COMBINATION THERAPY WITH COAGULATION FACTORS AND MULTISPECIFIC ANTIBODIES
20220119550 · 2022-04-21
Assignee
Inventors
Cpc classification
A61K38/4846
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61P7/04
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K38/4846
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
Abstract
The invention relates to therapies for a patient with bleeding disorders, comprising the application of certain blood coagulation (clotting) factors in combination with antibodies.
Claims
1-22. (canceled)
23. A method of treating hemophilia A, the method comprising administering to a subject in need thereof a therapeutically effective amount of (i) a multispecific antibody which comprises a first antigen-binding site that binds to coagulation factor IX (FIX) and/or activated FIX and a second antigen-binding site that binds to coagulation factor X (FX), and (ii) a FIX.
24. A method of treating a deficiency or malfunction of coagulation factor VIII, the method comprising administering to a subject in need thereof a therapeutically effective amount of (i) a multispecific antibody which comprises a first antigen-binding site that binds to FIX and/or activated FIX and a second antigen-binding site that binds to FX and (ii) a FIX.
25. A method of treating a subject with hemophilia A, the method comprising: (a) identifying a subject with hemophilia A who (i) is undergoing surgery or other invasive procedure or (ii) has suffered vascular injury, and (b) administering to the identified subject a therapeutically effective amount of (i) a multispecific antibody which comprises a first antigen-binding site that binds to FIX and/or activated FIX and a second antigen-binding site that binds to FX and (ii) a FIX.
26. A method of increasing or accelerating thrombin generation, increasing or accelerating thrombin generation at a site of vascular injury or at a site of tissue factor release, enhancing blood coagulation, enhancing fibrin clot formation; or preventing or treating bleeding, diseases accompanying bleeding, or diseases caused by bleeding in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of (i) a multispecific antibody which comprises a first antigen-binding site that binds to FIX and/or activated FIX and a second antigen-binding site that binds to FX and (ii) a FIX.
27. The method of any one of claims 23-26, wherein the subject is a patient.
28. The method of any one of claims 23-26, wherein the subject suffers from a congenital or acquired deficiency of coagulation factor VIII.
29. The method of claim 28, wherein the deficiency is acquired by antibodies, other inhibitors, consumption or dilution.
30. The method of any one of claims 23-26, wherein the FIX is administered in an amount of about 10 U/kg body weight to about 200 U/kg body weight.
31. The method of any one of claims 23-26, further comprising administering to the subject a therapeutically effective amount of one or more of the coagulation factors selected from the group consisting of: FX, coagulation factor II (FII), and coagulation factor VII (FVII).
32. The method of claim 31, wherein the FX is administered to the subject in an amount of about 10 U/kg body weight to about 200 U/kg body weight.
33. The method of claim 32, wherein the FX is administered to the subject in an amount of about 50 U/kg body weight to about 200 U/kg body weight.
34. The method of claim 31, wherein the FII is administered to the subject in an amount of about 10 U/kg body weight to about 200 U/kg body weight.
35. The method of any one of claims 23-26, wherein the FIX is comprised in a prothrombin complex concentrate (PCC).
36. The method of claim 35, wherein the PCC comprises FIX, FII, and FX.
37. The method of claim 36, wherein the PCC comprises FIX, FII, FX and FVII.
38. The method of any one of claims 23-26, wherein the multispecific antibody is a bispecific antibody.
39. The method of claim 38, wherein the first antigen-binding site comprises H chain complementarity determining region (CDR) 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107, respectively, and L chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158, respectively; and the second antigen-binding site comprises H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128, respectively, and L chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158, respectively.
40. The method of claim 38, wherein the first antigen-binding site comprises a H chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a L chain variable region comprising the amino acid sequence of SEQ ID NO: 62; and the second antigen-binding site comprises a H chain variable region comprising the amino acid sequence of SEQ ID NO: 52 and a L chain variable region comprising the amino acid sequence of SEQ ID NO: 62.
41. The method of claim 38, wherein the bispecific antibody is the bispecific antibody Q499-z121/J327-z119/L404-k comprising: a) a first H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a second H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) two L chains, each L chain consisting of the amino acid sequence of SEQ ID NO: 32.
42. The method of any one of claims 23-26, wherein the FIX administered to the subject is recombinant FIX.
43. The method of any one of claims 23-26, wherein the FIX administered to the subject is isolated from plasma.
44. The method of claim 31, wherein one or more of said coagulation factors is a recombinant coagulation factor.
45. The method of claim 31, wherein one or more of said coagulation factors is isolated from plasma.
46. The method of any one of claims 23-26, wherein said multispecific antibody and said FIX are co-administered simultaneously.
47. The method of any one of claims 23-26, wherein said multispecific antibody and said FIX are co-administered sequentially.
48. A pharmaceutical composition comprising: (i) a multispecific antibody which comprises a first antigen-binding site that binds to FIX and/or activated FIX and a second antigen-binding site that binds to FX, and (ii) a FIX.
49. The pharmaceutical composition of claim 47, wherein the multispecific antibody is a bispecific antibody.
50. The pharmaceutical composition of claim 49, wherein the first antigen-binding site comprises H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107, respectively, and L chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158, respectively; and the second antigen-binding site comprises H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128, respectively, and L chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158, respectively.
51. The pharmaceutical composition of claim 49, wherein the first antigen-binding site comprises a H chain variable region comprising the amino acid sequence of SEQ ID NO: 45 and a L chain variable region comprising the amino acid sequence of SEQ ID NO: 62; and the second antigen-binding site comprises a H chain variable region comprising the amino acid sequence of SEQ ID NO: 52 and a L chain variable region comprising the amino acid sequence of SEQ ID NO: 62.
52. The pharmaceutical composition of claim 49, wherein the bispecific antibody is the bispecific antibody Q499-z121/J327-z119/L404-k comprising: a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) two L chains, each consisting of the amino acid sequence of SEQ ID NO: 32.
Description
DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0118] The present invention provides a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for use in the treatment of hemophilia A, wherein the antibody is used in combination with a coagulation factor IX.
[0119] The present invention provides a coagulation factor IX for use in the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X.
[0120] The present invention provides a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the antibody is used in combination with a coagulation factor IX.
[0121] The present invention provides a coagulation factor IX for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X.
[0122] The present invention provides a combination of [0123] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, and [0124] ii) a coagulation factor IX, [0125] for the use in the treatment of hemophilia A.
[0126] The present invention provides a combination of [0127] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and [0128] a second antigen-binding site that binds to coagulation factor X, and ii) a coagulation factor IX [0129] for the use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII.
[0130] The present invention provides the use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for the manufacture of a medicament for the treatment of hemophilia A, [0131] wherein the treatment is in combination with a coagulation factor IX.
[0132] The present invention provides the use of a coagulation factor IX for the manufacture of a medicament for the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X.
[0133] The present invention provides the use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, [0134] wherein the treatment is in combination with a coagulation factor IX.
[0135] The present invention provides the use of coagulation factor IX for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X.
[0136] In one embodiment of the invention the patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, suffers from a congenital or acquired deficiency of coagulation factor VIII.
[0137] In one embodiment of the invention the deficiency is acquired by antibodies, other inhibitors, consumption or dilution.
[0138] The present invention provides a combination, antibody or use according to any one of the preceding embodiments, [0139] a) for use in increasing the thrombin generation; [0140] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0141] c) for use accelerating of the thrombin generation/formation; [0142] d) for use in increasing and accelerating the thrombin generation/formation; [0143] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0144] f) for use in enhancing blood coagulation; [0145] g) for use in enhancing fibrin clot formation; and/or [0146] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like.
[0147] The present invention provides a combination, antibody or use according to any one of the preceding embodiments [0148] a) wherein there exists an increased bleeding risk, [0149] b) during surgery or other invasive procedures, and/or [0150] c) after vascular injury.
[0151] One embodiment of the invention is the combination, antibody or use described above, wherein in addition a) a coagulation factor II or b) a coagulation factor X, c) a coagulation factors II and X; or d) coagulation factors II, X and VII is used in the combination.
[0152] One embodiment of the invention is the combination, antibody or use described above, wherein coagulation factors IX is comprised in a prothrombin complex concentrates (PCC).
[0153] In one embodiment of the invention such prothrombin complex concentrates comprises FIX, FII, and FX.
[0154] In one embodiment of the invention such prothrombin complex concentrates comprises FIX, FII, FX and FVII.
[0155] In one embodiment of the invention the antibody described above is bispecific and the first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX comprises a H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H chain CDRs of Q499)), respectively, and a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively. and the second antigen-binding site of comprises an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively.
[0156] In one embodiment of the invention the antibody described above is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32.
