Multispecific antigen-binding molecule, possessing alternative function to function of blood coagulation factor viii

FIELD: chemistry.

SUBSTANCE: claimed is bispecific antibody, which is bound with both blood coagulation factor IX/activated blood coagulation factor IX and with blood coagulation factor X and functionally replaced function of blood coagulation factor VIII. Described are nucleic acid, coding antibody by invention, vector, cell and method of obtaining antibody, as well as pharmaceutical composition and set for application in method of prevention and/or treatment of bleeding or diseases, associated with or induced by bleeding.

EFFECT: invention can be applied in therapy of diseases, associated with blood coagulation disorders.

16 cl, 2 ex, 6 dwg

 

The technical field

The present invention relates to multispecific antigen binding molecules that function instead of the coagulation factor VII, cofactor that promotes enzymatic reactions, and pharmaceutical compositions comprising such molecules as the active ingredient.

Prior art

Haemophilia A is a violation of bleeding caused by a congenital reduction or absence of the function of the factor of blood coagulation VIII (f VIII). Patients with hemophilia A is usually when the bleeding is assigned composition F. VIII (as needed). In recent years, F. VIII compositions are also prophylactic for the prevention of bleeding (preventive introduction; non-patent documents 1 and 2). The half-life of the composition F. VIII in the blood of approximately 12 to 16 hours. Thus, to prevent continuous composition F. VIII are introduced to patients three times per week (non-patent documents 3 and 4). As required composition F. VIII also entered when it is necessary, at regular intervals to prevent re-bleeding. In addition, the introduction of composition F. VIII carried out intravenously. Thus, there is an urgent need in the pharmaceutical agents is less than the load, what song F. VIII.

Sometimes in patients with hemophilia occur anti-F. VIII antibodies (inhibitors). Such inhibitors cancel the effects of the compositions of F. VIII. For bleeding in patients who form inhibitors (inhibiting patients), you can enter alternative composition. The mechanisms of their actions are not dependent on the function F. VIII, that is based on the activation factor of the blood coagulation X (f X) with activated coagulation factor IX (F. IXa). Thus, in some cases, substitute compositions are not able to stop the bleeding. In line with this, there is an urgent need for pharmaceutical agents that are not affected by inhibitors that can functionally replace F. VIII.

Recently, as means for solving this problem have been disclosed antibodies that functionally substitute for F. VIII, and their application (non-patent documents 1, 2 and 3). Antibodies may be effective for acquired hemophilia, which are anti-F. VIII autoantibodies and the disease von Willebrand's disease, which is caused by pathology or by failure of function of the factor a background of Villebranda (vWF), but the activity of functional substitution F. VIII is not sufficient. Thus, as pharmaceutical agents which exhibit a high hemost the political effect, are desirable antibodies with higher activity functional substitution F. VIII, than the above-mentioned antibodies.

The documents of the prior art

[Patent document]

[Patent document 1] WO 2005/035754

[Patent document 2] WO 2005/035756

[Patent document 3] WO 2006/109592

[Non-patent document]

[Non-patent document 1] Blood 58, 1-13 (1981)

[Non-patent document 2] Nature 312, 330-337 (1984)

[Non-patent document 3] Nature 312, 337-342 (1984)

[Non-patent document] Biochim. Biophys. Acta 871, 268-278 (1986)

Summary of the invention

[Problems, which are solved by the invention]

The present invention is to provide multispecificity antigen binding molecules that functionally replaces F. VIII cofactor that promotes enzymatic reactions.

[Means of solving problems]

As a result of a special study by the inventors in accordance with this application succeeded in discovering especifismo antibodies having improved activity, which promotes the formation F. Xa, compared with known antibodies among different bespecifically antibodies that specifically associated with F. IX/F. IXa, and F. X and replace the function of the cofactor F. VIII, that is based on the function facilitating the activation of f X with f IXa (function means the Oia education F. Xa).

In addition, the authors present invention has succeeded in solving the provisions of the amino acid sequence especifismo antibodies having the activity of a functional replacement F. VIII, which are important for improved activity towards the promotion of education F. Xa of these antibodies, and thus, they are successfully received bespecifically antibodies in which the activity of the functional replacement of F. VIII is additionally increased by replacing these amino acids. They also succeeded in obtaining bespecifically antibodies that are not only highly active functional replacement F. VIII, and also have low inhibitory activity against F. Hase. Satisfaction of these two conditions is very difficult.

In particular, the present invention relates to multispecific antigen binding molecules that functionally replaces F. VIII cofactor that promotes enzymatic reactions, and pharmaceutical compositions comprising such a molecule as an active ingredient, and, in particular, refers to the following:

[1] multispecific antigen binding molecule that functionally replaces the coagulation factor VIII, comprising a first binding site of the antigen, which recognizes blood coagulation factor IX and/and the activated coagulation factor IX, and the second binding site of the antigen, which recognizes blood coagulation factor X, where the functional replacement of coagulation factor VIII is a consequence of the activity in relation to the promotion of the formation of activated coagulation factor X (f Xa), higher than the activity of especifismo antibodies (hA69-KQ/hB26-PF/hAL-AQ), which comprises the H chain containing SEQ ID NO: 165 and 166, and a common L chain, which includes SEQ ID NO: 167;

[2] multispecific antigen binding molecule of [1], which comprises a first polypeptide containing the first binding site of the antigen, which recognizes blood coagulation factor IX and/or activated blood coagulation factor IX, and a third polypeptide comprising a third binding site of the antigen, which recognizes blood coagulation factor IX and/or activated blood coagulation factor IX, and a second polypeptide comprising a second binding site of the antigen, which recognizes blood coagulation factor X, and the fourth polypeptide comprising a fourth binding site of the antigen, which recognizes blood coagulation factor X;

[3] multispecific antigen binding molecule of [2], in which the first polypeptide third polypeptide, each comprising a binding site antigen H-chain or L-chain antibodies against coagulation factor IX or activated coagulation factor IX meet the but; and the second polypeptide and the fourth polypeptide each comprise a binding site antigen H-chain or L-chain antibodies against coagulation factor X, respectively;

[4] multispecific antigen binding molecule [3], in which the binding site of the antigen of the first polypeptide comprises the binding site of the antigen, which comprises the CDR of the H chain consisting of any one of the amino acid sequences selected from the following (a1)to(a11), or the binding site of the antigen, functionally equivalent, and the binding site of the antigen of the second polypeptide comprises the binding site of the antigen, which comprises the CDR of the H chain consisting of any one of the amino acid sequences selected from the following (b1)to(b11), or the binding site of the antigen, functionally equivalent them:

(a1) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 75, 76 and 77 (CDR N circuit Q1), respectively;

(a2) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 78, 79 and 80 (CDR N chain Q31), respectively;

(a3) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 81, 82 and 83 (CDR N chain Q64), respectively;

(a4) the binding site of the antigen, which comprises CDR 1, 2 and 3 of the H chain amino acid posledovatelno the s SEQ ID NO: 84, 85 and 86 (CDR N chain Q85), respectively;

(a5) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 87, 88 and 89 (CDR N chain Q153), respectively;

(a6) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 90, 91 and 92 (CDR N chain Q354), respectively;

(a7) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 93, 94 and 95 (CDR N chain Q360), respectively;

(a8) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 96, 97 and 98 (CDR H chain Q405), respectively;

(a9) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 99, 100 and 101 (CDR H chain Q458), respectively;

(a10) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 102, 103 and 104 (CDR H chain Q460), respectively;

(a11) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 105, 106 and 107 (CDR N chain Q499), respectively;

(b1) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 108, 109 and 110 (CDR N chain J232), respectively;

(b2) the binding site of the antigen, which comprises CDR 1, 2 and 3 of the H chain amino acid follow what telestai SEQ ID NO: 111, 112, and 113 (CDR H chain J259), respectively;

(b3) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 114, 115 and 116 (CDR N chain J268), respectively;

(b4) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 117, 118 and 119 (CDR N chain J300), respectively;

(b5) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 120, 121 and 122 (CDR N chain J321), respectively;

(b6) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 123, 124 and 125 (CDR N chain J326), respectively;

(b7) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 126, 127,and 128 (CDR N chain J327), respectively;

(b8) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 129, 130 and 131 (CDR N chain J339), respectively;

(b9) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 132, 133 and 134 (CDR N chain J344), respectively;

(b10) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences SEQ ID NO: 135, 136 and 137 (CDR N chain J346), respectively; and

(b11) the binding site of the antigen, which comprises CDR 1, 2 and 3 H circuit with linakis is now the sequences SEQ ID NO: 174, 175 and 176 (CDR N chain J142), respectively;

[5] multispecific antigen binding molecule [3], in which the binding site of the antigen of the first polypeptide comprises the binding site of the antigen, including variable section H chain consisting of any one of the amino acid sequences selected from the following (a1)to(a11), or the binding site of the antigen, functionally equivalent, and the binding site of the antigen of the second polypeptide comprises the binding site of the antigen, which includes variable section H chain consisting of any one of the amino acid sequences selected from the following (b1)to(b11), or the binding site of the antigen, functionally equivalent:

(a1) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 35 (variable block H chain Q1);

(a2) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 36 (variable block H chain Q31);

(a3) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 37 (variable block H chain Q1);

(a4) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 38 (variable block H chain Q85);

(a5) the site is of wyzwania antigen, which includes the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 39 (variable block H chain Q153);

(a6) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 40 (variable block H chain Q354);

(a7) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 41 (variable block H chain Q360);

(a8) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 42 (variable block H chain Q405);

(a9) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 43 (variable block H chain Q458);

(a10) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 44 (variable block H chain Q460);

(a11) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 45 (variable block H chain Q499);

(b1) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 46 (variable block H chain J232);

(b2) the binding site of the antigen, which comprises the amino acid sequence of VA is abulnaga plot H chain SEQ ID NO: 47 (variable block H chain J259);

(b3) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 48 (variable block H chain J268);

(b4) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 49 (variable block H chain J300);

(b5) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 50 (variable block H chain J321);

(b6) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 51 (variable block H chain J326);

(b7) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 52 (variable section H chain J327);

(b8) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 53 (variable block H chain J339);

(b9) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 54 (variable block H chain J344);

(b10) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 55 (variable block H chain J346); and

(b11) the binding site of the antigen, which includes AMI is kislotno sequence of the variable segment H chain SEQ ID NO: 172 (variable block H chain J142);

[6] multispecific antigen binding molecule [3], in which the binding sites of the antigen included in the third polypeptide and the fourth polypeptide, include the binding site of the antigen, which comprises the CDR of the L chain comprising any one of amino acid sequences selected from the following (c1)to(c10), or the binding site of the antigen that are functionally equivalent:

(c1) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 138, 139 and 140 (CDR L chain L2), respectively;

(c2) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 141, 142 and 143 (CDR L chain L45), respectively;

(c3) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 144, 145 and 146 (CDR L chain L248), respectively;

(c4) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 147, 148 and 149 (CDR L chain L324), respectively;

(c5) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 150, 151 and 152 (CDR L chain L334), respectively;

(c6) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 153, 154 and 155 (CDR L chain L377), respectively;

(c7) the binding site of the antigen, which comprises the CDR of the L chain amino acid is posledovatelnostei SEQ ID NO: 156, 157 and 158 (CDR L chain L404), respectively;

(c8) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 159, 160 and 161 (CDR L chain L406), respectively;

(c9) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 137, 138 and 139 (CDR L chain L408), respectively; and

(c10) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NO: 177, 178 and 179 (CDR L chain LI 80), respectively;

[7] multispecific antigen binding molecule [3], in which the binding sites of the antigen included in the third polypeptide and the fourth polypeptide, include the binding site of the antigen, which includes variable area L chain comprising any one of amino acid sequences selected from the following (c1)to(c10), or the binding site of the antigen that are functionally equivalent:

(c1) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 56 (variable plot L chain L2);

(c2) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 57 (variable plot L chain L45);

(c3) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 58 (variabeln the th land L chain L248);

(c4) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 59 (variable plot L chain L324);

(c5) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 60 (variable plot L chain L334);

(c6) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 61 (variable plot L chain L377);

(c7) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 62 (variable plot L chain L404);

(c8) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 63 (variable plot L chain L406);

(c9) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 64 (variable plot L chain L408); and

(c10) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 173 (variable plot L chain L180);

[8] multispecific antigen binding molecule [3], in which the first and second polypeptides further include a constant block H-chain antibodies, and the third and fourth polypeptides include con is tanty plot L chain of the antibody;

[9] multispecific antigen binding molecule [3], in which the first and second polypeptides comprise constant plot H-chain antibodies, and the third and fourth polypeptides include constant plot L chain antibodies, and where the third polypeptide and the fourth polypeptide have a common L chain;

[10] multispecific antigen binding molecule [8] or [9], where the first polypeptide includes a constant section H-chain antibodies comprising any one of amino acid sequences selected from the group which consists of the following (d1)to(d6), or from the group consisting of the following (d7)-(d9), and the second polypeptide includes a constant section H-chain antibodies, which consists of any one of the amino acid sequence selected from the group other than such of the above-mentioned first polypeptide:

(d1) const block H chain SEQ ID NO: 65 (G4k);

(d2) const block H chain SEQ ID NO: 66 (z7);

(d3) const block H chain SEQ ID NO: 67 (z55);

(d4) const block H chain SEQ ID NO: 68 (z106);

(d5) const block H chain of SEQ ID NO: 69 (z118);

(d6) const block H chain SEQ ID NO: 70 (z121);

(d7) const block H chain SEQ ID NO: 71 (G4h);

(d8) const block H chain SEQ ID NO: 72 (z107); and

(d9) const block H chain of SEQ ID NO: 73 (z119);

[11] multispecific antigen binding molecule [8] or [9], where the third and fourth the initial polypeptides include constant plot L chain antibodies, consisting of the following amino acid sequence:

(e) a constant area L chain antibody SEQ ID NO: 74 (k);

[12] multispecific antigen binding molecule [8] or [9], where the first polypeptide comprises any one H chain of an antibody selected from the following (a1)to(a14), the second polypeptide includes any one H chain of an antibody selected from the following (b1)to(b12), and the third polypeptide and the fourth polypeptide include any one L chain of an antibody selected from the following (c1)to(c10):

(a1) N chain antibody comprising amino acid sequence SEQ ID NO: 1 (Q1-G4k);

(a2) N chain antibody comprising amino acid sequence SEQ ID NO: 2 (Q31-z7);

(a3) N-chain antibody comprising amino acid sequence SEQ ID NO: 3 (Q64-z55);

(a4) N chain antibody comprising amino acid sequence SEQ ID NO: 10 (Q64-z7);

(a5) N chain antibody comprising amino acid sequence SEQ ID NO: 11 (Q85-G4k);

(a6) N chain antibody comprising amino acid sequence SEQ ID NO: 12 (Q153-G4k);

(a7) N chain antibody comprising amino acid sequence SEQ ID NO: 13 (Q354-z106);

(a8) N chain antibody comprising amino acid sequence SEQ ID NO: 14 (Q360-G4k);

(a9) N chain antibody comprising amino acid sequence SEQ ID NO: 15 (Q360-z118);

(a10) N chain antibodies comprising the amino acid consequently the particular SEQ ID NO: 16 (Q405-G4k);

(a11) N chain antibody comprising amino acid sequence SEQ ID NO: 17 (Q458-z106);

(a12) N chain antibody comprising amino acid sequence SEQ ID NO: 18 (Q460-z121);

(a13) N chain antibody comprising amino acid sequence SEQ ID NO: 19 (Q499-z118);

