F.Xase inhibitory effect is lower for clinical use. Therefore, further modifications were made to produce a bispecific antibody with further increased F.Xa production promoting activity without increasing the F.Xase inhibitory effect.

[0124] As a result, as bispecific antibodies with high FXa production-promoting activity and low FXase inhibitory activity, Q153-G4k / J232-G4h / L406-k, Q354-z106 / J259-z107 / L324-k, Q360-G4k / J232-G4h / L406-k, Q360-z118 / J300-z107 / L334-k, Q405-G4k / J232-G4h / L248-k, Q458-z106 / J346-z107 / L408-k, Q460-z121 / J327-z119 / L334-k, Q499-z118 / J327-z107 / L334-k, Q499-z118 / J327-z107 / L377-k, Q499-z118 / J346-z107 / L248-k, Q499-z121 / J327-z119 / L404-k, Q499-z121 / J339-z119 / L377-k, Q153-G4k / J142-G4h / L180-k were obtained. In addition, as the anti-human FIXa antibody heavy chain, Q153-G4k (SEQ ID NO: 12), Q354-z106 (SEQ ID NO: 13), Q360-G4k (SEQ ID NO: 14), Q360-z118 (SEQ ID NO: 15), Q405-G4k (SEQ ID NO: 16), Q458-z106 (SEQ ID NO: 17), Q460-z121 (SEQ ID NO: 18), Q499-z118 (SEQ ID NO: 19), Q499-z121 (SEQ ID NO: 20), as the anti-human FX antibody heavy chain with increased FXa production-promoting activity, J232-G4h (SEQ ID NO: 21), J259-z107 (SEQ ID NO: 22), J300-z107 (SEQ ID NO: 23), J327-z107 (SEQ ID NO: 24), J327-z119 (SEQ ID NO: 25), J339-z119 (SEQ ID NO: 26), J346-z107 (SEQ ID NO: 27), J142-G4h (SEQ ID NO: 170), and as the common antibody light chain, L248-k (SEQ ID NO: 28), L324-k (SEQ ID NO: 29), L377-k (SEQ ID NO: 31), L404-k (SEQ ID NO: 32), L408-k (SEQ ID NO: 34), L180-k (SEQ ID NO: 171) were found.

[0125] Since these antibodies have extremely high F.Xa production-promoting activity while the F.Xase inhibitory effect is suppressed, they are considered to have extremely useful properties even for patients maintaining the function of F.VIII or patients receiving treatment with F.VIII preparations. Antibodies generally have a long half-life and can be administered subcutaneously. Therefore, these bispecific antibodies are considered to be able to deliver high value to patients with hemophilia A compared to the regular replacement therapy by intravenous administration of existing FVIII preparations in hemophilia A.

[0126] The sequence comparison of the variable regions of each chain used in Example 1 and Example 2 is shown in FIGS. 6A to 6D. In order to improve the F.Xa production-promoting activity of the bispecific antibody, for example, the following amino acids have been found to be important. Isoleucine at position 34, asparagine or glutamine or serine at position 35, serine at position 49, arginine at position 61, glutamic acid at position 62, serine or threonine at position 96, lysine or arginine at position 98, serine or glutamic acid at position 99, phenylalanine or tyrosine at position 100, glycine at position 100b, tyrosine at position 102, etc. in the H chain of the anti-human F.IXa antibody. Aspartic acid at position 35, arginine at position 53, lysine at position 73, glycine at position 76, lysine or arginine at position 96, tyrosine at position 98, tyrosine at position 100, histidine at position 100a, etc. in the H chain of the anti-human F.X antibody. Lysine or arginine at position 27, glutamic acid at position 30, arginine at position 31, glutamine at position 32, arginine or glutamine at position 50, serine at position 52, arginine at position 53, lysine at position 54, glutamic acid at position 55, serine at position 92, serine at position 93, proline at position 94, proline at position 95, etc. in the common antibody L chain (the amino acid numbers of the variable regions are according to Kabat numbering (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH)).

Industrial Applicability

[0127] The present invention provides a multispecific antigen-binding molecule that is an antibody recognizing both an enzyme and a substrate of the enzyme and has high activity to substitute for the function of F.VIII. Further, the present invention provides a multispecific antigen-binding molecule that is an antibody recognizing both an enzyme and a substrate of the enzyme, has high activity to substitute for the function of F.VIII, and has low F.Xase inhibitory action. Since humanized antibodies are generally considered to have high stability and low immunogenicity in blood, the multispecific antibodies of the present invention are considered to be extremely promising as pharmaceuticals.

Claims

[Claim 1] The invention described herein.

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