[0157] In one embodiment of the invention the multispecific antibody comprises a first polypeptide comprising a first antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX and a third polypeptide comprising a third antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX, as well as a second polypeptide comprising a second antigen-binding site that binds to blood coagulation factor X and a fourth polypeptide comprising a fourth antigen-binding site that binds to blood coagulation factor X. In one embodiment such mutltispecific antibody comprises a first polypeptide to fourth polypeptide wherein the first polypeptide and the third polypeptide each comprises an antigen-binding site of an H chain or L chain of an antibody against blood coagulation factor IX or activated blood coagulation factor IX, respectively; and the second polypeptide and the fourth polypeptide each comprises an antigen-binding site of an H chain or L chain of an antibody against blood coagulation factor X, respectively.
[0158] In one embodiment of the invention the antigen-binding site of the first polypeptide comprises an antigen-binding site which comprises H chain CDRs consisting of any one of the amino acid sequences selected from the following (a1) to (a11), or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site which comprises H chain CDRs consisting of any one of the amino acid sequences selected from the following (b1) to (b11), or an antigen-binding site functionally equivalent thereto: (a1) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain CDRs of Q1), respectively; (a2) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 78, 79, and 80 (H chain CDRs of Q31), respectively; (a3) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain CDRs of Q64), respectively; (a4) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 84, 85, and 86 (H chain CDRs of Q85), respectively; (a5) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 87, 88, and 89 (H chain CDRs of Q153), respectively; (a6) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 90, 91, and 92 (H chain CDRs of Q354), respectively; (a7) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 93, 94, and 95 (H chain CDRs of Q360), respectively; (a8) an antigen-binding site comprising the of H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 96, 97, and 98 (H chain CDRs of Q405), respectively; (a9) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain CDRs of Q458), respectively; (a10) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 102, 103, and 104 (H chain CDRs of Q460), respectively; (a11) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H chain CDRs of Q499), respectively; (b1) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 108, 109, and 110 (H chain CDRs of J232), respectively; (b2) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H chain CDRs of J259), respectively; (b3) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 114, 115, and 116 (H chain CDRs of J268), respectively; (b4) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H chain CDRs of J300), respectively; (b5) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 120, 121, and 122 (H chain CDRs of J321), respectively; (b6) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H chain CDRs of J326), respectively; (b7) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively; (b8) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H chain CDRs of J339), respectively; (b9) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 132, 133, and 134 (H chain CDRs of J344), respectively; (b10) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H chain CDRs of J346), respectively; and (b11) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 174, 175, and 176 (H chain CDRs of J142), respectively.
[0159] In one embodiment of the invention the antigen-binding site of the first polypeptide comprises an antigen-binding site which comprises an H chain variable region consisting of any one of the amino acid sequences selected from the following (a1) to (a11), or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site which comprises an H chain variable region consisting of any one of the amino acid sequences selected from the following (b1) to (b11), or an antigen-binding site functionally equivalent thereto: (a1) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 35 (H chain variable region of Q1); (a2) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 36 (H chain variable region of Q31); (a3) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 37 (H chain variable region of Q1); (a4) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 38 (H chain variable region of Q85); (a5) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 39 (H chain variable region of Q153); (a6) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 40 (H chain variable region of Q354); (a7) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 41 (H chain variable region of Q360); (a8) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 42 (H chain variable region of Q405); (a9) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 43 (H chain variable region of Q458); (a10) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 44 (H chain variable region of Q460); (a11) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 45 (H chain variable region of Q499); (b1) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 46 (H chain variable region of J232); (b2) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 47 (H chain variable region of J259); (b3) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 48 (H chain variable region of J268); (b4) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 49 (H chain variable region of J300); (b5) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 50 (H chain variable region of J321); (b6) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 51 (H chain variable region of J326); (b7) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 52 (H chain variable region of J327); (b8) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 53 (H chain variable region of J339); (b9) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 54 (H chain variable region of J344); (b10) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 55 (H chain variable region of J346); and (b11) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 172 (H chain variable region of J142).
[0160] In one embodiment of the invention the antigen-binding sites included in the third polypeptide and the fourth polypeptide comprise an antigen-binding site which comprises L chain CDRs consisting of any one of the amino acid sequences selected from the following (c1 to (c10), or an antigen-binding site functionally equivalent thereto: (c1) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 138, 139, and 140 (L chain CDR of L2), respectively; (c2) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain CDR of L45), respectively; (c3) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 144, 145, and 146 (L chain CDR of L248), respectively; (c4) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain CDR of L324), respectively; (c5) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 150, 151, and 152 (L chain CDR of L3 34), respectively; (c6) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain CDR of L377), respectively; (c7) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively; (c8) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain CDR of L406), respectively; (c9) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 137, 138, and 139 (L chain CDR of L408), respectively; and (c10) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain CDR of L180), respectively.
[0161] In one embodiment of the invention the antigen-binding sites included in the third polypeptide and the fourth polypeptide comprise an antigen-binding site which comprises an L chain variable region consisting of any one of the amino acid sequences selected from the following (c1) to (c10), or an antigen-binding site functionally equivalent thereto: (c1) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 56 (L chain variable region of L2); (c2) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 57 (L chain variable region of L45); (c3) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 58 (L chain variable region of L248); (c4) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 59 (L chain variable region of L324); (c5) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 60 (L chain variable region of L334); (c6) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 61 (L chain variable region of L377); (c7) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 62 (L chain variable region of L404); (c8) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 63 (L chain variable region of L406); (c9) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 64 (L chain variable region of L408); and (c10) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 173 (L chain variable region of L180).
[0162] In one embodiment of the invention the first and second polypeptides further comprise an antibody H chain constant region, and the third and fourth polypeptides comprise an antibody L chain constant region.
[0163] In one embodiment of the invention the first and second polypeptides comprise an antibody H chain constant region, and the third and fourth polypeptides comprise an antibody L chain constant region, and wherein the third polypeptide and the fourth polypeptide are a commonly shared L chain.
[0164] In one embodiment of the invention the first polypeptide comprises any one antibody H chain selected from the following (a1) to (a14), the second polypeptide comprises any one antibody H chain selected from the following (b1) to (b12), and the third polypeptide and the fourth polypeptide comprise any one antibody L chain selected from the following (c1) to (c10): (a1) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 1 (Q1-G4 k); (a2) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 2 (Q31-z7); (a3) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 3 (Q64-z55); (a4) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 10 (Q64-z7); (a5) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 11 (Q85-G4k); (a6) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 12 (Q153-G4k); (a7) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 13 (Q354-z106); (a8) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 14 (Q360-G4k); (a9) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 15 (Q360-z118); (a10) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 16 (Q405-G4k); (a11) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 17 (Q458-z106); (a12) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 18 (Q460-z121); (a13) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 19 (Q499-z118); (a14) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 20 (Q499-z121); (b1) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 4 (J268-G4h); (b2) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 5 (J321-G4h); (b3) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 6 (J326-z107); (b4) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 7 (J344-z107); (b5) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 21 (J232-G4h); (b6) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 22 (J259-z107); (b7) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 23 (J300-z107); (b8) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 24 (J327-z107); (b9) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 25 (J327-z119); (b10) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 26 (J339-z119); (b11) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 27 (J346-z107); (b12) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 170 (J142-G4h); (c1) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 8 (L2-k); (c2) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 9 (L45-k); (c3) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 28 (L248-k); (c4) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 29 (L324-k); (c5) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 30 (L334-k); (c6) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 31 (L377-k); (c7) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 32 (L404-k); (c8) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 33 (L406-k); (c9) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 34 (L408-k); and (c10) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 171 (L180-k).
[0165] In one embodiment of the invention the antibody described above is a bispecific antibody of any one of the following (a) to (u): (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 9; (b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 9; (c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 8; (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 3, 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 are a commonly shared L chain of SEQ ID NO: 9; (e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 29; (k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (1) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 28; (n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 34; (o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein 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 a commonly shared L chain of SEQ ID NO: 30; (p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 31; (r) a bispecific antibody (Q499-z118/J346-z107/L248-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 28; (s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 32; (t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 31; and (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 171.
[0166] One embodiment of the invention is the combination, antibody or use described above, wherein the FIX is administered in an amount of 10 U-200 U FIX/kg body weight in a patient with hemophilia A treated with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. One embodiment of the invention is the combination, antibody or use described above, wherein additionally the FX is administered in an amount of 10 U-200 U FX/kg body weight. One embodiment of the invention is the combination, antibody or use described above, wherein additionally the FII is administered in an amount of 10 U-200 U FII/kg body weight. One embodiment of the invention is the combination, antibody or use described above, wherein additionally the FII and FX are administered in an amount of 10 U-200 U FII/kg body weight and 10 U-200 U FX/kg body weight. One embodiment of the invention is the combination, antibody or use described above, wherein prothrombin complex (PCC) is administered in amount of 10 U-200 U PCC/kg body weight in a patient with hemophilia A treated with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X.