(a14) N chain antibody comprising amino acid sequence SEQ ID NO: 20 (Q499-z121);

(b1) N chain antibody comprising amino acid sequence SEQ ID NO: 4 (J268-G4h);

(b2) N chain antibody comprising amino acid sequence SEQ ID NO: 5 (J321-G4h);

(b3) N chain antibody comprising amino acid sequence SEQ ID NO: 6 (J326-z107);

(b4) N chain antibody comprising amino acid sequence SEQ ID NO: 7 (J344-z107);

(b5) H chain antibody comprising amino acid sequence SEQ ID NO: 21 (J232-G4h);

(b6) (H-chain antibody comprising amino acid sequence SEQ ID NO: 22 (J259-z107);

(b7) H chain antibody comprising amino acid sequence SEQ ID NO: 23 (J300-z107);

(b8) H chain antibody comprising amino acid sequence SEQ ID NO: 24 (J327-z107);

(b9) H chain antibody comprising amino acid sequence SEQ ID NO: 25 (J327-z119);

(b10) H chain antibody comprising amino acid sequence SEQ ID NO: 26 (J339-z119);

(b11) H chain antibody comprising amino acid sequence SEQ ID NO: 27 (J346-z107);

(b12) H chain antibodies, consisting of the amino acid sequence of SEQ ID NO: 170 (J142-G4h);

(c1) L chain antibody comprising amino acid sequence SEQ ID NO: 8 (L2-k);

(c2) L chain antibody comprising amino acid sequence SEQ ID NO: 9 (L45-k);

(c3) L chain antibody comprising amino acid sequence SEQ ID NO: 28 (L248-k);

(c4) L chain antibody comprising amino acid sequence SEQ ID NO: 29 (L324-k);

(c5) L chain antibody comprising amino acid sequence SEQ ID NO: 30 (L334-k);

(c6) L chain antibody comprising amino acid sequence SEQ ID NO: 31 (L377-k);

(c7) L chain antibody comprising amino acid sequence SEQ ID NO: 32 (L404-k);

(c8) L chain antibody comprising amino acid sequence SEQ ID NO: 33 (L406-k);

(c9) L chain antibody comprising amino acid sequence SEQ ID NO: 34 (L408-k); and

(c10) L chain antibody comprising amino acid sequence SEQ ID NO: 171 (L180-k);

[13] multispecific antigen binding molecule of [1], in which the first polypeptide comprises the binding site of the antigen that binds to an epitope that overlaps with an epitope that binds to an antibody comprising the H chain of the antibody, which is any one of (a1)to(a14)and the L chain of the antibody, which is any one of (c1)to(c10) [12], and the second polypeptide of vkluchaetsia binding antigen, which binds to the epitope that overlaps with an epitope that binds to an antibody that comprises the H chain of the antibody, which is any one of (b1)to(b12), and L-chain antibodies, which represents any of (c1)to(c10) [12];

[14] multispecific antigen binding molecule [8] or [9], in which the first polypeptide comprises any one H chain of an antibody selected from the following (e1)-(e3), the second polypeptide includes any one H chain of an antibody selected from the following (f1)-(f3), and the third polypeptide and the fourth polypeptide include any one L chain of an antibody selected from the following (g1)-(g4):

(e1) N chain antibody, which binds to an epitope overlapping with an epitope that binds to an antibody comprising the H chain of the antibody, which is any one of (a1)to(a14), and L-chain antibodies, which represents any one of (c1)to(c10) [12];

(e2) H chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b and 102 in accordance with the Kabat numbering in any one H chain of an antibody selected from (e1), is substituted by another amino acid;

(e3) N chain antibodies, in which in accordance with the Kabat numbering, amino acid at position 34 is an isoleucine, the amino acid at position 35 submitted is an asparagine, glutamine or serine, the amino acid at position 49 is a serine, the amino acid at position 61 is an arginine, the amino acid at position 62 is a glutamic acid, the amino acid at position 96 is a serine or threonine, the amino acid at position 98 is a lysine or arginine, the amino acid at position 100 is a phenylalanine or tyrosine, the amino acid at position 100 is a glycine or an amino acid at position 102 is a tyrosine in any N-chain antibodies selected from (e1);

(f1) H chain antibody, which binds to an epitope overlapping with an epitope that binds to an antibody comprising the H chain of the antibody, which is any one of (b1)to(b12) [12], and the L chain of the antibody, which is any one of (c1)to(c10) [12];

(f2) N chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 35, 53, 73, 76, 96, 98, 100, and 100a in accordance with the Kabat numbering in any N-chain antibodies (f1), is substituted by another amino acid;

(f3) N chain antibodies, in which in accordance with the Kabat numbering amino acid at position 35 is an aspartic acid, the amino acid at position 53 is an arginine, the amino acid at position 73 represents whether the in, the amino acid in position 76 is a glycine amino acid at position 96 is a lysine or arginine, the amino acid at position 98 is a tyrosine, the amino acid at position 100 is tyrosine or amino acid position 100a is a histidine in any one H chain of an antibody selected from (f1);

(g1) L chain antibody, which binds to an epitope overlapping with an epitope that binds to an antibody comprising the H chain of the antibody, which is any one of (a1)to(a14), and L-chain antibodies, which represents any one of (c1)to(c10) [12];

(g2) L chain antibody, which binds to an epitope overlapping with an epitope that binds to an antibody comprising the H chain of the antibody, which is any one of (b1)to(b12), and L-chain antibodies, which represents any one of (c1)to(c10) [12];

(g3) L chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94 and 95 according to the Kabat numbering in the L chain of the antibody or (g1)or (g2), is substituted by another amino acid; and (g4) H chain antibodies, in which in accordance with the Kabat numbering amino acid at position 27 is a lysine or arginine, the amino acid in position 30 represents a glutamic acid, s is nakilat in position 31 is an arginine, the amino acid at position 32 is a glutamine, the amino acid at position 50 is an arginine or glutamine, the amino acid at position 52 is a serine, the amino acid at position 53 is an arginine, the amino acid at position 54 is lysine, the amino acid at position 55 is glutamic acid, the amino acid at position 92 is a serine, the amino acid at position 93 is serine, amino acid position 94 is a Proline or an amino acid at position 95 is a Proline in the L chain of the antibody of any of either (g1)or (g2);

[15] multispecific antigen binding molecule of any one of [1]to[14], where the specific antigen binding molecule is multispecific antibody;

[16] bespecifically antibody in accordance with one of the following (a)to(u):

(a) bespecifically antibody (Q1-G4k/J268-G4h/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(b) bespecifically antibody (Q1-G4k/J321-G4h/L45-k), in which the first polypeptide is an H chain consisting of aminotic is now the sequence of SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(c) bespecifically antibody (Q31-z7/J326-z107/L2-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 8;

(d) bespecifically antibody (Q64-z55/J344-z107/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 3, and the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(e) bespecifically antibody (Q64-z7/J326-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(f) bespecifically antibody (Q64-z7/J344-z107/L406-k), in which the first item is lipacid represents H chain, consisting of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(g) bespecifically antibody (Q85-G4k/J268-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(h) bespecifically antibody (Q85-G4k/J321-G4h/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(i) bespecifically antibody (Q153-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(k) bespecifically antibody (Q360-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(l) bespecifically antibody (Q360-z118/J300-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 23, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(m) bespecifically antibody (Q405-G4k/J232-G4h/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth is ipated have a common L chain sequence of SEQ ID NO: 28;

(n) bespecifically antibody (Q458-z106/J346-z107/L408-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 34;

(o) bespecifically antibody (Q460-z121/J327-z119/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(p) bespecifically antibody (Q499-z118/J327-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(q) bespecifically antibody (Q499-z118/J327-z107/L377-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence is lnasty SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31;

(r) bespecifically antibody (Q499-z118/J346-z107/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 28;

(s) bespecifically antibody (Q499-z121/J327-z119/L404-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 32;

(t) bespecifically antibody (Q499-z121/J339-z119/L377-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 26, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31; and

(u) bespecifically antibody (Q153-G4k/J142-G4h/L180-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ IDNO: 12, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 170, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 171;

[17] nucleic acid that encodes multispecific antigen binding molecule according to one of [1]to[15] or bespecifically antibody according to [16];

[18] a vector containing a nucleic acid according to [17];

[19] cell comprising a nucleic acid according to [17] or the vector according to [18];

[20] the method of obtaining multispecific antigen binding molecule according to any of [1]to[15] or especifismo antibody according to [16] by culturing cells [19];

[21] a pharmaceutical composition comprising multispecific antigen binding molecule according to one of [1]to[15] or bespecifically antibody according to [16] and a pharmaceutically acceptable carrier;

[22] the composition according to [21], which is a pharmaceutical composition used for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding;

[23] the composition according to [22], where the bleeding, disorder accompanied by bleeding, or disorder, ysanne bleeding, is a disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII;

[24] the composition according to [23], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII is a hemophilia A;

[25] the composition according to [23], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII, is a disease which shows the appearance of an inhibitor of coagulation factor VIII and/or activated blood coagulation factor VIII;

[26] the composition according to [23], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII, is an acquired hemophilia;

[27] the composition according to [23], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated factor with which artisania blood VIII, is a disease von Willebrand's disease;

[28] the method of preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding, which includes the step of introducing multispecific antigen binding molecule according to one of [1]to[15], or especifismo antibody according to [16], or the composition according to any one of [21]-[27]; and

[29] a kit for use in the method of prevention and/or treatment in accordance with [28], which includes at least multispecific antigen binding molecule according to one of [1]to[15], or bespecifically antibody according to [16], or the composition according to any one of[21]-[27].

In addition, the present invention relates to:

[30] use multispecific antigen binding molecule according to any of [1]to[15], especifismo antibody according to [16] or the composition according to any one of [21]-[27] in the production of an agent for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding; and

[31] multispecific antigen binding molecule according to any one of [1]to[15], bespecifically the antibody according to [16] or the composition according Lubim one of [21]-[27] for preventing and/or treating bleeding, diseases accompanied by bleeding, or a disease caused by bleeding.

The present invention also relates to bespecifically antibodies that functionally substitute for F. VIII cofactor that promotes enzymatic reactions, and pharmaceutical compositions comprising the antibody as an active ingredient, and, in particular, applies to:

[32] bespecifically the antibody that functionally substitutes for the coagulation factor VIII, comprising a first binding site of the antigen, which recognizes blood coagulation factor IX and/or activated blood coagulation factor IX, and a second binding site of the antigen, which recognizes blood coagulation factor X, which bespecifically antibody is any of the following (a)to(u):

(a) bespecifically antibody (Q1-G4k/J268-G4h/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(b) bespecifically antibody (Q1-G4k/J321-G4h/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 1, the second polypeptide is Cobain chain, consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(c) bespecifically antibody (Q31-z7/J326-z107/L2-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 8;

(d) bespecifically antibody (Q64-z55/J344-z107/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 3, and the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(e) bespecifically antibody (Q64-z7/J326-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(f) bespecifically antibody (Q64-z7/J344-z107/L406-k), in which the first polypeptide is an H chain consisting the Yu of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(g) bespecifically antibody (Q85-G4k/J268-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(h) bespecifically antibody (Q85-G4k/J321-G4h/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(i) bespecifically antibody (Q153-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(j) bespecifically antibody (Q354-z106/J259-z107/L324-k), where p is pout polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 13, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 22, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 29;

(k) bespecifically antibody (Q360-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(l) bespecifically antibody (Q360-z118/J300-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 23, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(m) bespecifically antibody (Q405-G4k/J232-G4h/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 28;

(n) bespecifically antibody (Q458-z106/J346-z107/L408-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 34;

(o) bespecifically antibody (Q460-z121/J327-z119/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(p) bespecifically antibody (Q499-z118/J327-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(q) bespecifically antibody (Q499-z118/J327-z107/L377-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid posledovatel the particular SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31;

(r) bespecifically antibody (Q499-z118/J346-z107/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 28;

(s) bespecifically antibody (Q499-z121/J327-z119/L404-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 32;

(t) bespecifically antibody (Q499-z121/J339-z119/L377-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 26, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31; and

(u) bespecifically antibody (Q153-G4k/J142-G4h/L180-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ IDNO: 12, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 170, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 171;

[33] the nucleic acid that encodes bespecifically antibody according to[32];

[34] the vector containing the nucleic acid according to [33];

[35] the cell comprising the nucleic acid according to [33] or the vector according to [34];

[36] the method of obtaining especifismo antibody according to [32] by culturing cells according to [35];

[37] pharmaceutical compositions comprising bespecifically antibody according to [32] and a pharmaceutically acceptable carrier;

[38] the composition according to [37], which is a pharmaceutical composition used for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding;

[39] the composition according to [38], where the bleeding, disorder accompanied by bleeding, or disorder caused by bleeding, is a disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII;

[40] com is osili in accordance with [39], where is the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII is a hemophilia A;

[41] the composition according to [39], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII, is a disease which shows the appearance of an inhibitor of coagulation factor VIII and/or activated blood coagulation factor VIII;

[42] the composition according to [39], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII, is an acquired hemophilia;

[43] the composition according to [39], where the disease that develops and/or progresses due to decrease or lack of activity of the factor of blood coagulation VIII and/or activated blood coagulation factor VIII, is a disease von Willebrand's disease;

[44] the method of preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding, which includes a step in which edenia especifismo antibody according to [32] or the composition according to any one of [37]-[43]; and

[45] the kit for use in the method of prevention and/or treatment in accordance with [44], which includes bespecifically antibody according to [32] or the composition according to any one of[37]-[43];

[46] use especifismo antibody according to [32] or the composition according to any one of [37]-[43] in the production of an agent for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding; and

[47] bespecifically the antibody according to [32] or the composition according to any one of [37] - [43] for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding.

[Effects of the invention]

The present invention provides antibodies that recognize both the enzyme and its substrate, which are multispecific antigen binding molecules that have a high functional activity substitution F. VIII. In addition, the present invention provides antibodies that recognize both the enzyme and its substrate, which are multispecific antigen binding molecules that have a high functional activity substitution F. VIII and low inhibitory activity against F. Hase. PQS is LCU humanized antibodies in General are assumed to be as such which have high stability in blood and low immunogenicity, multispecific antibodies in accordance with the present invention may be promising as pharmaceutical agents.

Brief description of drawings

Fig.1 describes the inhibitory effect against F. Hase.

(a) F. VIIIa forms a complex with f IXa (F-Hosoi) and activates F. X.

(b) Bespecifically antibody binds to F. IXa and F. X and activates F. X.

(c) As F. VIIIa and bespecifically antibody triggers F. X without competition.

(d) Binding especifismo antibodies to F. IXa and/or F. X inhibits formation of a complex which is formed between F. by Hosoi and F. X.

(e) Linking especifismo antibodies to F. IXa and/or F. X inhibits the activity of F-Haza.

Fig.2 describes the screening. Received approximately 200 types of each of the genes for antibodies against human F. IXa and human F. X, they were cloned into expression vectors for animal cells. Thus was temporarily downregulation of 40000 or more bespecifically antibodies in combination with anti-F. IXa antibody and anti-F. X antibodies. The activity in relation to the promotion of education F. Xa and inhibitory activity against F. Hase been evaluated for screening bespecifically antibodies that have high activity against the promotion of education F. Xa is low inhibitory activity against F. Hase. In addition, by replacing the amino acids, if necessary, were obtained prototypical antibody.