[0167] Multispecific antibodies and antigen-binding molecules described herein comprise a first antigen-binding site and a second antigen-binding site that can specifically bind to at least two different types of antigens. While the first antigen-binding site and the second antigen-binding site are not particularly limited as long as they have an activity to bind to FIX and/or FIXa, and FX, respectively, examples include sites necessary for binding with antigens, such as antibodies, scaffold molecules (antibody-like molecules) or peptides, or fragments containing such sites. Scaffold molecules are molecules that exhibit function by binding to target molecules, and any polypeptide may be used as long as they are conformationally stable polypeptides that can bind to at least one target antigen. Examples of such polypeptides include antibody variable regions, fibronectin (WO 2002/032925), protein A domain (WO 1995/001937), LDL receptor A domain (WO 2004/044011, WO 2005/040229), ankyrin (WO 2002/020565), and such, and also molecules described in documents by Nygren et al. (Current Opinion in Structural Biology, 7: 463-469 (1997); and Journal of Immunol Methods, 290: 3-28 (2004)), Binz et al. (Nature Biotech 23: 1257-1266 (2005)), and Hosse et al. (Protein Science 15: 14-27(2006)). Furthermore, as mentioned in Curr Opin Mol Ther. 2010 August; 12(4): 487-95 and Drugs. 2008; 68(7): 901-12, peptide molecules that can bind to target antigens may be used.
[0168] Herein, multispecific antigen-binding molecules are not particularly limited as long as they are molecules that can bind to at least two different types of antigens, but examples include polypeptides containing the above-mentioned antigen-binding sites, such as antibodies and scaffold molecules as well as their fragments, and aptamers comprising nucleic acid molecules and peptides, and they may be single molecules or multimers thereof. Preferred multispecific antigen-binding molecules include multispecific antibodies that can bind specifically to at least two different antigens. Particularly preferred examples of antibodies which have an activity of functionally substituting for FVIII of the present invention include bispecific antibodies (BsAb) that can bind specifically to two different antigens (they may also be called dual specific antibodies).
[0169] In the present invention, the term “commonly shared L chain” refers to an L chain that can link with two or more different H chains, and show binding ability to each antigen. Herein, the term “different H chain(s)” preferably refers to H chains of antibodies against different antigens, but is not limited thereto, and also refers to H chains whose amino acid sequences are different from each other. Commonly shared L chain can be obtained, for example, according to the method described in WO 2006/109592.
[0170] The multispecific antigen-binding molecules of the present invention (preferably bispecific antibodies) are antibodies having specificity to two or more different antigens, or molecules comprising fragments of such antibodies. The antibodies of the present invention are not particularly limited, but are preferably 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 modified gene recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.
[0171] Furthermore, the L chains of an antibody which will become a multispecific antigen-binding molecule of the present invention may be different, but preferably have commonly shared L chains.
[0172] Multispecific antigen-binding molecules of the present invention are preferably recombinant antibodies produced using genetic recombination techniques (See, for example, Borrebaeck CAK and Larrick J W, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies, can be obtained by cloning DNAs encoding antibodies from hybridomas or antibody-producing cells, such as sensitized lymphocytes, that produce antibodies, inserting them into suitable vectors, and then introducing them into hosts (host cells) to produce the antibodies.
[0173] Furthermore, antibodies of the present invention may include not only whole antibodies but also antibody fragments and low-molecular-weight antibodies (minibodies), and modified antibodies.
[0174] For example, antibody fragments or minibodies include diabodies (Dbs), linear antibodies, and single chain antibody (hereinafter, also denoted as scFvs) molecules. Herein, an “Fv” fragment is defined as the smallest antibody fragment that comprises a complete antigen recognition site and binding site.
[0175] An “Fv” fragment is a dimer (VH-VL dimer) in which an H chain variable region (VH) and an L chain variable region (VL) are strongly linked by non-covalent binding. The three complementarity determining regions (CDRs) of each of the variable regions interact with each other to form an antigen-binding site on the surface of the VH-VL dimer. Six CDRs confer the antigen-binding site to an antibody. However, one variable region (or half of the Fv comprising only three CDRs specific to an antigen) alone can recognize and bind to an antigen, though its affinity is lower than that of the entire binding site.
[0176] An Fab fragment (also called F(ab)) further comprises an L chain constant region and an H chain constant region (CH1). An Fab′ fragment differs from an Fab fragment in that it additionally comprises several residues derived from the carboxyl terminus of the H chain CH1 region, comprising one or more cysteines from the hinge region of the antibody. Fab′-SH refers to an Fab′ in which one or more cysteine residues of its constant region comprise a free thiol group. An F(ab′) fragment is produced by cleavage of disulfide bonds between the cysteine residues in the hinge region of F(ab′) 2 pepsin digest. Other chemically bound antibody fragments are also known to those skilled in the art.
[0177] Diabodies are bivalent minibodies constructed by gene fusion (Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097; WO 93/11161). Diabodies are dimers consisting of two polypeptide chains, in which each polypeptide chain comprises an L chain variable region (VL) and an H chain variable region (VH) linked with a linker short enough to prevent association of these two domains within the same chain, for example, a linker of preferably 2 to 12 amino acids, more preferably 3 to 10 amino acids, particularly about 5 amino acids. The polypeptide chain form a dimer since the linker between the VL and VH encoded on the same polypeptide is too short to form a single chain variable region fragment. Therefore, diabodies comprise two antigen-binding sites.
[0178] A single-chain antibody or an scFv antibody fragment comprises the VH and VL regions of an antibody, and these regions exist in a single polypeptide chain. In general, an Fv polypeptide further comprises a polypeptide linker between the VH and VL regions, and this enables an scFv to form a structure necessary for antigen binding (for a review on scFvs, see Pluckthun “The Pharmacology of Monoclonal Antibodies” Vol. 113 (Rosenburg and Moore ed. (Springer Verlag, New York) pp. 269-315, 1994). In the context of the present invention, linkers are not particularly limited so long as they do not inhibit the expression of the antibody variable regions linked at their ends.
[0179] IgG-type bispecific antibodies can be secreted from hybrid hybridomas (quadromas) produced by fusing two kinds of hybridomas that produce IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). They can also be secreted by taking the L chain and H chain genes constituting the two kinds of IgGs of interest, a total of four kinds of genes, and introducing them into cells to coexpress the genes.
[0180] In this case, by introducing suitable amino acid substitutions to the CH3 regions of the H chains, IgGs having a heterogeneous combination of H chains can be preferentially secreted (Ridgway J B et al. Protein Engineering 1996, 9: 617-621; Merchant A M et al. Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; Davis J H et al. Protein Eng Des Sel. 2010, 4: 195-202).
[0181] Regarding the L chains, since diversity of L chain variable regions is lower than that of H chain variable regions, commonly shared L chains that can confer binding ability to both H chains may be obtained. The antibodies of the present invention comprise commonly shared L chains. Bispecific IgGs can be efficiently expressed by introducing the genes of the commonly shared L chain and both H chains into cells.
[0182] Bispecific antibodies may be produced by chemically crosslinking Fab's. Bispecific F(ab′) 2 can be produced, for example, by preparing Fab′ from an antibody, using it to produce a maleimidized Fab′ with ortho-phenylenedi-maleimide (o-PDM), and then reacting this with Fab′ prepared from another antibody to crosslink Fab's derived from different antibodies (Keler T et al. Cancer Research 1997, 57: 4008-4014). The method of chemically linking an Fab′-thionitrobenzoic acid (TNB) derivative and an antibody fragment such as Fab′-thiol (SH) is also known (Brennan M et al. Science 1985, 229: 81-83).
[0183] Instead of a chemical crosslink, a leucine zipper derived from Fos and Jun may also be used. Preferential formation of heterodimers by Fos and Jun is utilized, even though they also form homodimers. Fab′ to which Fos leucine zipper is added, and another Fab′ to which Jun leucine zipper is added are expressed and prepared. Monomeric Fab′-Fos and Fab′-Jun reduced under mild conditions are mixed and reacted to form bispecific F(ab′) 2 (Kostelny S A et al. J. of Immunology, 1992, 148: 1547-53). This method can be applied not only to Fab's but also to scFvs, Fvs, and such.
[0184] Furthermore, bispecific antibodies including sc(Fv) 2 such as IgG-scFv (Protein Eng Des Sel. 2010 April; 23(4): 221-8) and BiTE (Drug Discov Today 2005 Sep. 15; 10(18): 1237-44), DVD-Ig (Nat Biotechnol. 2007 November; 25(11): 1290-7. Epub 2007 Oct. 14; and MAbs. 2009 July; 1(4): 339-47. Epub 2009 Jul. 10), and also others (IDrugs 2010, 13: 698-700) including two-in-one antibodies (Science. 2009 Mar. 20; 323(5921): 1610-4; and Immunotherapy 2009 September; 1(5): 749-51), Tri-Fab, tandem scFv, and diabodies are known (MAbs. 2009 November; 1(6): 539-547). In addition, even when using molecular forms such as scFv-Fc and scaffold-Fc, bispecific antibodies can be produced efficiently by preferentially secreting a heterologous combination of Fcs (Ridgway J B et al., Protein Engineering 1996, 9: 617-621; Merchant A M et al. Nature Biotechnology 1998, 16: 677-681; WO 2006/106905; and Davis J H et al., Protein Eng Des Sel. 2010, 4: 195-202).
[0185] A bispecific antibody may also be produced using a diabody. A bispecific diabody is a heterodimer of two cross-over scFv fragments. More specifically, it is produced by forming a heterodimer using VH(A)-VL(B) and VH(B)—VL(A) prepared by linking VHs and VLs derived from two kinds of antibodies, A and B, using a relatively short linker of about 5 residues (Holliger P et al. Proc Natl. Acad. Sci. USA 1993, 90: 6444-6448).
[0186] The desired structure can be achieved by linking the two scFvs with a flexible and relatively long linker comprising about 15 residues (single chain diabody: Kipriyanov S M et al. J. of Molecular Biology. 1999, 293: 41-56), and conducting appropriate amino acid substitutions (knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788; VH/VL interface engineering: Igawa T et al. Protein Eng Des Sel. 2010, 8: 667-77).
[0187] An sc(Fv) 2 that can be produced by linking two types ofscFvs with a flexible and relatively long linker, comprising about 15 residues, may also be a bispecific antibody (Mallender W D et al. J. of Biological Chemistry, 1994, 269: 199-206).
[0188] Examples of modified antibodies include antibodies linked to various molecules such as polyethylene glycol (PEG). The antibodies of the present invention include such modified antibodies. In the context of the present invention, the substance to which the modified antibodies are linked is not limited. Such modified antibodies can be obtained by chemically modifying obtained antibodies. Such methods are well established in the art.
[0189] The antibodies of the present invention include human antibodies, mouse antibodies, rat antibodies, or such, and their origins are not limited. They may also be genetically modified antibodies, such as chimeric or humanized antibodies.
[0190] Methods for obtaining human antibodies are known in the art. For example, transgenic animals carrying the entire repertoire of human antibody genes can be immunized with desired antigens to obtain desired human antibodies (see International Patent Application WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).
[0191] Genetically modified antibodies can also be produced using known methods. Specifically, for example, chimeric antibodies may comprise H chain and L chain variable regions of an immunized animal antibody, and H chain and L chain constant regions of a human antibody Chimeric antibodies can be obtained by linking DNAs encoding the variable regions of the antibody derived from the immunized animal, with DNAs encoding the constant regions of a human antibody, inserting this into an expression vector, and then introducing it into host cells to produce the antibodies.
[0192] Humanized antibodies are modified antibodies often referred to as “reshaped” human antibodies. A humanized antibody is constructed by transferring the CDRs of an antibody, derived from an immunized animal to the complementarity determining regions of a human antibody. Conventional genetic recombination techniques for such purposes are known (see European Patent Application Publication No. EP 239400; International Publication No. WO 96/02576; Sato K et al., Cancer Research 1993, 53: 851-856; International Publication No. WO 99/51743).
[0193] The multispecific antigen-binding molecules of the present invention are those that recognize FIX and/or FIXa, and FX, and functionally substitute for cofactor function of FVIII, and characterized in that the molecules have a higher FXa generation-promoting activity compared to hA69-KQ/hB26-PF/hAL-AQ (described in WO 2006/109592) which is known as a bispecific antibody that functionally substitutes for FVIII. Furthermore, antibodies of the present invention usually have a structure which comprises a variable region of an anti-FIXa antibody and a variable region of an anti-FX antibody.
[0194] A multispecific antigen-binding molecule of the present invention functionally substitutes for FVIII, which comprises a first antigen-binding site that recognizes FIX and/or FIXa and a second antigen-binding site that recognizes FX, wherein the function that substitutes for the function of FVIII is caused by a higher FXa generation-promoting activity compared to the activity of the bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises H chains consisting of SEQ ID NOs: 165 and 166, and a commonly shared L chain consisting of SEQ ID NO: 167.
[0195] A multispecific antigen-binding molecule of the present invention comprises a first polypeptide and a third polypeptide comprising an antigen-binding site that recognizes FIX and/or FIXa, and a second polypeptide and a fourth polypeptide comprising an antigen-binding site that recognizes FX. The first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide each include the antigen-binding site of the antibody H chain and the antigen-binding site of the antibody L chain.
[0196] For example, in a multispecific antigen-binding molecule of the present invention, the first polypeptide and the third polypeptide include an antigen-binding site of an H chain and L chain of an antibody against FIX or FIXa, respectively; and the second polypeptide and the fourth polypeptide comprise an antigen-binding site of an H chain and L chain of an antibody against FX, respectively.
[0197] At this time, the antigen-binding sites of the antibody L chain included in the first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide may be commonly shared L chains.
[0198] A polypeptide comprising an antigen-binding site of an antibody L chain in the present invention is preferably a polypeptide which comprises all or a part of the sequence of the antibody L chain which binds to FIX, FIXa and/or FX.
[0199] In the present invention, the phrase “functionally substitute for FVIII” means that FIX and/or FIXa, and FX is recognized, and activation of FX is promoted (FXa generation is promoted).
[0200] In the present invention, “FXa generation-promoting activity” can be confirmed by evaluating the multispecific antigen-binding molecules of the present invention using, for example, a measurement system comprising FXIa (FIX activating enzyme), FIX, FX, F synthetic substrate S-2222 (synthetic substrate of FXa), and phospholipids. This measurement system shows the correlation between the severity of the disease and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomback M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985, 54: 811-23). That is, in the present measurement system, test substances that show higher FXa generation-promoting activity are expected to show better hemostatic effects against bleeding episodes in hemophilia A. With these results, if a multispecific antigen-binding molecule having activity of functionally substituting for FVIII is a molecule having a higher activity than hA69-KQ/hB26-PF/hAL-AQ, it may yield excellent blood coagulation-promoting activity, and excellent effects may be obtained as a pharmaceutical component for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding. To obtain excellent effects as the above-mentioned pharmaceutical component, for example, FXa generation-promoting activity measured under the conditions described in Example 2 of US 2013/0330345 is preferably not less than that of hA69-KQ/hB26-PF/hAL-AQ, and in particular, the activity is more preferably the same as or not less than that of Q153-G4k/J142-G4h/L180-k. Herein, the “FXa generation-promoting activity” is the value obtained by subtracting the change in absorbance upon 20 minutes in a solvent from the change in absorbance upon 20 minutes in an antibody solution.
[0201] A preferred embodiment of the present invention is a multispecific antibody that functionally substitutes for FVIII, which recognizes FIX and/or FIXa, and FX.
[0202] The above-mentioned multispecific antibodies of the present invention are preferably antibodies which comprise H chain CDRs of anti-FIX/FIXa antibodies or CDRs functionally equivalent to them, and H chain CDRs of anti-FX antibodies or CDRs functionally equivalent to them.
[0203] In one aspect of the present invention, in the combination of the multispecific antibody with FIX a prothrombin complex concentrates containing FIX, FII, FVII and FX (4-component PCC), or prothrombin complex concentrates containing only or mainly FIX, FII and FX (3 component PCC) can be used. The 3-component PCCs is one preferred embodiment for the combination of the present invention due to the lack of FVII, which might form a competition for its active form FVIIa. In another aspect of the present invention. In addition to FIX in the combination with the multispecific antibody, FII, FX; or FII and FX can be used as prepared preparation mixtures. In addition to the aforementioned proteins also the structural protein fibrinogen, or a antifibrinolytic drug such as tranexamic acid or aprotinin could be added.
[0204] Blood coagulation factors maybe exist in their inactive precursor forms as zymogen (e.g. FIX) or as activated forms (e.g. FIXa). A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the configuration to reveal the active site) for it to become an active enzyme. The biochemical change usually occurs in a lysosome where a specific part of the precursor enzyme is cleaved in order to activate it. The activated blood coagulation factors are typically abbreviated as e.g FIXa, FXa etc. The activation mechanism of e.g. of coagulation factor IX is described in Biol Chem. 2009 May-June; 390(5-6):391-400.
[0205] The terms “blood coagulation factor”, “coagulation factor”, or “(blood) coagulation factor” or in abbreviated form only “F” before the respective blood coagulation factor number (e.g. FVIII, FIX, FX etc) as used herein are interchangeable and refer to respective human blood coagulation factors of the human coagulation system. In their activated form they are abbreviated e.g. as FVIIIa, FIXa, FXa. In their no-activated form they are abbreviated as FVIII, FIX, FX etc.
[0206] “Coagulation factor IX” (FIX) is a zymogen, an inactive precursor. It is processed to remove the signal peptide, and then cleaved by factor XIa (of the contact pathway) or factor VIIa (of the tissue factor pathway) to produce a two-chain form where the chains are linked by a disulfide bridge (Di Scipio R G, et al, J. Clin. Invest. 61 (1978) 1528-38; Taran LD Biochemistry Mosc. 62 (1997) 685-93). When activated into factor IXa, in the presence of Ca.sup.2+, membrane phospholipids, and a Factor VIII cofactor, it hydrolyses one arginine-isoleucine bond in Factor X to form factor Xa. Therefore the term “Coagulation factor IX” (“FIX”) as used herein refers to the non-activated coagulation factor IX.