Fig.3 shows the activity in relation to the promotion of education F. Xa for hA69-KQ/hB26-PF/hAL-AQ, 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. Solution concentration of antibodies was 300, 30 and 3 µg/ml (concentration after mixing of human factor IXa, Novact (registered trademark) M, human factor X and solution of the antibody was 100, 10 and 1 µg/ml), development of enzymatic reactions and staining was carried out for 10 minutes and 50 minutes respectively. As a result, these antibodies showed higher activity towards the promotion of education F. Xa compared with hA69-KQ/hB26-PF/hAL-AQ, as described in the application WO 2006/109592.

Fig.4 shows activity towards the promotion of education F. Xa for hA69-KQ/hB26-PF/hAL-AQ, antibodies of the prototype, and modified antibodies with amino acid substitutions. Solution concentration of antibodies was 300, 30 and 3 µg/ml (concentration after mixing of human factor IXa, Novact (registered trademark) M, human factor X and solution of the antibody was 100, 10 and 1 µg/ml), the enzymatic reaction and the development of staining was carried out for 2 minutes and 20 minutes respectively. The resulting modified antibodies is and showed higher activity towards the promotion of education F. Xa compared with antibodies prototype.

Fig.5 shows the inhibitory effect of F. Haza hA69-KQ/hB26-PF/hAL-AQ, antibodies of the prototype and modified antibodies with amino acid substitutions.

The figure shows the influence hA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k, Q31-z7/J326-z107/L2-k, Q1-G4k/J321-G4h/L45-k, Q64-z55/J344-z107/L45-k, Q85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k, Q64-z7/J344-z107/L406-k, Q64-z7/J326-z107/L334-k, Q153-G4k/J142-G4h/L180-k, Q405-G4k/J232-G4h/L248-k, Q360-G4k/J232-G4h/L406-k, Q153-G4k/J232-G4h/L406-k, Q458-z106/J346-z107/L408-k, Q360-z118/J300-z107/L334-k, Q499-z118/J327-z107/L377-k, Q499-z121/J327-z119/L404-k, Q499-z121/J339-z119/L377-k, Q499-z118/J346-z107/L248-k, Q354-z106/J259-z107/L324-k, Q460-z121/J327-z119/L334-k and Q499-z118/J327-z107/L334-k activation f X with f IXa in the presence of F. VIIIa. Such inhibitory effects F. Haza antibodies appear as the value obtained by subtracting the absorption of the reaction solution, free from antibodies of the absorption of the reaction solution containing the antibody. Solution concentration of antibodies was 300 and 30 µg/ml (concentration after mixing of human factor IXa, F. VIIIa, human factor X and solution of the antibody was 100 and 10 μg/ml), the enzymatic reaction and the development of staining was carried out for 6 minutes and 14 minutes respectively. The more positive the value of the inhibitory effects of F. Haza, shown on the horizontal axis, the weaker inhibitory effect F. Haza. As a result of this hA69-KQ/hB26-PF/hAL-AQ, described in WO 2006/109592, showed a stronger inhibitory effect F. Haza. All this the antibodies in accordance with the present invention showed a weak inhibitory effect F. Haza compared to hA69-KQ/hB26-PF/hAL-AQ or showed no inhibitory effect.

Fig.6A shows the amino acid sequence of prototype antibodies and modified antibodies with amino acid substitutions. When the name of the sequence is not specified in the column "Reference", it is mentioned sequence of the variable segment column "Name". A- (dash)" is shown when the amino acid sequence is not in the position in accordance with the numbering. A ". (dot)is shown when the amino acid sequence is compared with the variable plot columns "Name" and column "Reference", and amino acid sequence of the variable segment column "Name" is shown when amino acids are different. Amino acids that are found as important for activity in relation to the promotion of education F. Xa, denoted by capturing them in a frame.

Fig.6B is a continuation of Fig.6A.

Fig.6C is a continuation of Fig.6B.

Fig.6D is a continuation of Fig.6C.

The method of carrying out the invention

Multispecificity antigen binding molecules described in this application include the first binding site of the antigen and the second binding site of the antigen, which may, in particular, to contact at least two different types of antigens. While the first binding site of the antigen and the Torah the binding site of the antigen are not particularly limited as long while they have activity binding to F. IX and/or F. IXa and F. X, respectively, examples include sites required for binding to antigens, such as antibodies, molecules frame (similar to antibody molecules or peptides, or fragments containing such sites. Frame molecules are molecules that demonstrate its function by binding to target molecules, and can be any of the polypeptides as long as they are conformationally stable polypeptides that can bind at least one target antigen. Examples of such polypeptides include variable parts of the antibody, fibronectin (WO 2002/032925)domain of protein A (WO 1995/001937), And the domain of the LDL receptor (WO 2004/044011, WO 2005/040229), ankyrin (WO 2002/020565) and others, as well as molecules that are described in the documents Nygren and others (Current Opinion in Structural Biology, 7: 463-469 (1997); and Journal of Immunol Methods 290: 3-28 (2004), Binz and others (Nature Biotech 23: 1257-1266 (2005) and Hosse and others (Protein Science 15: 14-27(2006). Furthermore, as mentioned in Curr Opin Mol Ther. 2010 Aug; 12(4): 487-95 and Drugs. 2008; 68(7): 901-12, can be used peptide molecules that can communicate with the target antigens.

In this application multispecific antigen binding molecule are not particularly limited as long as they are molecules that can bind at least two different tiamenidine, but examples include polypeptides containing the above-mentioned binding sites of the antigen, such as antibodies, frame molecules as well as fragments thereof, aptamers, including molecules of nucleic acids and peptides, they can be a single molecule or multimer. Preferred multispecific antigen binding molecules include multispecific antibodies that may contact, in particular, at least two different antigens. Particularly preferred examples of antibodies that have activity functional substitution F. VIII, in accordance with the present invention include bespecifically antibodies (BsAb), which may contact, in particular, with two different antigens (they can be called antibodies with dual specificity).

In this invention, the term "jointly shared L chain" refers to the L-chain, which can communicate with two or more or different H chains and to demonstrate the ability of binding to each antigen. In this application, the term "variety(s) H chain(s)" preferably refers to the H chains of antibodies against different antigens, but is not limited as such, and also belongs to H chains, amino acid sequences which differ from each other. Shared L chain may be perceived by the and, for example, in accordance with the method described in WO 2006/109592.

Multispecific antigen binding molecule in accordance with the present invention (preferably bespecifically antibodies are antibodies having specificity to two or more different antigens or molecules, including fragments of such antibodies. Antibodies in accordance with the present invention are not particularly limited as such, but preferably are monoclonal antibodies. 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 include artificially derived recombinant antibodies on the basis of modified gene, such as chimeric antibodies, humanized antibodies and bespecifically antibodies.

In addition, L-chain antibodies, which will become multispecific antigen binding molecule in accordance with the present invention, can be different, but preferably are shared L chain.

Multispecific antigen binding molecule in accordance with the present invention preferably are recombinant antibodies obtained PR is the use of techniques of genetic recombination (see, for example, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOKLONAL ANTIBODIES, published in the United Kingdom, MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies can be obtained by cloning the DNA that encode antibodies from hybridomas or cells that produce the antibody, such as sensitized lymphocytes, which produce antibodies, by embedding them in an acceptable vectors, and the subsequent introduction in their hosts (host cell) to generate antibodies.

In addition, antibodies in accordance with the present invention may include not only whole antibodies, but also fragments of antibody, antibodies or low molecular weight (Minitel) and modified antibodies.

For example, fragments of antibodies or Minitel include diately (Dbs), linear antibodies, and molecules of single-stranded antibody (in this application is hereinafter also referred to as scFvs). In this application "Fv" fragment is defined as the smallest fragment of the antibody, which includes a full site of antigen recognition and binding site.

"Fv" fragment is a dimer (VH-VL dimer), in which the variable section H chain (VH) and variable plot L chain (VL) are closely associated with non-covalent binding. Three sites, complementarity determining (CDR) of each of the variable regions, interact with each other with education customers tie is of the antigen on the surface of the VH-VL dimer. Six CDR provides the binding site of the antigen with the antibody. However, one variable plot (or half of an Fv comprising only three CDRs specific for an antigen), taken separately, can learn and connect with the antigen, although its affinity is lower than that for a single binding site.

Fab fragment (also called F(ab)additionally includes a constant section L chain and a constant block H chain (CH1). Fab' fragment differs from the Fab fragment of the fact that it additionally includes several residues, originating from carboxyterminal the end of the H chain CH1 plot involving one or more cysteine residues of the hinge area of the antibodies. Fab'-SH is for Fab'in which one or more cysteine residues of constant area include a free thiol group. F(ab') fragment obtained by cleavage of disulfide bonds between cysteine residues in the hinge area F(ab')2when using digestion by pepsin. Other chemically related fragments of antibodies are also known qualified specialist in this field of technology.

Diately represent a bivalent Minitel constructed by gene fusion (Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO 93/11161). Diately represent dimers, which consist of two p is dipeptidic circuits, where each polypeptide chain comprises variable plot L chain (VL) and variable block H chain (VH)linked using linker, short enough to prevent the Association of the two domains on the same chain, for example, linker, consisting preferably of 2-12 amino acids, more preferably from 3 to 10 amino acids, in particular about 5 amino acids. The polypeptide chain forms a dimer, because the linker between the VL and VH encoded on the same polypeptide, is very short in order to form a single-chain fragment variable segment. Thus, diately include two binding site of the antigen.

Single-chain antibody or scFv fragment of the antibody comprises the VH and VL plots antibodies, and these areas exist as a single polypeptide chain. In the General case, the Fv polypeptide further includes a polypeptide linker between the VH and VL plots, and this enables the scFv to form the structure required for binding of the antigen (for a review on scFvs, see Pluckthun "The Pharmacology of Monoclonal Antibodies" vol 113 (Rosenburg and Moore ed. (Springer Verlag, New York) pages 269-315, 1994). In the context of the present invention, the linkers are not particularly limited as long as they do not inhibit the expression of the variable regions of the antibodies attached to their ends.

Bespecifically antibodies of IgG type can before aromatica from hybrid hybridomas (cardroom), obtained by merging two kinds of hybridomas that produce IgG antibodies (Milstein C and other Nature 1983, 305: 537-540). They can also secretariats by capturing genes of the L chain and H chain comprising two types of IgG that are of interest, a total of eight kinds of genes, and embed them in cages for the joint gene expression.

In this case, by introducing acceptable amino acid substitutions in the CH3 plots of N chains of IgG with heterogeneous combination of N circuits can be mainly secreted (Ridgway JB and other Protein Engineering, 1996, 9: 617-621; Merchant AM and other Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; Davis JH and other Protein Eng Des Sel. 2010, 4: 195-202).

As for the L chain, the diversity of the variable regions of the L chain is lower than that for the variable regions of H chain can be obtained shared L chains, which can give the ability to bind both H chains. Antibodies in accordance with the present invention include shared L chain. Bespecifically IgG can be effectively expressed through the introduction of genes shared L chain and the two H chains in the cells.

Bespecifically antibodies can be obtained by chemical formation of cross-links Fab'. Bespecifically F(ab')2 can be obtained, for example, through the preparation of Fab' antibody using it to get maleimide the new Fab' using ortho-phenylenedimaleimide (o-PDM), and then through reaction with Fab'derived from another antibody for cross-linking of Fab', originating from different antibodies (Keler T and other Cancer Research 1997, 57: 4008-4014). The way chemical binding of the Fab'-dinitrobenzoic acid (TNB) derivative and fragments of antibodies such as Fab'-thiol (SH), is also known (Brennan M and other Science 1985, 229: 81-83).

Instead of chemical cross-linking can be used lacinova clasp, originating from the Fos and Jun. Used preferred education heterodimers with Fos and Jun, even though they also form homodimer. Fab', to which is attached Fos lacinova the clasp, and the other Fab', to which is attached Jun lacinova clasp, Express and receive. Monomeric Fab'-Fos and Fab'-Jun, restored under mild conditions, are mixed and subjected to the reaction with the formation of especifismo F(ab')2(Kostelny SA and others J. of Immunology, 1992, 148: 1547-53). This method can be used not only for Fab', but also for scFvs, Fvs, and others.

In addition, bespecifically antibodies, including sc(Fv)2such as IgG-scFv (Protein Eng Des Sel. 2010 Apr; 23(4): 221-8) and 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.; and MAbs. 2009 Jul; 1(4): 339-47. Epub 2009 Jul 10.), and other (IDrugs 2010, 13: 698-700), including antibodies "two in one" (Science. 2009 Mar 20; 323(5921): 1610-4; and Immunotherapy. 2009 Sep; 1(5): 749-51.), Tri-Fab, tandas the intelligent scFv and diately are also known (MAbs. 2009 November; 1(6): 539-547). In addition, even when used molecular forms, such as scFv-Fc and Fc frame, bespecifically antibodies can be effectively obtained through the preferential secretion of a heterologous combination of Fcs (Ridgway JB, et al., Protein Engineering, 1996, 9: 617-621; Merchant AM and other Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; and Davis JH, et al., Protein Eng Des Sel. 2010, 4: 195-202.).

Bespecifically antibody can also be obtained from the use of diately. Bespecifically ditelo is heterodimer two crossovers scFv fragments. In particular, it is obtained through the formation of heterodimer using VH(A)-VL(B) and VH(B)-VL(A), obtained by linking VH and VL, which are the origin of two kinds of antibodies, A and B, when using a relatively short linker, which consists of approximately 5 residues (Holliger P, etc. Proc Natl. Acad. Sci. USA 1993, 90: 6444-6448).

The desired structure can be achieved by linking two scFvs with a flexible and relatively long linker comprising about 15 residues (single-chain antibodies: Kipriyanov SM, etc. J. of Molecular Biology. 1999, 293: 41-56), and the creation of acceptable amino acid substitutions (the tabs in depression: Zhu Z and other Protein Science. 1997, 6: 781-788; VH/VL engineering boundaries between the two systems, Igawa T, and other Protein Eng Des Sel. 2010, 8: 667-77).

Sc(Fv)2that can be obtained by linking two types of scFvs with flexible and otnositelnogo linker, comprising about 15 residues, can also be bespecifically antibody (Mallender WD, etc. J. of Biological Chemistry, 1994, 269: 199-206).

Examples of the modified antibodies include antibodies associated with various molecules such as polyethylene glycol (PEG). Antibodies in accordance with the present invention include modified antibodies. In the context of the present invention the substance is bound to the modified antibody is not limited. Such modified antibodies can be produced by chemical modification of the obtained antibodies. Such methods are well known in the field of engineering.

Antibodies in accordance with the present invention include human antibodies, mouse antibodies, rat antibodies or other, and their origin is not limited as such. They may represent a genetically engineered antibodies, such as chimeric or humanized antibodies.

The method of obtaining human antibodies are well known in the art. For example, transgenic animals that carry a complete set of genes of human antibodies can be subjected to immunization with the desired antigen to obtain the desired human antibody (see international patent application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, O 96/34096 and WO 96/33735).

Genetically modified antibodies can also be obtained using known methods. In particular, for example, chimeric antibodies can include variable regions of H chain and L chain of the antibody immunized animal and constant plot of H chain and L chain of a human antibody. Chimeric antibodies can be obtained by linking the DNA encoding the variable parts of the antibodies that are derived from the immunized animal with DNA encoding the constant parts of the human antibodies, embedding it in the expression vector, and then introducing into the host cell to generate antibodies.

Humanized antibodies that are a modified antibody, often referred to as "reconstructed" human antibodies. Humanitariannet antibody design by migrating CDR antibody, originating from the immunized animal, on areas that define complementarity, human antibodies. Traditional methods of genetic recombination for such purposes are known (see the publication of European patent application no EP 239400 and international publication WO 96/02576; Sato K and others, Cancer Research 1993, 53: 851-856; international publication WO 99/51743).