[0207] Deficiency of factor IX causes Christmas disease (hemophilia B) (Biggs, R; et al British Medical Journal 2 (4799) 1952 1378-82). Over 100 mutations of factor IX have been described; some cause no symptoms, but many lead to a significant bleeding disorder. The original Christmas disease mutation was identified by sequencing of Christmas' DNA, revealing a mutation which changed a cysteine to a serine (Taylor, S. A.; et al, Thrombosis and haemostasis 67 (1992) 63-65. Recombinant factor IX is used to treat Christmas disease, and is commercially available as “BeneFIX®” “Alprolix®”, and “Rixubis®” (all brand names for a recombinant Factor IX products). Some rare mutations of factor IX result in elevated clotting activity, and can result in clotting diseases, such as deepvein thrombosis (Simioni P, et al, N. Engl. J. Med. 361 (2009) 1671-5).
[0208] FIX is synthesized as a single polypeptide chain 415 amino acids in length. FIX is present in blood as an inactive precursor molecule that consists of (1) a gamma-carboxyglutamic acid containing domain (“Gla domain”), (2) and (3) two epidermal growth factor-like domains (“EGF-1 domain”, “EGF-2 domain”), (4) an activation peptide region (“AP region”), and (5) a serine protease domain. FIX undergoes extensive post-translational modification during transit through the endoplasmatic reticulum and Golgi apparatus: removal of the signal sequence; gamma-carboxylation of twelve Glu residues in the Gla domain by vitamin K dependent gamma-glutamyl carboxylase, a hepatic microsomal enzyme; N-glycosylation of N-157 and N-167 in the AP region; O-glycosylation of S-53 and S-61 in the Gla domain and T-159, T-169, T-172 and T-179 in the AP region; beta-hydroxylation at Asp-64 in the EGF-1 domain; sulfation of Tyr-155 and phosphorylation of Ser-158, both in the AP region.
[0209] In Haemophilia B, the deficiency is either in the amount or in the function of FIX. This disease is successfully treated by replacement therapy consisting of the administration of preparations of human plasma derived (pdFIX) or recombinant coagulation factor IX (rFIX). Plasma derived products are either prothrombin complex concentrates (which have been used in the past for the treatment of Haemophilia B) or purified FIX concentrates (mainly affinity purified factor IX). rFIX has been extensively characterised with respect to post-translational modifications. Despite minor differences to the pdFIX, specific activities and pharmacological effectiveness are comparable.
[0210] Biochemical comparison between pdFIX and CHO derived rFIX showed an indistinguishable secondary/tertiary structure as measured by fluorescence, circular dichroism or analytical ultracentrifugation. Minor differences were detected in post-translational modifications. Whereas in pdFIX all 12 Glu residues in the Gla domain are occupied (i.e. transformed to Gla), only 10 of the 12 sites are fully occupied in rFIX (“undercarboxylation” of Gla-40 or Gla-40 and Gla-36, respectively). N-linked glycans are fully sialylated and show high heterogeneity in pdFIX (however, this may also be due to the fact that pdFIX is prepared from plasma pools having diverse plasma donations); low hetereogeneity and often incomplete sialysation in rFIX. Ser-53 is Xyl-Xyl-Gic-glycosylated in rFIX whereas in pdFIX Ser-53 contains additional Xyl-Glc-glycosylation (Ser-61 contains NeuAc-Gal-GlcNAc-Fuc-in both forms). rFIX from CHO cells exhibits glycosylation with carbohydrates capped with sialic acid alpha(2-3)-galactose groups (CHO cells lack alpha(2-6)-sialyltransferase) whereas pdFIX contains terminal sialic acid alpha(2-6)-galactose moieties. Human host cells for expressing rFIX (such as HEK 293 cells) contain alpha(2-3)- and alpha(2-6)-sialyltransferases; accordingly HEK 293 derived rFIX differs in this respect from commercial CHO-derived rFIX (White et al., Thromb. Haemost. 78(1) (1997), 261-265; Bond et al., Sem. Hematol. 35 (2) (1998), Suppl. 2, 11-17; Bebgie et al., Thromb. Haemost. 94 (2005), 1138-1147).
[0211] It has been speculated whether a lower degree of phosphorylation of Ser-155 in the AP region and the lower degree of sulfation of Tyr-158 are responsible for the lower in-vivo recovery of rFIX (37.81+−14.0% of rFIX compared to 52.61+−12.36 for pdFIX purified with monoclonal antibodies (White et al. (1997)). Griffith et al. (J. Thromb. Haemost. 5 (2007), Suppl. 2: P-M-043) reported that N-Glycan sialylation is important for in vivo recovery of rFIX. In WO 2007/101681 A1 rFIX products with improved in vivo recovery are provided comprising at least 25% and less than 98% of fully phosphorylated and sulfated rFIX.
[0212] Elimination half life of CHO expressed rFIX and immunopurified pdFIX are comparable (18.10+−5.10 hours and 17.66+−5.31 hours, respectively (White et al., 1997)). Based on a report that deletion of the AP region (a del(155-177) mutant showed a terminal catabolic half life increase of 45% compared to the wild-type form (Bebgie et al. (2005)), Chang et al. (J. Thromb. Haemost. 5 (2007), Suppl. 2: O-M-088) treated FIX with neuraminidase and N- and O-glycanase to remove both, the N- and O-linked carbohydrates. De-glycosylated FIX had a significantly lower recovery than untreated FIX, whereas recovery of the de-glycosylated form were not statistically different in rFIX and pdFIX. It was therefore concluded that this suggested that glycosylation plays a major role in determining the recovery of FIX. It was further concluded that the role of sulfation/phosphorylation play a “relatively minor” role in in vivo recovery. Half life or activity data were not reported for the de-glycosylated forms of rFIX and pdFIX in Chang et al.
[0213] In clinical studies, rFIX has been shown to be safe and effective, but a 20 to 50% higher dosage than for pdFLX is needed for successful treatment. This is due to a 30 to 50% lower in vivo recovery for CHO derived rFIX than for pdFIX (as described above), as also revealed by pharmacokinetic data collected from preclinical and clinical studies, where pdFIX and rFIX are compared in different animal models, and clinical studies in haemophilia B patients. However, the circulating half-life of rFIX is not distinguishable from pdFIX preparations.
[0214] There had been various attempts to improve FIX drugs, e.g. (for rFIX) increased mRNA production, reduced binding to collagen IV, increasing the specific activity and improving the recovery by making rFIX more similar to pdFIX (Pipe, Sem. Thromb. Hemost. 30 (2) (2004), 227-237; WO 2007/101681 A1); (for pdFIX) enrichment and specific purification (U.S. Pat. No. 5,639,857 A). However, there is still a strong need for improved FIX preparations which can be administered in a lower dosage or in larger time intervals than conventional FIX preparations for a successful treatment. Whereas most strategies in the prior art concentrate on improving recovery and increasing FIX activity, strategies which aim at prolongation of half life of the protein are rare, mainly because half life of rFIX and pdFIX are the same. This is mainly due to the known sensibility of the FIX protein against (even minor) chemical modification or mutations and the potential immunological effects of introducing mutations into a human protein (Bebgie et al. (2005); Kaufman, Thromb. Haemost. 79 (1998), 1068-1079; Hansson et al., J Thromb. Haemost. 3 (2005), 2633-2648; Wojcik et al., Biochem. J. 323 (1997), 629-636)
[0215] The term “coagulation factor IX” (FIX) as used herein shall be any form of factor IX molecule with the typical characteristics of blood coagulation factor IX. FIX shall include FIX from plasma (pdFIX) and any form of rFIX which is capable of curing bleeding disorders in a patient which are caused by deficiencies in FIX (e.g. haemophilia B). FIX is comprised of the GIa domain, two EGF domains (EGF-1 and EGF-2), an AP region and a serine protease domain. FIX according to the present invention shall have the same amino acid sequence as human pdFIX and human rFIX and all functional variations thereof, i.e. variations (both, in amino acid sequence and post-translational modifications) which provide a comparable or improved in vivo activity of FIX. For curing the respective FIX related bleeding disorders in animals, the corresponding FIX sequences may be applied or those FIX forms which show sufficient cross-activity in related animal species. Furthermore, FIX according to the present invention shows all post-translational modifications necessary for a proper functioning of the protein in vivo. Ample literature is available describing functional forms of FIX, for example a naturally occurring Ala/Thr exchange at position 148; suitable FIX molecules which can be covalently coupled to the water-soluble hydrophilic polymers according to the present invention are described e.g. in White et al. (1997); Pipe (2004); WO 2007/101681 A1; U.S. Pat. No. 5,639,857 A; Bebgie et al. (2005); Kaufman (1998); Hansson et al. (2005); Wojcik et al. (1997). Preferably, the FIX according to the present invention is a recombinantly produced FIX. The term “recombinant” when used with reference to FIX indicates that FIX has been produced by the introduction of a heterologous or non-naturally occurring nucleic acid or protein into a host cell, or the alteration of a native nucleic acid or protein in a host cell. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express wild type and variant genes that are not in the native position in the genome of the cell, or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. The term “biologically produced” FIX covers all FIX forms being produced by organisms or cells without further chemical modification (not performable by such organisms or cells) after FIX has been isolated from such organisms or cells.
[0216] Commercially available recombinant factor IX products include “BeneFIX®”, “Alprolix® (recombinant Factor IX Fc fusion protein with elongated halflife)” and “Rixubis®” (all brand names for a recombinant Factor IX product Benefix™).
[0217] Commercially available recombinant factor IX products are often manufactured by using stable transfected Chinese hamster ovary (CHO) cells. CHO cells provide capacity for glycosylation and other post-translational modifications. With these cells, large-scale suspension cultures can be maintained without the addition of animal- or human-derived raw material. In the manufacture of one of these commercial products (marketed under the trade name Benefix™) rFIX is co-expressed with the endopeptidase PACE/furin and is highly purified via multiple filtration and chromatographic steps.
[0218] The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. In one example, this term refers to a nucleic acid that is not in its native position in the genome. In another example, the nucleic acid is recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g. a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in na-ture (e.g. a fusion protein), or that it is a protein derived from a heterologous nucleic acid.
[0219] Any biologically active derivative of FIX may be modified thereby including any derivative of FIX having qualitatively the same functional and/or biological properties of FIX such as binding properties, and/or the same structural basis, such as a peptidic backbone. Minor deletions, additions and/or substitutions of amino acids of the polypeptide sequence of FIX which are not abolishing the biological activity of said polypeptide (i.e. reducing the activity to below 10% or even below 5% of the wild type form (=100%)) are also included in the present application as biologically active derivatives, especially those with improved specific activity (above 100% activity of the wild-type form). The FIX according to the present invention may be derived from any vertebrate, e.g. a mammal. In one specific example of the present invention, the FIX is human FIX. The FIX according to the present invention may be produced by any method known in the art. This may include any method known in the art for the production of recombinant DNA by genetic engineering, e.g. via reverse transcription of RNA and/or amplification of DNA. Additionally, the recombinant DNA coding for FIX, e.g. a plasmid, may also contain a DNA sequence encoding a selectable marker for selecting the cells which have been successfully transfected with the plasmid. In an example of the present invention, the plasmid may also confer resistance to a selectable marker, e.g. to the antibiotic drug G418, by delivering a resistance gene, e.g. the neo resistance gene conferring resistance to G418.
[0220] The production of rFIX may include any method known in the art for the introduction of recombinant DNA into eukaryotic cells by transfection, e.g. via electroporation or microinjection. For example, the recombinant expression of human FIX can be achieved by introducing an expression plasmid containing the human FIX encoding DNA sequence under the control of one or more regulating sequences such as a strong promoter, into a suitable host cell line by an appropriate transfection method resulting in cells having the introduced sequences stably integrated into the genome. The calcium-phosphate co-precipitation method is an example of a transfection method which may be used according to the present invention.
[0221] The term “amino acid” within the scope of the present invention is meant to include all naturally occurring L.alpha.-amino acids. The one and three letter abbreviations for naturally occurring amino acids are used herein (Lehninger, Biochemistry, 2d ed., Worth Publishers, New York, 1995: 71-92).
[0222] The term“coagulation factor II” (FII, prothrombin) as used herein refers to any form of factor II molecule with the typical characteristics of blood coagulation factor II. Blood coagulation factor II is a zymogen also known as prothrombin and is proteolytically cleaved to form the activated blood coagulation factor II (FIIa) also known as thrombin in the coagulation cascade, which ultimately results in the reduction of blood loss. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.
[0223] “Prothrombin complex concentrate” (PCC, trade names Beriplex®, Octaplex®, Kcentra®, Cofact®, among others) is a combination of blood coagulation factors II, VII, IX and X, as well as protein C and S, prepared from fresh-frozen human blood plasma. It is used to reverse the effects of oral anticoagulation therapy when bleeding occurs (e.g. in the brain or gut) requiring rapid action to accelerate coagulation. It is available as a powder and solvent for solution for injection.
[0224] “Coagulation factor X”, also known by the eponym Stuart-Prower factor or as prothrombinase, thrombokinase or thromboplastin, is an enzyme of the coagulation cascade. The term “coagulation factor X” (FX) as used herein shall be any form of factor X molecule with the typical characteristics of blood coagulation factor X. Factor X is synthesized in the liver and requires vitamin K for its synthesis. Factor X is activated into factor Xa by both factor IX (with its cofactor, factor VIII in a complex known as intrinsic Xase) and factor VII with its cofactor, tissue factor (a complex known as extrinsic Xase). It acts by cleaving prothrombin in two places (an arg-thr and then an arg-ile bond), which yields the active thrombin. This process is optimized when factor Xa is complexed with activated co-factor V in the prothrombinase complex. Factor Xa is inactivated by protein Z-dependent protease inhibitor (ZPI), a serine protease inhibitor (serpin). The affinity of this protein for factor Xa is increased 1000-fold by the presence of protein Z, while it does not require protein Z for inactivation of factor XI. Defects in protein Z lead to increased factor Xa activity and a propensity for thrombosis. The half life of factor X is 40-45 hours. Factor X is part of fresh frozen plasma and Prothrombin complex and Prothrombin complex concentrates. A commercially available concentrate is Factor X P Behring′ manufactured by CSL Behring. Bio Products Laboratory has a high purity Factor X currently in development.
[0225] Factor VII (blood-coagulation factor VII) is one of the proteins that causes blood to clot in the coagulation cascade. It is an enzyme of the serine protease class. The term “coagulation factor VII” (FVII) as used herein shall be any form of factor VII molecule with the typical characteristics of blood coagulation factor VII. A recombinant form of its activated form human factor VIIa (eptacog alfa [activated], NovoSeven) is approved for the treatment of uncontrolled bleeding in hemophilia patients. There have been safety concerns when used in severe uncontrollable bleeding.
[0226] The main role of factor VII (FVII) is to initiate the process of coagulation in conjunction with tissue factor (TF/coagulation factor III/FIII). Tissue factor is found on the outside of blood vessels—normally not exposed to the bloodstream. Upon vessel injury, tissue factor is exposed to the blood and circulating factor VII. Once bound to TF, FVII is activated to FVIIa by different proteases, among which are thrombin (factor IIa), factor Xa, IXa, XIIa, and the FVIIa-TF complex itself The complex of factor VIIa with TF catalyzes the conversion of factor IX and factor X into the active proteases, factor IXa and factor Xa, respectively (Wajima T, et al, Clin Pharmacol Ther 86 (2009). 290-8). The action of the factor is impeded by tissue factor pathway inhibitor (TFPI), which is released almost immediately after initiation of coagulation. Factor VII is vitamin K dependent; it is produced in the liver. Use of warfarin or similar anticoagulants decreases hepatic synthesis of FVII.
[0227] The terms “bind to”, “recognize”, “specifically bind to” or “anti-” as used herein are interchangeable and refer to refer to multispecific antibody or its antigen binding site that is capable of binding the respective antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting. Preferably the multispecific antibody as described herein is bispecific and binds to FIX and/or FIXa (activated form of FIX) as the first antigen, and to FX as the second antigen, respectively In one embodiment, the extent of binding of an anti-Bsab FIX/FX, antibody to an unrelated, non-FIX, non-FIXa, non-FX protein is less than about 10% of the binding of the antibody to FIX, FIXa, FX, respectively, as measured, e.g., by a radioimmunoassay (RIA).
[0228] The term “antigen-binding site” as used herein denotes the region(s) of an antibody molecule to which a ligand actually binds. The term “antigen-binding site” include antibody heavy chain variable domains (VH) and/or an antibody light chain variable domains (VL), or pairs of VH/VL, and can be derived from whole antibodies or antibody fragments such as single chain Fv, a VH domain and/or a VL domain, Fab, or (Fab)2. In one embodiment of the current invention each of the antigen-binding sites comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), and preferably is formed by a pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH), wherein antibody light chain variable domain (VL) is preferably part of a commonly shared L chain.
[0229] The term “wherein the treatment is in combination with a coagulation factor IX” refers to the combined treatment of the relevant disorder with a) a multispecific antibody which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, and b) a coagulation factor IX. The combined treatment can be simultaneous or sequential wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Said multispecific antibody and FIX are co-administered either simultaneously or sequentially (e.g. via an intravenous (i.v.) through a continuous infusion).