Multispecific antigen binding molecule in accordance with the present invention which are those they learn the F. IX and/or F. IXa and F. X and functionally replaces the function of the cofactor F. VIII, and are characterized by the fact that molecules have a higher activity towards the promotion of education F. Xa compared with hA69-KQ/hB26-PF/hAL-AQ (described in WO 2006/109592), which is known as especifismo antibodies that functionally replaces F. VIII. In addition, antibodies in accordance with the present invention typically have a structure that includes a variable area anti-F. IXa antibody and variable plot anti-F. X antibodies.

In particular, the present invention provides multispecific antigen binding molecule that functionally replaces F. VIII and includes the first binding site of the antigen, which learns F. IX and/or F. IXa, and the second binding site of the antigen, which learns F. X, where the function that replaces the function F. VIII, occurs as a result of higher activity towards the promotion of education F. Xa compared with the activity especifismo antibodies (hA69-KQ/hB26-PF/hAL-AQ), which includes the H chain consisting of SEQ ID nos: 165 and 166, and shared L chain, which consists of SEQ ID NO: 167.

Multispecificity antigen binding molecule in accordance with the present invention includes a first polypeptide third polypeptide comprising the binding site of the antigen, which learns F. IX or F. IXa, and the second polypeptide and the fourth polypeptide comprising the binding site of the antigen, which learns F. X. First polypeptide third polypeptide and the second polypeptide and the fourth polypeptide each comprise a binding site antigen H chain of the antibody and the binding site of the antigen L chain of the antibody.

For example, in multispecific antigen binding molecule in accordance with the present invention, the first polypeptide third polypeptide include the binding site of the antigen H chain and L chain antibodies against F. IX or F. IXa, respectively; and the second polypeptide and the fourth polypeptide include the binding site of the antigen H chain and L chain antibodies against F. X, respectively.

At the same time, the binding sites of the antigen L chain of the antibody contained in the first polypeptide and the second polypeptide and the second polypeptide and the fourth polypeptide can be a shared L chain.

The polypeptide comprising the binding site of the antigen L chain antibodies, in the present invention preferably is a polypeptide that includes all or part of the sequence L chain antibody, which binds to f IX, f IXa and/or F. X.

The preferred embodiment of the binding site of the first antigen polypeptide antibodies in accordance with the present invention, in particular, include the binding sites of the anti-Christ. Jena, comprising the amino acid sequence:

Q1 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 75, 76 and 77, respectively);

Q31 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 78, 79 and 80, respectively);

Q64 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 81, 82 and 83, respectively);

Q85 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 84, 85 and 86, respectively);

Q153 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 87, 88 and 89, respectively);

Q354 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 90, 91 and 92, respectively);

Q360 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 93, 94 and 95, respectively);

Q405 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 96, 97 and 98, respectively);

Q458 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 99, 100 and 101, respectively);

Q460 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 102, 103 and 104, respectively); and

Q499 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 105, 106 and 107,respectively)

mentioned in the following examples, or the binding sites of the antigen, which are functionally equivalent.

The preferred embodiment of the binding site of the antigen of the second polypeptide, in particular, include, for example, the binding sites of the antigen comprising the amino acid sequence:

J232 sequence of each is of the CDR1, 2 and 3 of the H chain (SEQ ID NOs: 108, 109 and 110, respectively);

J259 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 111, 112 and 113, respectively);

J268 sequence of each of CDR1, 2 and 3 of the H chain (SEQ ID NOs: 114, 115, and 116, respectively);

J300 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 117, 118 and 119, respectively);

J321 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 120, 121 and 122, respectively);

J326 sequence of each CDR 1, 2 and 3 And chain (SEQ ID NOs: 123, 124 and 125, respectively);

J327 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 126, 127 and 128, respectively);

J339 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 129, 130 and 131, respectively);

J344 sequence of each CDR 1, 2 and 3 And chain (SEQ ID NOs: 132, 133 and 134, respectively);

J346 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 135, 136, and 137, respectively); and

J142 sequence of each CDR 1, 2 and 3 of the H chain (SEQ ID NOs: 174, 175 and 176,respectively)

mentioned in the following examples, or the binding sites of the antigen, which are functionally equivalent.

In particular, the present invention provides multispecific molecule that binds an antigen, in which the binding site of the antigen of the first polypeptide comprises the binding site of the antigen, which comprises the CDR of the H chain consisting of any one of the amino acid sequences selected from the following (a1)to(a11), or the binding site of the antigen, functionally equivalent, and the binding site of the antigen of the second polypeptide comprises the binding site of the antigen, which comprises the CDR of the H chain consisting of any one of the amino acid sequences selected from the following (b1)to(b11), or the binding site of the antigen that are functionally equivalent:

(a1) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 75, 76 and 77 (CDR N circuit Q1), respectively;

(a2) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 78, 79 and 80 (CDR N chain Q31), respectively;

(a3) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 81, 82 and 83 (CDR N chain Q64), respectively;

(a4) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 84, 85 and 86 (CDR N chain Q85), respectively;

(a5) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 87, 88 and 89 (CDR N chain Q153), respectively;

(a6) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 90, 91 and 92 (CDR N chain Q354), respectively;

(a7) the binding site of the antigen, which comprises CDR 1, 2 and 3 of the H chain amino acid placenta is valinoti SEQ ID NOs: 93, 94, and 95 (CDR N chain Q360), respectively;

(a8) the binding site of the antigen, which consists of CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 96, 97 and 98 (CDR N chain Q405), respectively;

(a9) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 99, 100 and 101 (CDR N chain Q458), respectively;

(a10) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 102, 103 and 104 (CDR N chain Q460), respectively;

(a11) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 105, 106 and 107 (CDR N chain Q499), respectively;

(b1) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 108, 109 and 110 (CDR N chain J232), respectively;

(b2) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 111, 112 and 113 (CDR N chain J259), respectively;

(b3) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 114, 115 and 116 (CDR N chain J268), respectively;

(b4) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 117, 118 and 119 (CDR N chain J300), respectively;

(b5) the binding site of the antigen, which comprises CDR 1, 2 and 3 of the H chain with aminoxy the pilot sequences of SEQ ID NOs: 120, 121 and 122 (CDR N chain J321), respectively;

(b6) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 123, 124, and 125 (CDR N chain J326), respectively;

(b7) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 126, 127 and 128 (CDR N chain J327), respectively;

(b8) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 129, 130 and 131 (CDR N chain J339), respectively;

(b9) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 132, 133 and 134 (CDR N chain J344), respectively;

(b10) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 135, 136, and 137 (CDR N chains J346), respectively; and

(b11) the binding site of the antigen, which comprises CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs: 174, 175 and 176 (CDR N chain J142), respectively.

The preferred embodiment of the binding site of the antigen of the third and fourth polypeptides, in particular, include, for example, the binding sites of the antigen comprising the amino acid sequence:

L2 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 138, 139 and 140, respectively);

L45 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 141, 142 and 143, respectively;

L248 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 144, 145 and 146, respectively);

L324 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 147, 148 and 149, respectively);

L334 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 150, 151 and 152, respectively);

L377 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 153, 154 and 155, respectively);

L404 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 156, 157 and 158, respectively);

L406 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 159, 160 and 161, respectively);

L408 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 162, 163 and 164, respectively); and

L180 sequence of each of CDR1, 2 and 3 of the L chain (SEQ ID NOs: 177, 178, and 179,respectively)

mentioned in the following examples, or the binding sites of the antigen, which are functionally equivalent.

In particular, the present invention provides multispecific molecule that binds an antigen, in which the binding sites of the antigen contained in the third polypeptide and the fourth polypeptide, include the binding site of the antigen, which comprises the CDR of the L chain comprising any one of amino acid sequences selected from the following (c1)to(c10), or the binding site of the antigen that are functionally equivalent:

(c1) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid follow what telestai SEQ ID NOs: 138, 139 and 140 (CDR L chain L2), respectively;

(c2) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 141, 142 and 143 (CDR L chain L45), respectively;

(c3) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 144, 145 and 146 (CDR L chain L248), respectively;

(c4) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 147, 148 and 149 (CDR L chain L324), respectively;

(c5) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 150, 151 and 152 (CDR L chain L334), respectively;

(c6) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 153, 154 and 155 (CDR L chain L377), respectively;

(c7) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 156, 157 and 158 (CDR L chain L404), respectively;

(c8) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 159, 160 and 161 (CDR L chain L406), respectively;

(c9) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 137, 138 and 139 (CDR L chain L408), respectively; and

(c10) the binding site of the antigen, which comprises the CDR of the L chain with the amino acid sequences of SEQ ID NOs: 177, 178 and 179 (CDR L chain LI 80, respectively.

Amino acid sequences of variable regions of H chain Q1, Q31, Q64, Q85, Q153, Q354, Q360, Q405, Q458, Q460 and Q499 in accordance with the present invention are represented by the following SEQ ID nos respectively.

Q1: SEQ ID NO: 35

Q31: SEQ ID NO: 36

Q64: SEQ ID NO: 37

Q85: SEQ ID NO: 38

Q153: SEQ ID NO: 39

Q354: SEQ ID NO: 40

Q360: SEQ ID NO: 41

Q405: SEQ ID NO: 42

Q458: SEQ ID NO: 43

Q460: SEQ ID NO: 44

Q499: SEQ ID NO: 45

Amino acid sequences of variable regions of H chain J232, J259, J268, J300, J321, J326, J327, J339, J344, J346 and J142 in accordance with the present invention are represented by the following SEQ ID NOs respectively.

J232: SEQ ID NO: 46

J259: SEQ ID NO: 47

J268: SEQ ID NO: 48

J300: SEQ ID NO: 49

J321: SEQ ID NO: 50

J326: SEQ ID NO: 51

J327: SEQ ID NO: 52

J339: SEQ ID NO: 53

J344: SEQ ID NO: 54

J346: SEQ ID NO: 55

J142: SEQ ID NO: 172

In particular, the present invention provides multispecific molecule that binds an antigen, in which the binding site of the antigen of the first polypeptide comprises the binding site of the antigen, which includes variable section H chain consisting of any one of the amino acid sequences selected from the following (a1)to(a11), or the binding site of the antigen, functionally equivalent, and the binding site of the antigen of the second polypeptide comprises the binding site of the antigen, which includes variable section H chain consisting of any one of amino the PCI-e slot sequences, selected from the following (b1)to(b11), or the binding site of the antigen that are functionally equivalent:

(a1) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 35 (variable section H chain Q1);

(a2) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 36 (variable block H chain Q31);

(a3) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 37 (variable block H chain Q1);

(a4) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 38 (variable block H chain Q85);

(a5) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 39 (variable block H chain Q153);

(a6) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 40 (variable section H chain Q354);

(a7) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 41 (variable block H chain Q360);

(a8) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 42 (in Realny plot H chain Q405);

(a9) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 43 (variable block H chain Q458);

(a10) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 44 (variable block H chain Q460);

(a11) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 45 (variable block H chain Q499);

(b1) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 46 (variable block H chain J232);

(b2) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 47 (variable block H chain J259);

(b3) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 48 (variable block H chain J268);

(b4) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 49 (variable block H chain J300);

(b5) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 50 (variable plot B chain J321);

(b6) the binding site of the antigen, which includes Amin is acid sequence of the variable segment of the H chain of SEQ ID NO: 51 (variable block H chain J326);

(b7) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 52 (variable block H chain J327);

(b8) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 53 (variable block H chain J339);

(b9) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 54 (variable block H chain J344);

(b10) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 55 (variable block H chain J346); and

(b11) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 172 (variable block H chain J142).

In addition, amino acid sequences of variable regions of the L chain L2, L45, L248, L324, L334, L377, L404, L406, L408 and L180 in accordance with the present invention are represented by the following SEQ ID nos respectively.

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: SEQ ID NO: 173

In particular, the present invention provides multispecific molecule that binds an antigen, in which the binding sites of the antigen contained in rethem the polypeptide and the fourth polypeptide include the binding site of the antigen, which includes variable area L chain comprising any one of amino acid sequences selected from the following (c1)to(c10), or the binding site of the antigen that are functionally equivalent:

(c1) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 56 (variable plot L chain L2);

(c2) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 57 (variable plot L chain L45);

(c3) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 58 (variable plot L chain L248);

(c4) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 59 (variable plot L chain L324);

(c5) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 60 (variable plot L chain L334);

(c6) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 61 (variable plot L chain L377);

(c7) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 62 (var is abelly plot L chain L404);

(c8) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 63 (variable plot L chain L406);

(c9) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 64 (variable plot L chain L408); and

(c10) the binding site of the antigen, which includes variable area L chain with the amino acid sequence SEQ ID NO: 173 (variable plot L chain L180).

Amino acid sequences of CDRs 1-3 and FR 1-4 in each of these sequences are as described in Fig.3A-D.

Upon receipt of a full-sized antibodies using variable regions disclosed in this invention, without particular restrictions can be used const areas, a well-known qualified specialist in this field of technology. For example, can be used constant plots, described in "Sequences of proteins of immunological interest", (1991), U. S. Department of Health and Human Services. Public Health Service National Institutes of Health, or "An efficient route to human bispecific IgG", (1998). Nature Biotechnology vol. 16, 677-681. Preferred examples of constant sites of the antibodies in accordance with the present invention include a constant plots IgG antibodies. When using a constant area of IgG antibodies from its type is not limited, while mo is should be used const plot of IgG subclass, such as IgGI, IgG2, IgG3 or IgG4. In addition, can be introduced amino acid mutations in the constant section of these IgG subclasses. Amino acid mutations that are introduced may constitute, for example, those that increase or decrease binding to Fc - receptors (Proc Nati Acad Sci USA. 2006 Mar 14; 103(11): 4005-10; and MAbs. 2009 Nov; 1(6): 572-9), increase or decrease 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), but they are not limited as such. Two types of H chains must be heterologic associated to obtain especifismo antibodies. The method of "tabs " in the trench" (J Immunol Methods. 2001 Feb 1; 248(1-2): 7-15; J Biol Chem. 2010 Jul 2; 285(27): 20850-9), the technique of electrostatic repulsion (WO 2006/106905), the method SEEDbody (Protein Eng Des Sel. 2010 Apr; 23(4): 195-202) and others can be used for heterologous Association of two types of H chains using snz domain. In addition, antibodies in accordance with the present invention may be those with a modified sugar or defective circuit. Examples of antibodies having modified sugar chains include antibodies constructed using a modified glycosylation (for example, WO 99/54342), antibodies with deforsirovannym sugar chains (WO 00/61739, WO 02/31140, WO 2006/067847, WO 2006/067913, and so on) and antibody having a sugar chain with bisected GlcNAc for example, WO 02/79255). Examples of known methods to obtain the IgG antibodies are defective sugar chain include a method of introducing a mutation to asparagine at position 297 using EU numbering (J Clin Pharmacol. 2010 May; 50(5): 494-506), as well as the way to obtain IgG using Escherichia coli (J Immunol Methods. 2002 May 1; 263(1-2): 133-47; J Biol Chem. 2010 Jul 2; 285(27): 20850-9). In addition, deletion, accompanied by the heterogeneity of the C-terminal lysine IgG, and improper mating, accompanied by the heterogeneity of disulfide bonds in the hinge area IgG2, can be reduced by introducing amino acid deletions/substitutions (WO 2009/041613).

The present invention provides, for example, multispecificity molecule that binds an antigen, in which the first and second polypeptides comprise constant block H-chain antibodies, and the third and fourth polypeptides include constant plot L chain of the antibody.