[0230] Multispecific antigen-binding molecules described herein comprise a first antigen-binding site and a second antigen-binding site that can specifically bind to at least two different types of antigens. While the first antigen-binding site and the second antigen-binding site are not particularly limited as long as they bind to FIX and/or FIXa, and FX, respectively, examples include sites necessary for binding with antigens, such as antibodies, scaffold molecules (antibody-like molecules) or peptides, or fragments containing such sites. Scaffold molecules are molecules that exhibit function by binding to target molecules, and any polypeptide may be used as long as they are conformationally stable polypeptides that can bind to at least one target antigen. Examples of such polypeptides include antibody variable regions, fibronectin (WO 2002/032925), protein A domain (WO 1995/001937), LDL receptor A domain (WO 2004/044011, WO 2005/040229), ankyrin (WO 2002/020565), and such, and also molecules described in documents by Nygren et al. (Current Opinion in Structural Biology, 7: 463-469 (1997); and Journal of Immunol Methods, 290: 3-28 (2004)), Binz et al. (Nature Biotech 23: 1257-1266 (2005)), and Hosse et al. (Protein Science 15: 14-27(2006)). Furthermore, as mentioned in Curr Opin Mol Ther. 2010 August; 12(4): 487-95 and Drugs. 2008; 68(7): 901-12, peptide molecules that can bind to target antigens may be used.
[0231] Herein, multispecific antigen-binding molecules are not particularly limited as long as they are molecules that can bind to at least two different types of antigens, but examples include polypeptides containing the above-mentioned antigen-binding sites, such as antibodies and scaffold molecules as well as their fragments, and aptamers comprising nucleic acid molecules and peptides, and they may be single molecules or multimers thereof. Preferred multispecific antigen-binding molecules include multispecific antibodies that can bind specifically to at least two different antigens. Particularly preferred examples of antibodies which have an activity of functionally substituting for FVIII of the present invention include bispecific antibodies (BsAb) that can bind specifically to two different antigens (they may also be called dual specific antibodies).
[0232] In the present invention, the term “commonly shared L chain” refers to an L chain (light chain) of an antibody that can link with two or more different H chains (heavy chains) of antibody, and show binding ability to each antigen. Herein, the term “different H chain(s)” preferably refers to H chains of antibodies against different antigens, but is not limited thereto, and also refers to H chains whose amino acid sequences are different from each other. Commonly shared L chain can be obtained, for example, according to the method described in WO 2006/109592.
[0233] The multispecific antigen-binding molecules of the present invention (preferably bispecific antibodies) are antibodies having specificity to two or more different antigens, or molecules comprising fragments of such antibodies. The antibodies of the present invention are not particularly limited, but are preferably 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 modified gene recombinant antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies.
[0234] Furthermore, the L chains of an antibody which will become a multispecific antigen-binding molecule of the present invention may be different, but preferably have commonly shared L chains.
[0235] Multispecific antigen-binding molecules of the present invention are preferably recombinant antibodies produced using genetic recombination techniques (See, for example, Borrebaeck CAK and Larrick J W, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies, can be obtained by cloning DNAs encoding antibodies from hybridomas or antibody-producing cells, such as sensitized lymphocytes, that produce antibodies, inserting them into suitable vectors, and then introducing them into hosts (host cells) to produce the antibodies.
[0236] Furthermore, antibodies of the present invention may include not only whole antibodies but also antibody fragments and low-molecular-weight antibodies (minibodies), and modified antibodies.
[0237] The multispecific antigen-binding molecules of the present invention are those that recognize FIX and/or FIXa, and FX, and functionally substitute for cofactor function of FVIII, and characterized in that the molecules have a higher FXa generation-promoting activity compared to hA69-KQ/hB26-PF/hAL-AQ (described in WO 2006/109592) which is known as a bispecific antibody that functionally substitutes for FVIII. Furthermore, antibodies of the present invention usually have a structure which comprises a variable region of an anti-FIXa antibody and a variable region of an anti-FX antibody.
[0238] More specifically, the present invention provides a multispecific antigen-binding molecule that functionally substitutes for FVIII, which comprises a first antigen-binding site that recognizes FIX and/or FIXa and a second antigen-binding site that recognizes FX, wherein the function that substitutes for the function of FVIII is caused by a higher FXa generation-promoting activity compared to the activity of the bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises H chains consisting of SEQ ID NOs: 165 and 166, and a commonly shared L chain consisting of SEQ ID NO: 167.
[0239] A multispecific antigen-binding molecule of the present invention comprises a first polypeptide and a third polypeptide comprising an antigen-binding site that recognizes FIX and/or FIXa, and a second polypeptide and a fourth polypeptide comprising an antigen-binding site that recognizes FX. The first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide each include the antigen-binding site of the antibody H chain and the antigen-binding site of the antibody L chain.
[0240] For example, in a multispecific antigen-binding molecule of the present invention, the first polypeptide and the third polypeptide include an antigen-binding site of an H chain and L chain of an antibody against FIX or FIXa, respectively; and the second polypeptide and the fourth polypeptide comprise an antigen-binding site of an H chain and L chain of an antibody against FX, respectively.
[0241] At this time, the antigen-binding sites of the antibody L chain included in the first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide may be commonly shared L chains.
[0242] A polypeptide comprising an antigen-binding site of an antibody L chain in the present invention is preferably a polypeptide which comprises all or a part of the sequence of the antibody L chain which binds to FIX, FIXa and/or FX.
[0243] In the present invention, the phrase “functionally substitute for FVIII” means that FIX and/or FIXa, and FX is recognized, and activation of FX is promoted (FXa generation is promoted).
[0244] In the present invention, “FXa generation-promoting activity” can be confirmed by evaluating the multispecific antigen-binding molecules of the present invention using, for example, a measurement system comprising FXIa (FIX activating enzyme), FIX, FX, F synthetic substrate S-2222 (synthetic substrate of FXa), and phospholipids. This measurement system shows the correlation between the severity of the disease and clinical symptoms in hemophilia A cases (Rosen S, Andersson M, Blomback M et al. Clinical applications of a chromogenic substrate method for determination of FVIII activity. Thromb Haemost 1985, 54: 811-23). That is, in the present measurement system, test substances that show higher FXa generation-promoting activity are expected to show better hemostatic effects against bleeding episodes in hemophilia A. With these results, if a multispecific antigen-binding molecule having activity of functionally substituting for FVIII is a molecule having a higher activity than hA69-KQ/hB26-PF/hAL-AQ, it may yield excellent blood coagulation-promoting activity, and excellent effects may be obtained as a pharmaceutical component for preventing and/or treating bleeding, a disease accompanying bleeding, or a disease caused by bleeding. To obtain excellent effects as the above-mentioned pharmaceutical component, for example, FXa generation-promoting activity measured under the conditions described in Example 2 of US 2013/0330345 is preferably not less than that of hA69-KQ/hB26-PF/hAL-AQ, and in particular, the activity is more preferably the same as or not less than that of Q153-G4k/J142-G4h/L180-k. Herein, the “FXa generation-promoting activity” is the value obtained by subtracting the change in absorbance upon 20 minutes in a solvent from the change in absorbance upon 20 minutes in an antibody solution (see also US 2013/0330345).
[0245] In one embodiment of antibodies, combination or use of the present invention, since the antibodies used in the present invention functionally substitute for factor FVIII, they are expected to become effective pharmaceutical agents against diseases resulting from decrease in activity (function) of this cofactor. Examples of the above-mentioned diseases include bleeding, diseases accompanying bleeding, or a disease caused by bleeding. In particular, there may have excellent therapeutic effects on hemophilias, in which bleeding disorders are caused by a deficiency or decrease of FVIII/FVIIIa function. Among the hemophilias, they are expected to become excellent therapeutic agents for hemophilia A, in which bleeding disorders are caused by a hereditary deficiency or decrease of FVIII/FVIIIa function.
[0246] In the context of the present invention, bleeding, diseases accompanying bleeding, and/or diseases caused by bleeding preferably refer to diseases that develop and/or progress due to reduction or deficiency in activity of FVIII and/or activated coagulation factor VIII (F.VIIIa). Such diseases include the above-described hemophilia A, diseases in which an inhibitor against FVIII/FVIIIa appear, acquired hemophilia, von Willebrand's disease, and such, but are not particularly limited thereto.