In addition, the present invention provides multispecific molecule that binds the antigen, where the first polypeptide includes a constant section H-chain antibodies, which consists of any one of the amino acid sequence selected from the group which consists of the following (d1)to(d6), or from a group that consists of the following (d7)-(d9), and the second polypeptide includes a constant section H-chain antibodies, which consists of any one of the amino acid p is sledovatelnot, selected from the group different from that of the above-mentioned first polypeptide:

(d1) const block H chain SEQ ID NO: 65 (G4k);

(d2) const block H chain SEQ ID NO: 66 (z7);

(d3) const block H chain SEQ ID NO: 67 (z55);

(d4) const block H chain SEQ ID NO: 68 (z106);

(d5) const block H chain of SEQ ID NO: 69 (z118);

(d6) const block H chain SEQ ID NO: 70 (z121);

(d7) const block H chain SEQ ID NO: 71 (G4h);

(d8) const block H chain SEQ ID NO: 72 (z107); and

(d9) const block H chain of SEQ ID NO: 73 (z119).

In addition, the present invention provides multispecific molecule that binds the antigen, where the third and fourth polypeptides include constant plot L chain antibodies, which consists of the following amino acid sequence:

(e) a constant area L chain antibody SEQ ID NO: 74 (k).

In the present invention, the phrase "functionally replaces F. VIII" means that f IX, and/or F. IXa, and f X are learned and enhanced activation of F. X (promoterwise development F. Xa).

In the present invention the activity in relation to the promotion of education F. Xa" can be confirmed by assessing multispecificity antigen binding molecules according to the present invention using, for example, measurement system, which includes F. XIa (F. IX activating enzyme), f IX, f X, F synthetic substrate S-2222 (SinTe the practical substrate F. Xa) and phospholipids. This measurement system shows the correlation between disease severity and clinical symptoms in cases of hemophilia A (Rosen S, Andersson M, Blomba"ck M and other Clinical applications of a chromogenic substrate method for determination ofFVIII activity. Thromb Haemost 1985, 54: 811-23). To have in this system of measurement of the analyte, which show higher activity towards the promotion of education F. Xa, are assumed such that demonstrate the best hemostatic effects against bleeding episodes in hemophilia A. In accordance with these results, if multispecificity antigen binding molecule, which has the activity of functional substitution F. VIII, is a molecule that has a higher activity than hA69-KQ/hB26-PF/hAL-AQ, it can provide an excellent activity promotion of blood clotting, and can be obtained excellent effects for the pharmaceutical component, intended for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding. To obtain excellent effects as mentioned above pharmaceutical component, for example, the activity in relation to the promotion of education F. Xa, which is measured under the conditions described in [Example 2], preferably is not less than that is new for hA69-KQ/hB26-PF/hAL-AQ, and, in particular, the activity more preferably is the same or not less than Q153-G4k/J142-G4h/L180-k. In this application "the promoting activity in relation to education F. Xa" is a value obtained by subtracting the changes in absorbance after 20 minutes the solvent from the changes in absorbance after 20 minutes in a solution of antibodies.

A preferred embodiment in accordance with the present invention is multispecificity antibody that functionally substitutes F. VIII, recognizing F. IX and/or F. IXa and F. X.

The above-mentioned multispecificity antibodies in accordance with the present invention preferably are antibodies that comprise the CDR of the H chain of anti-F. IX/F. IXa antibody or CDRs functionally equivalent thereto, and the CDR of the H chain of anti-F. X antibodies or CDRs functionally equivalent.

In addition, antibodies in accordance with the present invention preferably are antibodies that comprise the binding site of the antigen with:

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 75, 76 and 77 (CDR N circuit Q1), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 78, 79 and 80 (CDR N chains Q31), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 81, 82 and 83 (CDR N chains Q64), respectively;

CDR 1, 2 and 3 of the H chain aminokislotnye sequences of SEQ ID NOs 84, 85 and 86 (CDR N chains Q85), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 87, 88 and 89 (CDR N chain Q153), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 90, 91 and 92 (CDR N chains Q354), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 93, 94 and 95 (CDR N chains Q360), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 96, 97 and 98 (CDR N chains Q405), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 99, 100 and 101 (CDR N chains Q458), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 102 103 and 104 (CDR N chains Q460), respectively; or

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 105 106 and 107 (CDR N chains Q499), respectively,

in anti-F. IX/IXa antibody, or the binding site of the antigen, functionally equivalent, and the binding site of the antigen, including:

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 108, 109 and 110 (CDR N chains J232), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 111, 112 and 113 (CDR H circuits J259), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 114, 115 and 116 (CDR H circuits J268), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 117, 118 and 119 (CDR H circuits J300), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 120, 121 and 122 (CDR H circuits J321), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 123, 124 and 125 (CDR H circuits J326), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 126, 127 and 128 (CDR H circuits J327), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 129, 130 and 131 (CDR H circuits J339), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 132, 133 and 134 (CDR H circuits J334), respectively;

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 135, 136 and 137 (CDR H circuits J346), respectively; or

CDR 1, 2 and 3 H chains with amino acid sequences of SEQ ID NOs 174, 175 and 176 (CDR H circuits J142), respectively,

in anti-F. IX/IXa antibody, or the binding site of the antigen, are functionally equivalent.

In the present invention the binding sites of the antigen are functionally equivalent" means that the activity of functional substitution F. VIII, which have multispecificity antigen binding molecules with the binding sites of the antigen, are equivalent.

In this invention, the term "equivalent" with the need should not mean the same degree of activity, and the activity may be increased, or the activity may be reduced as long as there is activity higher than that hA69-KQ/hB26-PF/hAL-AQ in accordance with what Stamou measurement, described above, or preferably the activity in relation to the promotion of education F. Xa, which is measured under the conditions described in [Example 2], is equivalent to or not less than for Q153-G4k/J142-G4h/L180-k.

The above-mentioned antibodies may have one or more amino acid substitutions, deletions, additions and/or insertions in variable plot (sequence of CDR and/or sequence FR) amino acid sequences as long as they have the activity higher than that hA69-KQ/hB26-PF/hAL-AQ in accordance with the measurement system described above on page 35, lines 11-30, or preferably the activity in relation to the promotion of education F. Xa, which is measured under the conditions described in [Example 2], is equivalent to or not less than for Q153-G4k/J142-G4h/L180-k. The method of introducing mutations into proteins is a well-known qualified in the art as a way of introducing one or more amino acid substitutions, deletions, additions and/or insertions in the amino acid sequence. For example, a qualified expert in the art can obtain the desired mutant is functionally equivalent to multispecificity the polypeptide multimer, which has the activity of functional substitution F. VIII, by introducing appropriate mutations in AMI is kislotno sequence using site-directed mutagenesis (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 ofDNA 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; and Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Nati Acad Sci USA. 82: 488-492), etc.

As such, antibodies in accordance with the present invention also include antibodies with one or more amino acid mutations in the variable section and with activity higher than that for hA69-KQ/hB26-PF/hAL-AQ in accordance with the measurement system described above on page 35, lines 11-30, or preferably the activity in relation to the promotion of education F. Xa, which is measured under the conditions described in [Example 2], is equivalent to or not less than for Q153-G4k/J142-G4h/L180-k.

When changing amino acid residue, the amino acid is preferably subjected to mutation with the change to the other(s) amino acid(s), which preserves the properties of amino acid side chain. Examples of properties of amino acid side chains are: hydrophobic amino acids (A, I, L, M, F, P, W, Y and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S and T), amino acids, containing aliphatic the ski side chains (G, A, V, L, I, and P), amino acids having a side chain containing a hydroxyl group (S, T, and Y), amino acids having sulfur-containing side chains (C and M), amino acids with side chains containing carboxylic acid and amide (D, N, E, and Q), amino acids containing basic side chains (R, K, and H), and amino acids containing aromatic side chains (H, F, Y, and W) (amino acids are represented in single-letter code in parentheses). Amino acid substitutions in each group are referred to as conservative substitutions. It is also known that a polypeptide containing a modified amino acid sequence in which one or more amino acid residues in this amino acid sequences are delegated, inserted and/or replaced by other amino acids, can retain the original biological activity (Mark, D. F., and others, Proc. Natl. Acad. Sci. USA; (1984) 81: 5662-6; Zoller, M. J. and Smith, M., Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. and others, Science (1984) 224: 1431-3; Dalbadie-McFarland, G., and others, Proc. Natl. Acad. Sci. USA (1982) 79: 6409-13). Such mutants have amino acid identity, at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95%, with variable areas (for example, CD sequences, FR sequences or whole variable plots) in accordance with the present invention. In this application, the sequence identity is defined as the percentage of residues identical to the original amino acid sequence of the variable segment of the heavy chain or the variable segment light chain determined after aligning the sequences and acceptable introducing gaps to maximize sequence identity, if it is necessary. The identity of amino acid sequences can be determined using the methods described below.

Alternatively, the amino acid sequence of the variable regions, which have a substitution, deletion, insertion, and/or insertion of one or more amino acids in the amino acid sequences of variable regions (CDR sequence and/or sequence FR) and have a higher activity than that hA69-KQ/hB26-PF/hAL-AQ in accordance with the measurement system described above on page 35, lines 11-30, or preferably the activity in relation to the promotion of education F. Xa, which is measured under the conditions described in [Example 2] is equivalent to that or not less than such Q153-G4R/J142-G4h/L180-k, can be obtained from nucleic acids, which can gibridizatsiya when W is strict conditions with nucleic acid, which consists of a nucleotide sequence that encodes the amino acid sequence of the variable regions. The stringent conditions of hybridization for isolating a nucleic acid that hybridizes under stringent conditions with a nucleic acid that includes a nucleotide sequence encoding the amino acid sequence of the variable regions include, for example, conditions of 6 M urea, 0.4% of SDS and 0.5 × SSC, 37°C, or conditions of hybridization with a stiffness equivalent to that described above. Under more severe conditions, for example under conditions of 6 M urea, 0.4% of SDS in 0.1 × SSC, 42°C, can be expected isolation of nucleic acids with greater homology. The sequence of the isolated nucleic acids can be determined using known methods, described below. The overall homology of the nucleotide sequence of the isolated nucleic acid is at least 50% or higher sequence identity, preferably 70% or higher, more preferably 90% or higher (for example, 95%, 96%, 97%, 98%, 99% or above).

Nucleic acids that hybridize under stringent conditions with a nucleic acid which comprises a nucleotide sequence that encodes the amino acid sequence of the variable regions may also be isolated when IP is the use, instead of the above-described methods, using techniques of hybridization, gene amplification methods such as polymerase chain reaction (PCR) using primers synthesized based on the information of nucleotide sequence that encodes the amino acid sequence of the variable regions.

The identity of a single nucleotide sequence or amino acid sequence to the other can be determined using the algorithm BLAST, Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7). Programs such as BLASTN and BLASTX have been developed based on this algorithm (Altschul and others, J. Mol. Biol. (1990) 215: 403-10). For analysis of the nucleotide sequences in accordance with BLASTN based on BLAST set parameters, such as score =100 and word length =12. On the other hand, the parameters used for the analysis of amino acid sequences by using BLASTX based on BLAST, include score=50 and word length =3. Settings are applied by default when you use programs BLAST and Gapped BLAST. Specific methods for carrying out such assays are known in the art (see the website of the National center for biotechnology information (BLAST); http://www.ncbi.nlm.nih.gov).

The present invention also provides antibodies that bind to an epitope overlapping with an epitope, which is first bound antibody, above.

The ability of antibodies to recognize an epitope overlapping with an epitope that is recognized by another antibody can be confirmed using a competition between the two antibodies against this epitope. Competition between antibodies can be assessed using analysis of competitive binding when using tools, such as the enzyme-linked immunosorbent assay (ELISA), a method of energy transfer fluorescence (FRET) and the method of fluorescence analysis in a small volume (FMAT (registered trademark)). The number of antibodies bound to the antigen, directly correlates with binding ability candidatesa competitive antibodies (investigational antibodies), which competitively binds to an overlapping epitope. In other words, while the number or the affinity of the investigated antibodies against overlapping epitope increases, the number of antibodies bound to the antigen is reduced, and the number of tested antibodies bound to the antigen increases. In particular, in an acceptable manner labeled antibodies and antibodies that are estimated simultaneously added to antigens, and thus the bound antibodies define when using labels with pre-labeling of antibodies. This label is not particularly limited, and SPO is about tagging selected in accordance with the methodology used in the analysis. The method includes tagging fluorescent label, radioactive label, enzyme label, and so on

For example, a fluorescently labeled antibody and its antibodies or investigational antibodies simultaneously added to the beads immobilized with F. IX, F. IXa or F. X, and labeled antibody is determined using the method of fluorescence analysis in a small volume.

In this application the "antibody that binds to an overlapping epitope" refers to an antibody that can reduce binding of the labeled antibody, at least 50% at a concentration, which is usually 10 times higher, preferably 80 times higher, more preferably 50 times higher, even more preferably 30 times higher, and even more preferably 10 times higher than the concentration at which its antibody reduces the binding of labeled antibody by 50% (IC50).

Multispecificity molecule that binds the antigen, which are the binding sites of the antigen of antibodies that bind to epitopes overlapping with the epitope associated with the above-mentioned antibodies, can provide an excellent activity functional replacement F. VIII. In addition, the binding sites of the antigen of antibodies that bind to epitopes overlapping with the epitope associated with the above-mentioned antibodies, one or more amino acids can b shall be changed for the better activity of functional substitution F. VIII. Multispecificity antigen binding molecules having the best activity functional substitution F. VIII can be obtained by modifying the amino acids of the binding sites of the antigen and selection multispecificity antigen binding molecules with activity higher than that for hA69-KQ/hB26-PF/hAL-AQ, in accordance with the measurement system described above, or preferably, with activity in relation to the promotion of education F. Xa, which are measured under the conditions described in [Example 2], and which is equivalent or no lower than for Q153-G4R/J142-G4h/L180-k. To obtain the excellent activity of functional substitution of f VIII in accordance with the present invention the following changes of amino acids are most preferred.

(1) At least one amino acid residue selected from amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b and 102 in accordance with the Kabat numbering in the H chain of the antibody, which recognizes F. IX and/or F. IXa, is substituted with an excellent amino acid.

(2) At least one amino acid residue selected from amino acid residues at positions 35, 53, 73, 76, 96, 98, 100 and 100a in accordance with the Kabat numbering in the H chain of the antibody, which recognizes F. X., is substituted with an excellent amino acid.

(3) At least one amine is an acid residue, selected from amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94 and 95 according to the Kabat numbering in the L chain of the antibody is substituted with an excellent amino acid.

In addition, in the present invention, the preferred amino acids of the antibody for the best activity of functional substitution F. VIII include those referred to in paragraphs (4)to(6)below. In respect of these amino acids can be said that the H chain of the antibody may the source be such amino acids, or amino acids H-chain antibodies can be modified to have such consistency. (4) N chain antibody, which recognizes F. IX and/or F. IXa, where in accordance with the Kabat numbering amino acid in position 34 is an isoleucine, the amino acid at position 35 is an asparagine, glutamine or serine, the amino acid at position 49 is a serine, the amino acid at position 61 is an arginine, the amino acid at position 62 is a glutamic acid, the amino acid at position 96 is a serine or threonine, the amino acid at position 98 is a lysine or arginine, the amino acid at position 100 is a phenylalanine or tyrosine, the amino acid in position 100b is a glycine or an amino acid at position 102 represents the t of a tyrosine.

(5) the H chain of the antibody, which recognizes F. X, where according to the Kabat numbering amino acid at position 35 is an aspartic acid, the amino acid at position 53 is an arginine, the amino acid at position 73 is a lysine, the amino acid at position 76 is a glycine amino acid at position 96 is a lysine or arginine, the amino acid at position 98 is a tyrosine, the amino acid at position 100 is a tyrosine, or the amino acid at position 100a is a histidine.

(6) the L chain of the antibody, where in accordance with the Kabat numbering amino acid at position 27 is a lysine or arginine, the amino acid at position 30 is a glutamic acid, the amino acid at position 31 is an arginine, the amino acid at position 32 is a glutamine, the amino acid at position 50 is an arginine or glutamine, the amino acid at position 52 is a serine, the amino acid at position 53 is an arginine, the amino acid at position 54 is a lysine, the amino acid at position 55 is a glutamic acid, the amino acid at position 92 is a serine, the amino acid at position 93 is a serine, the amino acid at position 94 is own the th Proline, or the amino acid at position 95 is a Proline.

Among the above-mentioned amino acid residues of the antibody of paragraphs (1)to(6) favorable position of amino acid residues to obtain a particularly good F. VIII-like activity are presented in the following paragraphs(1)-(3).

(1) Amino acid residues at positions 34, 35, 61, 98, 100 and 100b, especially amino acid residues at positions 61 and 100 in accordance with the Kabat numbering in the H chain of the antibody, which recognizes F. IX and/or F. IXa.

(2) Amino acid residues at positions 35, 53, 73, 96, 98, 100 and 100a in accordance with the Kabat numbering in the H chain of the antibody, which recognizes F. X.

(3) Amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 93, 94 and 95, especially amino acid residues at positions 27, 30, 31, 50, 53, 94 and 95 according to the Kabat numbering in the L chain of the antibody.

In particular, the present invention provides multispecific molecule that binds an antigen, in which the first polypeptide includes any of the H chain of an antibody selected from the following (a1)to(a14), and any of the L chain of an antibody selected from the following (c1)to(c10), and the second polypeptide includes any of the H chain of an antibody selected from the following (b1)to(b12), and any of the L chain of an antibody selected from the following (c1)to(c10):

(a1) H chain antibody comprising amino acid sequence SEQ ID NO: 1 (Q1-G4k);

(a2) H-chain antibodies, consisting the th of the amino acid sequence of SEQ ID NO: 2 (Q31-z7);

(a3) H chain antibody comprising amino acid sequence SEQ ID NO: 3 (Q64-z55);

(a4) H chain antibody comprising amino acid sequence SEQ ID NO: 10 (Q64-z7);

(a5) H chain antibody comprising amino acid sequence SEQ ID NO: 11 (Q85-G4k);

(a6) H chain antibody comprising amino acid sequence SEQ ID NO: 12 (Q153-G4k);

(a7) H chain antibody comprising amino acid sequence SEQ ID NO: 13 (Q354-z106);

(a8) N chain antibody comprising amino acid sequence SEQ ID NO: 14 (Q360-G4k);

(a9) H chain antibody comprising amino acid sequence SEQ ID NO: 15 (Q360-z118);

(a10) H chain antibody comprising amino acid sequence SEQ ID NO: 16 (Q405-G4k);

(a11) H chain antibody comprising amino acid sequence SEQ ID NO: 17 (Q458-z106);

(a12) H chain antibody comprising amino acid sequence SEQ ID NO: 18 (Q460-z121);

(a13) H chain antibody comprising amino acid sequence SEQ ID NO: 19 (Q499-z118);

(a14) H chain antibody comprising amino acid sequence SEQ ID NO: 20 (Q499-z121);

(b1) H chain antibody comprising amino acid sequence SEQ ID NO: 4 (J268-G4h);

(b2) H chain antibody comprising amino acid sequence SEQ ID NO: 5 (J321-G4h);

(b3) H chain antibody comprising amino acid sequence SEQ ID NO: 6 (J326-z107);

(b4) H chain is nitela, consisting of the amino acid sequence of SEQ ID NO: 7 (J344-z107);

(b5) H chain antibody comprising amino acid sequence SEQ ID NO: 21 (J232-G4h);

(b6) (H-chain antibody comprising amino acid sequence SEQ ID NO: 22 (J259-z107);

(b7) H chain antibody comprising amino acid sequence SEQ ID NO: 23 (J300-z107);

(b8) H chain antibody comprising amino acid sequence SEQ ID NO: 24 (J327-z107);

(b9) H chain antibody comprising amino acid sequence SEQ ID NO: 25 (J327-z119);

(b10) H chain antibody comprising amino acid sequence SEQ ID NO: 26 (J339-z119);

(b11) H chain antibody comprising amino acid sequence SEQ ID NO: 27 (J346-z107);

(b12) H chain antibody comprising amino acid sequence SEQ ID NO: 170 (J142-G4h);

(c1) L chain antibody comprising amino acid sequence SEQ ID NO: 8 (L2-k);

(c2) L chain antibody comprising amino acid sequence SEQ ID NO: 9 (L45-k);

(c3) L chain antibody comprising amino acid sequence SEQ ID NO: 28 (L248-k);

(c4) L chain antibody comprising amino acid sequence SEQ ID NO: 29 (L324-k);

(c5) L chain antibody comprising amino acid sequence SEQ ID NO: 30 (L334-k);

(c6) L chain antibody comprising amino acid sequence SEQ ID NO: 31 (L377-k);

(c7) the L chain of the antibody consisting of amino the PCI-e slot sequence SEQ ID NO: 32 (L404-k);

(c8) L chain antibody comprising amino acid sequence SEQ ID NO: 33 (L406-k);

(c9) L chain antibody comprising amino acid sequence SEQ ID NO: 34 (L408-k); and

(c10) L chain antibody comprising amino acid sequence SEQ ID NO: 171 (L180-k).

The present invention also provides multispecificity molecule that binds an antigen, in which the first polypeptide comprises the binding site of the antigen to bind to the epitope that binds to an antibody comprising the H chain according to any one of items (a1)to(a14) and the L chain of the antibody according to any one of items (c1)to(c10), described above, and the second polypeptide comprises the binding site of the antigen to bind to the epitope that binds to an antibody comprising the H chain of the antibody according to any one of items (b1)-(b12) and the L chain of the antibody according to any one of items (c1)to(c10), described above.

In addition, the present invention provides multispecific molecule that binds an antigen, in which the first polypeptide comprises any one H chain of an antibody selected from the following (e1)-(e3), the second polypeptide includes any one H chain of an antibody selected from the following (f1)-(f3), a third polypeptide and the fourth polypeptide include any one L chain of an antibody selected from the following (g1)-(g4):

(e1) H chain of the antibody, waywayway epitope, which overlaps with the epitope that binds an antibody that comprises the H chain of the antibody according to any one of items (a1)to(a14)and the L chain of the antibody according to any one of items (c1)to(c10), described above

(e2) H chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b and 102 in accordance with the Kabat numbering in any one H chain of an antibody selected from (e1), as described above, is substituted by another amino acid;

(e3) H chain antibodies, in which in accordance with the Kabat numbering amino acid in position 34 is an isoleucine, the amino acid at position 35 is an asparagine, glutamine or serine, the amino acid at position 49 is a serine, the amino acid at position 61 is an arginine, the amino acid at position 62 is a glutamic acid, the amino acid at position 96 is a serine or threonine, the amino acid at position 98 is a lysine or arginine, the amino acid at position 100 is a phenylalanine or tyrosine, the amino acid at position 100b is a glycine or an amino acid in position 102 is a tyrosine at any H chain of an antibody selected from (E1), as described above;

(f1) N chain antibodies, binding the rpm die epitope, which overlaps with the epitope that binds an antibody that comprises the H chain of the antibody according to any one of items (b1)to(b12), as described above, and the L chain of the antibody according to any of items (c1)to(c10), as described above;

(f2) H chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 35, 53, 73, 76, 96, 98, 100 and 100a in accordance with the Kabat numbering in any N-chain antibodies (f1), as described above, is substituted by another amino acid;

(f3) H chain antibodies, in which in accordance with the Kabat numbering, amino acid at position 35 is an aspartic acid, the amino acid at position 53 is an arginine, the amino acid at position 73 is a lysine, the amino acid at position 76 is a glycine amino acid at position 96 is a lysine or arginine, the amino acid at position 98 is a tyrosine, the amino acid at position 100 is a tyrosine, or the amino acid at position 100a is a histidine in any one H chain of an antibody selected from (f1), as described above;

(g1) L chain antibody, which binds to an epitope overlapping with an epitope that binds an antibody that comprises the H chain of the antibody according to any of items (a1)to(a14) and L CE and antibodies in accordance with any of paragraphs (c1)to(c10), as described above;

(g2) L chain antibody, which binds to an epitope overlapping with an epitope that binds an antibody that comprises the H chain of the antibody according to any of items (b1)to(b12) and the L chain of the antibody according to any of items (c1)to(c10), as described above;

(g3) L chain antibodies, in which at least one amino acid residue selected from amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94 and 95 according to the Kabat numbering in the L chain of the antibody or (g1)or (g2), as described above, is substituted by another amino acid; and

(g4) L chain antibodies, in which in accordance with the Kabat numbering amino acid at position 27 is a lysine or arginine, the amino acid at position 30 is a glutamic acid, the amino acid at position 31 is an arginine, the amino acid at position 32 is a glutamine, the amino acid at position 50 is an arginine or glutamine, the amino acid at position 52 is a serine, the amino acid at position 53 is an arginine, the amino acid at position 54 is a lysine, the amino acid at position 55 is a glutamic acid, the amino acid at position 92 is a serine, the amino acid at position 93 is a serine, the amino acid in point is the situation 94 represents a Proline, or the amino acid at position 95 is a Proline in the L chain of the antibody or (g1)or (g2), as described above.

Amino acid substitutions can be made in antibodies (clones) in accordance with the present invention, in order to avoid desametasone, oxidation of methionine and so on, or to structurally stabilize the antibody.

Methods of obtaining multispecificity antigen binding molecules according to the present invention are not particularly limited and may be any method. Bespecifically antibodies can be obtained according to methods described in WO 2006/109592, WO 2005/035756, WO 2006/106905 or WO 2007/114325, which are known as examples of the method of obtaining bespecifically antibodies; after that can be selected and obtained the desired antibodies having the activity of substitution function of the cofactor.

For example, in accordance with the present invention is provided bespecifically antibody described in any of the following items (a)to(u):

(a) bespecifically antibody (Q1-G4k/J268-G4h/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth is the first polypeptide have a common L chain sequence of SEQ ID NO: 9;

(b) bespecifically antibody (Q1-G4k/J321-G4h/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 1, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(c) bespecifically antibody (Q31-z7/J326-z107/L2-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 2, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 8;

(d) bespecifically antibody (Q64-z55/J344-z107/L45-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 3, and the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 9;

(e) bespecifically antibody (Q64-z7/J326-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO 6, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(f) bespecifically antibody (Q64-z7/J344-z107/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 10, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 7, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(g) bespecifically antibody (Q85-G4k/J268-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 4, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(h) bespecifically antibody (Q85-G4k/J321-G4h/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 11, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 5, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(i) bespecifically antibody (Q153-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 12, the WTO is the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(j) bespecifically antibody (Q354-z106/J259-z107/L324-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 13, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 22, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 29;

(k) bespecifically antibody (Q360-G4k/J232-G4h/L406-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 14, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 33;

(l) bespecifically antibody (Q360-z118/J300-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 15, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 23, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(m) bespecifically antibody (Q405-G4k/J232-G4h/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 16, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 21, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 28;

(n) bespecifically antibody (Q458-z106/J346-z107/L408-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 17, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 34;

(o) bespecifically antibody (Q460-z121/J327-z119/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(p) bespecifically antibody (Q499-z118/J327-z107/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 30;

(q) bespecifically antibody (Q499-z118/J327-z107/L377-k), in which the ohms of the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 24, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31;

(r) bespecifically antibody (Q499-z118/J346-z107/L248-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 19, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 27, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 28;

(s) bespecifically antibody (Q499-z121/J327-z119/L404-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 32;

(t) bespecifically antibody (Q499-z121/J339-z119/L377-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 26, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 31 et

(u) bespecifically antibody (Q153-G4k/J142-G4h/L180-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 12, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 170, and the third polypeptide and the fourth polypeptide have a common L chain sequence of SEQ ID NO: 171.

Amino acid sequence, molecular weight, isoelectric point, the presence or absence and shape of the sugar chains of antibodies in accordance with the present invention may vary depending on the cells or the owners that produce antibodies, or cleaning methods described below. However, because these antibodies have functions equivalent to the antibodies in accordance with the present invention, they are included in the present invention. For example, when the antibody in accordance with the present invention is expressed in prokaryotic cells such as E. coli, a methionine residue is added to the N terminal end of the amino acid sequence of the original antibody. Antibodies in accordance with the present invention also include antibodies.

Bespecifically antibodies in accordance with the present invention can be obtained using methods known to the skilled technicians who Ista in this field of technology.

Based on the obtained sequence of anti-F. IX/F. IXa antibody or anti-F. X antibodies can be obtained anti-F. IX/F. IXa antibody or anti-F. X antibody, for example, using the techniques of genetic recombination, known qualified specialist in this field of technology. In particular, polynucleotide encoding the antibody can be constructed based on a series of anti-F. IX/F. IXa antibody or anti-F. X antibodies, built-in expression vector and subsequent expression acceptable in the host cells (see, for example. Co, M. S., and others, 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. and others, Methods Enzymol. (1986) 121, 663-669; Bird, R. E. and Walker, W. W., Trends Biotechnol. (1991) 9, 132-137).

Such vectors include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Alternatively, when the goal is to subclinical and cut cDNA, these vectors include, for example, pGEM-T, pDIRECT, and RT, in addition to the vectors described above. Expression vectors are particularly useful when using vectors to obtain antibodies in accordance with the present invention. For example, when targeting the expression in E. coli, such as JM109, DH5α, NV and XL I-Blue, expression vectors not only have features that allow amplification of the vector in E. coli, but mouttaki be promoter, which allows efficient expression in E. coli, for example, lacZ promoter (Ward and others, Nature (1989) 341: 544-546; FASEB J. (1992) 6: 2422-2427), Agabus promoter (Better et, Science (1988) 240: 1041-1043), T7 promoter or the other. Such vectors include pGEX-5X-l (Pharmacia), "QIAexpress system" (Qiagen), pEGFP, or pet (in this case, the host preferably represents BL21, which expresses RNA polymerase T7) in addition to the vectors described above.

Expression plasmid vectors may contain a signal sequence for secretion of antibodies. As a signal sequence for secretion of antibodies can be used pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169: 4379) when the protein is secreted into periplasm E. coli. The vector may be introduced into the host cell using, for example, methods based on calcium chloride or electroporation.

In addition to the vectors for E. coli, vectors to obtain antibodies in accordance with the present invention include expression vectors mammals (for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990, 18(17): p5322), pEF, and pCDM8), expression vectors, which are derived from insect cells (e.g., "baculovirus expression system Bac-to-BAC" (Gibco-BRL) and Rusnak), expression vectors based on the plants (for example, RMN and RMN), expression vectors, which are the origin is of an unforgettable from animal viruses (e.g., pHSV, pMV, and pAdexLcw), retroviral expression vectors (for example, pZIPneo), yeast expression vectors (e.g., "Pichia expression kit" (Invitrogen), pNV11, and SP-Q01), and expression vectors based on Bacillus subtilis (for example, pPL608 and RCT).

Aiming expression in animal cells such as CHO, COS, and NIH3T3 cells, the expression plasmid vector must have a promoter that is essential for expression in cells, for example, SV40 promoter (Mulligan and others, Nature (1979) 277: 108), MMLV-LTR promoter, EF1α promoter (Mizushima and other Nucleic Acid Res. (1990) 18: 5322) and CMV promoter, and more preferably they contain a gene for selection of transformed cells (for example, the gene of resistance to medicines that enables evaluation using agent (neomycin, G418, or other). Vectors with such characteristics include rmam, pDR2, pBK-RSV, pBK-CMV, pOPRSV and RoR, for example.

Furthermore, for stable gene expression and gene amplification in cells can be used in the following ways: Cho cells defective in the synthesis pathway nucleic acid is introduced into a vector that carries the gene for DHFR, which compensates for the deficiency (e.g., pSV2-dhfr (Molecular Cloning 2thed., Cold Spring Harbor Laboratory Press, 1989)) and amplified vector when using methotrexate (MTX). Alternatively, you may use the following method for temporal gene expression: COS cells with the gene, expr siraudin SV40 T antigen on their chromosomes, transformed into a vector with the SV40 replication origin (pcD, etc.). Point replication had origin from virus polyoma, adenovirus, papillomavirus cattle (BPV), while the other may also be used. To amplify the number of copies of a gene in a host cell expression vectors may optionally bear selective markers, such as gene aminoglycoside transferase (ARN), gene timedancing (TS), gene xanthine-guanine phosphoribosyltransferase E. coli (Ecogpt) gene dehydropeptidase (dhfr).

Antibodies in accordance with the present invention, obtained by using the methods as described above can be isolated from the cells or from the medium surrounding the cells (culture medium or other), and then purified to homogeneity. Antibodies can be isolated and purified using methods that are typically used for isolation and purification of antibodies, and type of method is not limited. For example, antibodies can be isolated and purified using the acceptable selection and parallel column chromatography, filtration, ultrafiltration, vysalivaniya, deposition solvent, solvent extraction, distillation, thus, electrophoresis in SDS-polyacrylamide gel, isoelectrofocusing, dialysis, recrystallization, and others.

Ways chromatography on the hunger for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, chromatography reverse phase and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak, etc., Cold Spring Harbor Laboratory Press, 1996). Chromatographic methods described above may be carried out using liquid chromatography, such as HPLC and EHBR. Columns that are used for affinity chromatography include column with protein A and column with protein g Columns that use A protein include, for example, Hyper D, POROS and Sepharose FF (GE Amersham Biosciences). The present invention includes antibodies that are highly purified when using these cleaning methods.

The resulting antibodies can be purified to homogeneity. Separation and purification of antibodies can be performed using traditional methods of separation and purification, which are used for normal proteins. For example, antibodies can be separated and purified by appropriate selection and application of chromatography, such as affinity chromatography, filtration, ultrafiltration, vysalivaniya, dialysis, electrophoresis in SDS polyacrylamide gel, isoelectric focusing, or other means, without limitation, (Antibodies: A Laboratory Manual, ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Columns that use the I for affinity chromatography, include, for example, speakers with protein A and column with protein G.

In one embodiment, the antibodies in accordance with the present invention, since the antibodies in accordance with the present invention functionally replaces cofactor F. VIII, they are assumed to be as effective pharmaceutical agents against diseases that occur as a result of lower activity (function) of this cofactor. Examples of the above-mentioned diseases include bleeding, a disease accompanying bleeding, and diseases caused by bleeding. In particular, they can provide excellent therapeutic effects in cases of hemophilia, in which bleeding disorders are caused by a deficiency or reduced function F. VIII/F. VIIIa. They are assumed as an excellent therapeutic agents for hemophilia A, in which bleeding disorders are caused by a hereditary deficiency or reduced function F. VIII/F. VIIIa.

The present invention provides a (pharmaceutical) composition comprising antibodies in accordance with the present invention, and pharmaceutically acceptable carriers. For example, antibodies in accordance with the present invention, which are recognized as F. IX, and F. IXa and F. X, and functionally replaces F. VIII, assumed as such, which will become a pharmaceutical (pharmacist is ical compositions or pharmaceutical agents for preventing and/or treating bleeding, diseases accompanied by hemorrhage, and diseases caused by bleeding, and the like.

In the context of the present invention bleeding, a disease accompanying bleeding, and/or diseases caused by bleeding, preferably related to the disease that develops and/or progresses due to decrease or lack of activity of F. VIII and/or activated coagulation factor VIII (F. VIIIa). Such diseases include the above hemophilia A, is a disease in which is formed an inhibitor against F. VIII/F. VIIIa, acquired hemophilia, a disease von Willebrand's disease and others, but without such restrictions.

The pharmaceutical composition used for therapeutic or preventive purposes, include antibodies in accordance with the present invention as active ingredients, can be receptionby by mixing, if it is necessary, available with pharmaceutically acceptable carriers, fillers, those which are inactive against antibodies. For example, use sterilized water, saline, stabilizers, fillers, antioxidants (such as ascorbic acid), buffers (such as phosphate, citrate, his-tag and other organic acids), antiseptics, surfactants (which such as PEG and twin), chelating agents (such as EDTA) and binding agents. They may also include other polypeptides of low molecular weight proteins, such as serum albumin, gelatin and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, sugar alcohols such as mannitol and sorbitol. When preparing an aqueous solution for injection, it can be used saline and isotonic solutions, which include glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and if it is necessary, in combination with acceptable solubilizers agents such as alcohol (e.g. ethanol), a polyalcohol such as propylene glycol and PEG), and non-ionic surface-active agents (such as Polysorbate 80, Polysorbate 20, poloxamer 188 and HCO-50). By adding hyaluronidase in the composition increased fluids may be injected subcutaneously (Expert Opin Drug Deliv. 2007 Jul; 4(4): 427-40).

If it is necessary, antibodies in accordance with the present invention may be encapsulated in microcapsules (for example, those made of hydroxymethylcellulose, gelatin and poly(methylmethacrylate)), or introduced in Kacha is the firmness of the components in the colloidal system of drug delivery (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) (see, for example, "Remington''s Pharmaceutical Science 16th-oe ed.", Oslo Ed. (1980)). Methods of obtaining pharmaceutical agents in the form of pharmaceutical agents, controlled release are also known, and such methods can be applied to the antibodies in accordance with the present invention (Langer and others, J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105 (1982); U.S. patent No. 3,773,919; publication of the European patent application EP 58,481; Sidman and others, Biopolymers 22: 547-556 (1983); EP 133,988).

The dose of the pharmaceutical composition in accordance with the present invention can be reasonably identified with regard to dosage form, method of administration, patient's age and body weight, the patient's symptoms, the type of disease and the degree of development of the disease, and ultimately determined by the treating physician. In General, the daily dose for an adult is from 0.1 mg to 2000 mg in one step or in several portions. More preferably, the dose ranges from 0.2 to 1000 mg/day, even more preferably from 0.5 to 500 mg/day, more preferably from 1 to 300 mg/day, more preferably from 3 to 100 mg/day and most preferably from 5 to 50 mg/day. These doses may vary, depending on age and body weight of the patient, and the route of administration; however, choosing the acceptable dosage is within the competence of a person skilled in the art. Similarly, the period of introduction of dosages can be reasonably determined depending on therapeutic progress.

In addition, the present invention provides genes or nucleic acids encoding the antibodies in accordance with the present invention. In addition, gene therapy can be carried out by incorporating the genes or nucleic acids encoding the antibodies in accordance with the present invention, vectors in gene therapy. In addition, for direct injection of naked plasmids, methods of introduction include introduction after packaging into liposomes, etc, the formation of a variety of viral vectors, such as retroviral vectors, adenoviral vectors, the vectors of virus-based vaccine vectors based on poxvirus vectors based on adeno-associated viruses and HVJ vectors (see: Adolph "Viral Genome Methods, CRC Press, Florida (1996)), or coating with the use of balls of media, such as particles of colloidal gold (WO 93/17706 and others). However, up until antibodies are expressed in vivo and is manifested their activity, for injection can be used any way. Preferably, a sufficient dose can be administered with acceptable parenteral route of administration (such as intravenous injection or infusion, administered intraperitoneally, subcutaneously, intradermally, intramuscularly, in the LM the new tissue or mammary glands; by inhalation; gazoprovodnaya bombardment by particles (using electron guns and similar); or by insertion through mucous membranes, such as when using nasal drip.

Alternatively, the genes encoding the antibodies in accordance with the present invention, may be introduced into blood cells, bone marrow cells, and can be introduced ex vivo using liposomal transfection, particle bombardment (U.S. patent No. 4,945,050) or viral infection, and these cells can be re-introduced to the patients. Any gene that encodes the antibody in accordance with the present invention may be used in gene therapy, and these examples include the genes containing the nucleotide sequence encoding the CDR 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 and L180, as described above.

The present invention also provides methods for preventing and/or treating bleeding, a disease accompanying bleeding, and/or diseases caused by bleeding, such methods include the step of introducing the antibodies or compositions in accordance with the present invention. Antibodies or compositions can be administered, for example, using the above-mentioned methods.

In addition, the present invention provides kits that IP is result above mentioned ways, such kits include at least the antibody or composition according to the present invention. In addition, the kits can include packaging with a syringe, a needle for injection, pharmaceutically acceptable Wednesday, alcohol-soaked cotton wool, lipoplasty bandage, instructions describing the method of application, and the like.

The present invention also relates to the use of multispecificity molecule that binds the antigen, especifismo antibody or composition according to the present invention in the manufacture of an agent for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding.

In addition, the present invention relates to multispecific molecule that binds the antigen, bespecifically the antibody or composition according to the present invention for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding.

All references to the documents of the prior art, which are contained in this application are entered in this description by reference.

Examples

Hereinafter the present invention will be particularly described with reference to Examples, but they should not be interpreted as such that limit.

[the example 1] Get bespecifically antibodies possessing activity against the promotion of education F. Xa

In WO 2006/109592, hA69-KQ/hB26-PF/hAL-AQ was obtained as bespecifically antibody having the activity of a functional replacement F. VIII. However, there is a possibility that the antibody has an inhibitory effect on the reaction in which F. IXa activates F. X when using F. VIIIa as cofactor.

As shown in Fig.1, antibodies that are associated with F. IX/F. IXa or F. X, can inhibit the formation F. IXa-F. VIIIa complex (Factor of haze (F. Hase)), or to inhibit the activity of F. Haza (activate f X). Next will be noted that the inhibition of education F. Haza and/or inhibition of activity of F-Haza will be referred to as the inhibitory effect of F. Haza. The inhibitory effect of F. Haza represents the inhibition of the coagulation reaction, in which F. VIIIa serves as a cofactor, which can supressive the residual function f VIII in a patient or function of the introduced composition F. VIII. Thus, it is desirable that the activity in relation to the promotion of education F. Xa, which is a task especifismo antibodies was high, while the inhibitory effect of F. Haza was low. In particular, for patients, support F. VIII function, and patients receiving treatment using the composition F. VIII, is more than W is undesirable, to the activity in relation to the promotion of education F. Xa and the inhibitory effect of F. Haza were separated as much as possible.

However, the inhibitory effect of F-Haza is achieved by binding to the antigen (F. IXa and/or F. X), which is a fundamental property of antibodies. On the other hand, bespecifically antibody with activity against the promotion of education F. Xa (functional substitution F. VIII), also requires binding to antigens (F. IXa) and (f X). Thus, it is assumed that it is extremely difficult to get bespecifically antibodies, which have no inhibitory effects F. Haza, but have activity in relation to the promotion of education F. Xa (functional substitution F. VIII). Likewise assumes that it is extremely difficult to reduce the inhibitory effect of F. Haza, while increasing activity in relation to the promotion of education F. Xa by introducing amino acid substitutions in bespecifically antibody.

The authors in accordance with the present invention received the genes for approximately 200 types of antibodies against human F. IXa and human F. X, respectively, when using a method known to a qualified specialist in this field of technology, which is a way recip is of the genes of the antibody-producing cells of animals, immunized with antigen (human F. IXa or human f (X), and the introduction of amino acid substitutions, if necessary. Each gene antibodies have been built into the expression vector for animal cells.

40000 or more bespecifically antibodies, such as combinations of anti-F. IXa antibody and anti-F. X antibodies temporarily expressed by simultaneous transfection with expression vector H chain antibodies against F. IXa human expression vector H chain antibodies against F. X person and expression vector shared L chain antibody in animal cells, such as NACN cells. As a control for comparison was obtained bespecifically antibody hA69-KQ/hB26-PF/hAL-AQ (SEQ ID NOs: 165/166/167), described in WO 2006/109592.

As the mutations mentioned in WO 2006/106905 or WO 1996/027011, were introduced in the CH3 domain of each H chain, it is assumed that mainly expressed bespecifically antibodies. The antibodies in the culture supernatant of the cells was purified using the method known to the skilled technician in the art, when using protein A.

The inventors in accordance with the present invention was measured activity in relation to the promotion of education F. Xa of these antibodies using the method described below. All reactions were carried out at room temperature.

Five μl of the antibody solution, diluted the CSOs with Tris buffered saline, containing 0.1% serum albumin bovine forth in the application referred to as TBSB), was mixed with 2.5 µl of 27 ng/ml human factor a beta (Enzyme Research Laboratories) and 25 μl of 6 IU/ml Novact (registered trademark) M (Kaketsuken) and then incubated in the plate for 384 cells at room temperature for 30 minutes.

The enzymatic reaction in the mixed solution was initiated by adding 5 μl of 24.7 µg/ml of human factor X (Enzyme Research Laboratories) and in ten minutes was added 5 μl of 0.5 M EDTA to stop the reaction. The color reaction was initiated by adding 5 μl of a solution of coloring substrate. After 50 minutes of holding color reaction changes in absorption at a wavelength of 405 nm was measured using SpectraMax 340PC384(Molecular Devices). The activity in relation to the promotion of education F. Xa indicated as a value obtained by subtracting the absorption of the reaction solution, free from antibodies of the absorption of the reaction solution containing the antibody.

TSR (TBSB containing 93,75 μm solution of phospholipid (SYSMEX CO.), 7.5 mm CaCl2and 1.5 mm MgCl2used as a solvent for the human factor IXa, Novact (registered trademark) M, and the human factor X. the Solution of coloring substrate S-2222™ (CHROMOGENIX) was dissolved in purified water to the concentrations of 1.47 mg/ml and then used in the analysis.

To evaluate inhibitory effects F. Haza antibodies inventors in accordance with the present invention measured the effect on the activation of f X with f IXa in the presence of F. VIIIa by using the following method. All reactions were carried out at room temperature.

Five μl of the antibody solution, diluted with Tris buffered saline containing 0.1% serum albumin bovine forth in the application 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 in the plate for 384 cells at room temperature for 30 minutes.

Addition was added to 2.5 μl of 1.8 IU/ml F. VIIIa (manner of receipt will be described below) and after 30 seconds the enzymatic reaction in the mixed solution was initiated by adding 5 μl of 24.7 µg/ml of human factor X (Enzyme Research Laboratories). Six minutes were added 5 μl of 0.5 M EDTA to stop the reaction. The color reaction was initiated by adding 5 μl of a solution of coloring substrate. After 14 minutes of holding color reaction changes in absorption at a wavelength of 405 nm was measured using SpectraMax 340PC384(Molecular Devices). The inhibitory effect of F. Haza antibodies indicated as a value obtained by subtracting the absorption of the reaction solution, free otential, from the absorption of the reaction solution containing the antibody.

F. VIIIa was prepared by mixing of 5.4 IU/ml Kohinata (registered trademark) FS (Bayer HealthCare) and 1,11 μg/ml of human alpha thrombin (Enzyme Research Laboratories) at a volume ratio of 1:1, subjecting incubation at room temperature for one minute and then adding to 7.5 units/ml Hirudin (Merck KgaA) with the number that is half of the volume of the mixed solution. The prepared solution was defined as one that has a concentration of 1.8 IU/ml FVIIIa, and after one minute after the addition of Hirudin was used for analysis.

TSR (TBSB containing 93,75 μm solution of phospholipid (SYSMEX CO.), 7.5 mm CaCl2and 1.5 mm MgCl2used as a solvent for the human factor IXa, human factor X, Kohinata (registered trademark) FS, human alpha thrombin and Hirudin. The solution of coloring substrate S-2222™ (CHROMOGENIX) was dissolved in purified water at a concentration of 1.47 mg/ml and then used in the analysis.

Activity in relation to the promotion of education F. Xa of each of bespecifically antibodies indicated in Fig.3 and 4, and the inhibitory vozdeistviem F. Haza each of bespecifically antibodies is shown in Fig.5. Discovered various amino acid substitutions that increase the activity in respect of the promotion of education F. Xa, but, as expected, the majority of amino acid substitutions that increase the activity in relation to the promotion of education F. Xa, also increased the inhibitory effect of F. Haza, and suppression of the inhibitory effect of F. Haza with increasing activity towards the promotion of education F. Xa was daunting.

In such circumstances, the inventors in accordance with the present application received Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k, Q31-z7/J326-z107/L2-k, Q64-z55/J344-z107/L45-k as bespecifically antibodies with high activity in relation to the promotion of education F. Xa and low inhibitory effect F. Haza. In addition, Q1-G4k (SEQ ID NO: 1), 031-z7 (SEQ ID NO: 2) and Q64-z55 (SEQ ID NO: 3) were obtained as H chain antibodies against human F. IXa, J268-G4h (SEQ ID NO: 4), J321-G4h (SEQ ID NO: 5), J326-z107 (SEQ ID NO: 6, and J344-z107 (SEQ ID NO: 7) were obtained as a prototype H chain antibodies against human F. X, and L2-k (SEQ ID NO: 8) and L45-k (SEQ ID NO: 9) were obtained as a prototype shared L chain antibody. The character before the hyphen in the title sequence indicates variable plot, and the character after the hyphen denotes a constant area. Each name especifismo antibodies is indicated by changing the names of the sequences of each chain, which is subjected to transfection.

Most bespecifically antibodies, the possession is the corresponding activity in regard to the promotion of education F. Xa, close to that for hA69-KQ/hB26-PF/hAL-AQ, had a high inhibitory effect of P. Haza, as expected, but these bespecifically 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 detected as such, which have a higher activity towards the promotion of education F. Xa and lower inhibitory effect of F. Haza than hA69-KQ/hB26-PF/hAL-AQ, as described in the application WO 2006/109592. The inventors in accordance with the present application have conducted additional research on the increased activity in relation to the promotion of education F. Xa and reduce the inhibitory effects of F. Haza using these four antibodies as a prototype antibodies. Screening bespecifically antibodies, which have increased activity in relation to the promotion of education F. Xa and reduced the inhibitory effect of F. Haza, shown in Fig.2.

[Example 2] Obtaining a modified antibody

The inventors in accordance with the present application have carried out the introduction of various combinations of amino acid mutations that affect the activity in relation to the promotion of education F. Xa and the inhibitory effect of F. Haza found in Example 1 for each of the chains prototype antibodies using the method known to the skilled technician in the art, such as PCR, for the introduction of the of utazi and evaluation of combinations of modified circuits in large volume for screening amino acid substitutions, which will further increase the activity in relation to the promotion of education F. Xa and reduce inhibitory effects F. Haza four prototype antibodies.

Each of the modified bespecifically antibodies with amino acid substitutions temporarily expressed and purified using the methods similar to this prototype for antibodies. Activity in relation to the promotion of education F. Xa antibodies were measured using the following method. All reactions were carried out at room temperature.

Five μl of the antibody solution, diluted with Tris buffered saline containing 0.1% serum albumin bovine forth in the application referred to as TBSB), was mixed with 2.5 µl of 27 ng/ml human factor a beta (Enzyme Research Laboratories), and then mixed with 2.5 ál of 27 ng/ml human factor a beta (Enzyme Research Laboratories) and 2.5 μl of 6 IU/ml Novact (registered trademark) M (Kaketsuken), incubated in the plate for 384 cells at room temperature for 30 minutes.

The enzymatic reaction in the mixed solution was initiated by adding 5 μl of 24.7 µg/ml of human factor X (Enzyme Research Laboratories) and in two minutes was added 5 μl of 0.5 M EDTA to stop the reaction. The color reaction was initiated by adding 5 μl of a solution of coloring substr the TA. After 20 minutes of holding color reaction changes in absorption at a wavelength of 405 nm was measured using SpectraMax 340PC384(Molecular Devices). Activity in relation to the promotion of education F. Xa indicated as a value obtained by subtracting the absorption of the reaction solution, free from antibodies of the absorption of the reaction solution containing the antibody.

TSR (TBSB containing 93,75 μm solution of phospholipid (SYSMEX CO.), 7.5 mm CaCl2and 1.5 mm MgCl2used as a solvent for the human factor IXa Novact (registered trademark) M and the human factor X. the Solution of coloring substrate S-2222™ (CHROMOGENIX) was dissolved in purified water at a concentration of 1.47 mg/ml and then used in the analysis.

The inhibitory effect of F. Haza antibodies was also subjected to evaluation using the methods described above.

Activity in relation to the promotion of education F. Xa of each of the modified bespecifically antibodies indicated in Fig.4, and the inhibitory effect of F. Haza each of bespecifically antibodies are shown in Fig.5.

The inventors in accordance with the present application received 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 bespecifically antibodies with high activity in relation to the promotion of education F. Xa is low inhibitory effect F-Haza. In addition, they found Q64-z7 (SEQ ID NO: 10) and Q85-G4k (SEQ ID NO: 11) as H chain antibodies against F. IXa person and L334-k (SEQ ID NO: 30) and L406-k (SEQ ID NO: 33) as a shared L chains of the antibodies with increased activity in relation to the promotion of education F. Xa. It is assumed that the inhibitory effects of F. Haza slightly increased activity in relation to the promotion of education F. Xa increased significantly for 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. Since these modified antibodies had a very high activity towards the promotion of education F. Xa compared with increasing inhibitory effects of F. Haza, activity in relation to the promotion of education F. Xa and the inhibitory effect of F. Haza can be further subdivided in comparison with the prototype antibodies. Thus, were found combinations that cupressinum inhibitory effect of F. Haza and increase activity in relation to the promotion of education F. Xa.

While higher activity towards the promotion of education F. Xa is preferred for the open prototype antibodies for functional substitution F. VIII bespecifically antibodies, a lower inhibitory effect F. Haza was regarded as favorable for clinical use in patie the tov, which support the functions of F. VIII, or patients who receive treatment when using F. VIII composition. Thus, were made additional modifications to obtain bespecifically antibodies, in which the inhibitory effect of F. Haza not increased, while the activity in relation to the promotion of education F. Xa increases additionally.

The results were obtained 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 and Q153-G4k/J142-G4h/L180-k as bespecifically antibodies with high activity in relation to the promotion of education F. Xa and low inhibitory effect F. Haza. In addition, the inventors have found 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) and Q499-z121 (SEQ ID NO: 20) as H chain antibodies against F. IXa person, 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) as H chain antibodies against F. X person with increased activity in relation to the promotion of education F. Xa, and 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), and L180-k (SEQ ID NO: 171) as a shared L C the drink antibodies.

Since these antibodies have a very high activity in relation to the promotion of education F. Xa, while have supressirovano inhibitory effect of F-Haza, they can have very beneficial effects for patients, supporting the function F. VIII, who receive the treatment when using F. VIII composition. Because antibodies have a longer half-life and can be administered subcutaneously, these bespecifically antibodies can be of great value for patients with hemophilia A in comparison with the existing replacement therapy by intravenous existing F. VIII compositions for the treatment of hemophilia A.

Comparison of sequences of the variable regions of each of the circuits used in Example 1 and Example 2 is shown in Fig.6A-D. for Example, to improve the activity in relation to the promotion of education F. Xa especifismo antibodies the following amino acids were identified as important: H-chain antibodies against F. IXa person isoleucine at position 34, asparagine, 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, the glycine at position 100b, tyrosine put in the and 102, and others; in the H-chain antibodies against F. X human aspartic acid at position 35, arginine at position 53, the lysine at position 73, the glycine at position 76, lysine or arginine at position 96, the tyrosine at position 98, the tyrosine at position 100, the histidine at position 100a and others; shared L chain antibody lysine or arginine at position 27, a 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, a glutamic acid at position 55, serine at position 92, the serine at position 93, a Proline at position 94, the Proline at position 95, and other amino acids of the variable segment are numbered according to the Kabat numbering (Kabat EA, etc. 1991. Sequences of Proteins of Immunological Interest. NIH).

Industrial applicability

The present invention provides multispecific antigen binding molecules that have a high functional activity substitution F. VIII, which are antibodies that recognize both the enzyme and its substrate. In addition, the present invention provides multispecific antigen binding molecules with high functional activity substitution F. VIII and low inhibitory effect F. Haza, where the antibodies recognize both the enzyme and its substrate.

Because humanitarian antibodies can generally have a high stability in blood and low immunogenicity, multispecificity antibodies in accordance with the present invention can be very promising.

1. Bespecifically antibody that recognizes blood coagulation factor IX and/or activated blood coagulation factor IX and recognizes blood coagulation factor X, in which the H chain of the first antibody comprises the binding site of the antigen, which contains variable section H chain consisting of the following amino acid sequence (a10), the H chain of the second antibody comprises the binding site of the antigen containing variable section H chain consisting of the following amino acid sequence (b7), and each of the first and second L chain of the antibody comprise the binding site of the antigen containing variable plot L chain consisting of the following amino acid sequence (C5):
(a10) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 44 (variable block H chain Q460),
(b7) the binding site of the antigen, which comprises the amino acid sequence of the variable segment of the H chain of SEQ ID NO: 52 (variable block H chain J327);
(C5) the binding site of the antigen, which comprises the amino acid sequence of the variable segment L chain SEQ ID NO: 60 (variable plot L chain L334),
and L chains of the first and second antibodies form Paris H chain of the first antibody, and H chain of the second antibody, respectively, and bespecifically antibody activates the clotting of blood.

2. Bespecifically antibody under item 1, in which the H chain of the first antibody comprises a constant block H chain of the antibody consisting of the following amino acid sequence (d6) or (d9), H chain of the second antibody comprises a constant block H chain of the antibody consisting of the following amino acid sequence (d6) or (d9), and includes an amino acid sequence that is different from that of the above-mentioned N the chain of the first antibody, and each of the first and second L chain of the antibody comprise the constant region of L chain consisting of the following amino acid sequence (s):
(d6) const block H chain SEQ ID NO: 70 (z121);
(d9) const block H chain of SEQ ID NO: 73 (z119) and
(e) a constant area L chain antibody SEQ ID NO: 74 (k).

3. Bespecifically antibody under item 2, in which the H chain of the first antibody comprises the following N chain antibody (A12), the H chain of the second antibody comprises the following N chain antibodies (b9) and each of the first and second L chain antibodies include the following L chain antibody (C5):
(A12) N chain antibody comprising amino acid sequence SEQ ID NO: 18 (Q460-z121);
(b9) N chain antibody comprising amino acid sequence SEQ ID NO: 25 (J327-z119); and
(C5) L chain antibody comprising amino acid sequence SEQ ID NO: 30 (L334-k).

4. Bispecific the prioritization of the antibody according to any one of paragraphs.1-3, where the first and second L chain L are circuits with a common sequence.

5. Bespecifically antibody (o):
(o) bespecifically antibody (Q460-z121/J327-z119/L334-k), in which the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 18, the second polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide are L circuits with a common sequence of SEQ ID NO: 30;
where the first polypeptide and the second polypeptide are associated with the third polypeptide and the fourth polypeptide, respectively, and bespecifically antibody activates the clotting of blood.

6. Nucleic acid encoding bespecifically antibody according to any one of paragraphs.1-5.

7. The vector for the expression of especifismo antibody according to any one of paragraphs.1-5, where the vector is integrated nucleic acid under item 6.

8. Cell to obtain especifismo antibody according to any one of paragraphs.1-5, where the cell comprises a nucleic acid on p. 6 or vector under item 7.

9. The method of obtaining especifismo antibodies, where the method includes culturing cells under item 8 and secretion of antibodies.

10. Pharmaceutical composition for preventing and/or treating bleeding, a disease associated with bleeding, or disorder caused by bleeding, including bespecifically antibody is of any one of paragraphs.1-5 and a pharmaceutically acceptable carrier.

11. The composition according to p. 10, where the disease develops and/or progresses due to decrease or deficiency in the activity factor of the blood coagulation VIII and/or activated blood coagulation factor VIII.

12. The composition according to p. 11, where the disease that develops and/or progresses due to decrease or deficiency in the activity factor of the blood coagulation VIII and/or activated blood coagulation factor VIII is a hemophilia A.

13. The composition according to p. 11, where the disease that develops and/or progresses due to decrease or deficiency in the activity factor of the blood coagulation VIII and/or activated blood coagulation factor VIII, is a disorder that demonstrates the emergence of inhibitors against coagulation factor VIII and/or activated blood coagulation factor VIII.

14. The composition according to p. 11, where the disease that develops and/or progresses due to decrease or deficiency in the activity factor of the blood coagulation VIII and/or activated blood coagulation factor VIII, is an acquired hemophilia.

15. The composition according to p. 11, where the disease that develops and/or progresses due to decrease or deficiency in the activity factor of the blood coagulation VIII and/or activated blood coagulation factor VIII, is a disease von Willebrand's disease.

16. Set to use the program method of preventing and/or treating bleeding, diseases associated with bleeding, or disorder caused by bleeding, which includes at least bespecifically antibody according to any one of paragraphs.1-5.



 

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35 cl, 15 tbl, 5 ex, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a recovered human integrin α5β1 monoclonal antibody. The antibody is characterised by the fact that it contains 6 CDR, 3 CDR from a light chain and 3 CDR from a heavy chain. A nucleic acid (NA) coding the antibody according to the invention, an expression vector containing a NA molecule, a host cell containing the vector, and a method for preparing the antibody on the basis of the cell are described. There are disclosed: a composition and a method for growth inhibition of the tumour cells expressing human integrin α5β1 on the basis of the antibody. What is described is a version of the method for growth inhibition of the tumour cells expressing human integrin α5β1 using the composition.

EFFECT: invention provides the new antibodies with high (approximately nm, as measured by FACS) binding affinity for human integrin α5β1 that can find application in medicine in therapy of the tumours mediated by integrin α5β1 expression.

13 cl, 36 dwg, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and biotechnology. There are presented variants of antagonist antibodies binding to the interleukin-7 receptor (IL-7R). There are described: variants of nucleic acids coding the antibodies; a host cell recombinant producing the antibody; a pharmaceutical composition inhibiting the human IL-7R function, and methods of treating and/or preventing: an autoimmune disease specified in the group of type 1 diabetes, lupus, multiple sclerosis, rheumatoid arthritis; type 2 diabetes or graft-versus-host disease based on using the antibody.

EFFECT: invention provides the antagonist anti-IL-7R antibodies that can find application in medicine.

17 cl, 15 dwg, 8 tbl, 13 ex

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