In the following embodiments of the invention are listed. [0247] 1. A multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for use in the treatment of hemophilia A, wherein the antibody is used in combination with a (non-activated) IX coagulation factor IX. [0248] 2. A (non-activated) coagulation factor IX for use in the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0249] 3. A multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the antibody is used in combination with a (non-activated) coagulation factor IX. [0250] 4. A (non-activated) coagulation factor IX for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0251] 5. A combination of [0252] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, and [0253] ii) a (non-activated) coagulation factor IX, for the use in the treatment of hemophilia A. [0254] 6. A combination of [0255] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, and [0256] ii) a (non-activated) coagulation factor IX for the use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII. [0257] 7. Use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for the manufacture of a medicament for the treatment of hemophilia A, wherein the treatment is in combination with a (non-activated) coagulation factor IX. [0258] 8. Use of a (non-activated) coagulation factor IX for the manufacture of a medicament for the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0259] 9. Use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the treatment is in combination with a (non-activated) coagulation factor IX. [0260] 10. Use of (non-activated) coagulation factor IX for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0261] 11. The combination, antibody or use according to embodiments 3, 4, 6, 9 or 10, wherein the patient suffers from a congenital or acquired deficiency of coagulation factor VIII. [0262] 12. The combination, antibody or use according to embodiment 11, wherein the deficiency is acquired by antibodies, other inhibitors, consumption or dilution. [0263] 13. The combination, antibody or use according to any one of the preceding embodiments, [0264] a) for use in increasing the thrombin generation; [0265] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0266] c) for use accelerating of the thrombin generation/formation; [0267] d) for use in increasing and accelerating the thrombin generation/formation; [0268] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0269] f) for use in enhancing blood coagulation; [0270] g) for use in enhancing fibrin clot formation; and/or [0271] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like. [0272] 14. The combination, antibody or use according to any one of the preceding embodiments [0273] a) wherein there exists an increased bleeding risk, [0274] b) during surgery or other invasive procedures, and/or [0275] c) after vascular injury. [0276] 15. The combination, antibody or use according to any one of the preceding embodiments, wherein in addition a) a coagulation factor II or b) a coagulation factor X, c) coagulation factors II and X; or d) coagulation factors II, X and VII is used in the combination. [0277] 16. The combination, antibody or use according to any one of the preceding embodiments, wherein coagulation factor IX is comprised in a prothrombin complex concentrates (PCC). [0278] 17. The combination, antibody or use according to embodiment 16, wherein the prothrombin complex concentrates comprises FIX, FII, and FX. [0279] 18. The combination, antibody or use according to embodiment 16, wherein the prothrombin complex concentrates comprises FIX, FII, FX and FVII. [0280] 19. The combination, antibody or use according to any one of the preceding embodiments, wherein the antibody is bispecific and the first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX comprises a H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H chain CDRs of Q499)), respectively, and a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively. and the second antigen-binding site of comprises an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively. [0281] 20. The combination, antibody or use according to any one of the preceding embodiments, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0282] 21. The combination, antibody or use according to any one of embodiments 1 to 18, wherein the antibody comprises a first polypeptide comprising a first antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX and a third polypeptide comprising a third antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX, as well as a second polypeptide comprising a second antigen-binding site that binds to blood coagulation factor X and a fourth polypeptide comprising a fourth antigen-binding site that binds to blood coagulation factor X. [0283] 22. The combination, antibody or use according to embodiment 21, wherein the first polypeptide and the third polypeptide each comprises an antigen-binding site of an H chain or L chain of an antibody against blood coagulation factor IX or activated blood coagulation factor IX, respectively; and the second polypeptide and the fourth polypeptide each comprises an antigen-binding site of an H chain or L chain of an antibody against blood coagulation factor X, respectively. [0284] 23. The combination, antibody or use according to any one of embodiments 21 to 22, wherein the antigen-binding site of the first polypeptide comprises an antigen-binding site which comprises H chain CDRs consisting of any one of the amino acid sequences selected from the following (a1) to (a11), or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site which comprises H chain CDRs consisting of any one of the amino acid sequences selected from the following (b1) to (b11), or an antigen-binding site functionally equivalent thereto: (a1) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain CDRs of Q1), respectively; (a2) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 78, 79, and 80 (H chain CDRs of Q31), respectively; (a3) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain CDRs of Q64), respectively; (a4) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 84, 85, and 86 (H chain CDRs of Q85), respectively; (a5) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 87, 88, and 89 (H chain CDRs of Q153), respectively; (a6) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 90, 91, and 92 (H chain CDRs of Q354), respectively; (a7) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 93, 94, and 95 (H chain CDRs of Q360), respectively; (a8) an antigen-binding site comprising the of H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 96, 97, and 98 (H chain CDRs of Q405), respectively; (a9) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain CDRs of Q458), respectively; (a10) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 102, 103, and 104 (H chain CDRs of Q460), respectively; (a11) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H chain CDRs of Q499), respectively; (b1) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 108, 109, and 110 (H chain CDRs of J232), respectively; (b2) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H chain CDRs of J259), respectively; (b3) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 114, 115, and 116 (H chain CDRs of J268), respectively; (b4) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H chain CDRs of J300), respectively; (b5) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 120, 121, and 122 (H chain CDRs of J321), respectively; (b6) an antigen-binding site comprising the H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H chain CDRs of J326), respectively; (b7) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively; (b8) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H chain CDRs of J339), respectively; (b9) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 132, 133, and 134 (H chain CDRs of J344), respectively; (b10) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H chain CDRs of J346), respectively; and (b11) an antigen-binding site comprising an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 174, 175, and 176 (H chain CDRs of J142), respectively. [0285] 24. The combination, antibody or use according to any one of embodiments 21 to 23, wherein the antigen-binding site of the first polypeptide comprises an antigen-binding site which comprises an H chain variable region consisting of any one of the amino acid sequences selected from the following (a1) to (a11), or an antigen-binding site functionally equivalent thereto, and the antigen-binding site of the second polypeptide comprises an antigen-binding site which comprises an H chain variable region consisting of any one of the amino acid sequences selected from the following (b1) to (b11), or an antigen-binding site functionally equivalent thereto: (a1) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 35 (H chain variable region of Q1); (a2) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 36 (H chain variable region of Q31); (a3) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 37 (H chain variable region of Q1); (a4) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 38 (H chain variable region of Q85); (a5) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 39 (H chain variable region of Q153); (a6) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 40 (H chain variable region of Q354); (a7) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 41 (H chain variable region of Q360); (a8) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 42 (H chain variable region of Q405); (a9) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 43 (H chain variable region of Q458); (a10) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 44 (H chain variable region of Q460); (a11) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 45 (H chain variable region of Q499); (b1) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 46 (H chain variable region of J232); (b2) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 47 (H chain variable region of J259); (b3) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 48 (H chain variable region of J268); (b4) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 49 (H chain variable region of J300); (b5) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 50 (H chain variable region of J321); (b6) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 51 (H chain variable region of J326); (b7) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 52 (H chain variable region of J327); (b8) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 53 (H chain variable region of J339); (b9) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 54 (H chain variable region of J344); (b10) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 55 (H chain variable region of J346); and (b11) an antigen-binding site comprising an H chain variable region amino acid sequence of SEQ ID NO: 172 (H chain variable region of J142). [0286] 25. The combination, antibody or use according to any one of embodiments 21 to 24, wherein the antigen-binding sites included in the third polypeptide and the fourth polypeptide comprise an antigen-binding site which comprises L chain CDRs consisting of any one of the amino acid sequences selected from the following (c1 to (c10), or an antigen-binding site functionally equivalent thereto: (c1) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 138, 139, and 140 (L chain CDR of L2), respectively; (c2) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain CDR of L45), respectively; (c3) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 144, 145, and 146 (L chain CDR of L248), respectively; (c4) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain CDR of L324), respectively; (c5) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 150, 151, and 152 (L chain CDR of L3 34), respectively; (c6) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain CDR of L377), respectively; (c7) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively; (c8) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain CDR of L406), respectively; (c9) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 137, 138, and 139 (L chain CDR of L408), respectively; and (c10) an antigen-binding site comprising an L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain CDR of L180), respectively. [0287] 26. The combination, antibody or use according to any one of embodiments 21 to 25, wherein the antigen-binding sites included in the third polypeptide and the fourth polypeptide comprise an antigen-binding site which comprises an L chain variable region consisting of any one of the amino acid sequences selected from the following (c1) to (c10), or an antigen-binding site functionally equivalent thereto: (c1) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 56 (L chain variable region of L2); (c2) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 57 (L chain variable region of L45); (c3) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 58 (L chain variable region of L248); (c4) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 59 (L chain variable region of L324); (c5) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 60 (L chain variable region of L334); (c6) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 61 (L chain variable region of L377); (c7) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 62 (L chain variable region of L404); (c8) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 63 (L chain variable region of L406); (c9) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 64 (L chain variable region of L408); and (c10) an antigen-binding site comprising an L chain variable region amino acid sequence of SEQ ID NO: 173 (L chain variable region of L180). [0288] 27. The combination, antibody or use according to any one of embodiments 21 to 26, wherein the first and second polypeptides further comprise an antibody H chain constant region, and the third and fourth polypeptides comprise an antibody L chain constant region. [0289] 28. The combination, antibody or use according to any one of embodiments 21 to 26, wherein the first and second polypeptides comprise an antibody H chain constant region, and the third and fourth polypeptides comprise an antibody L chain constant region, and wherein the third polypeptide and the fourth polypeptide are a commonly shared L chain. [0290] 29. The combination, antibody or use according to any one of embodiments 21 to 28, wherein the first polypeptide comprises any one antibody H chain selected from the following (a1) to (a14), the second polypeptide comprises any one antibody H chain selected from the following (b1) to (b12), and the third polypeptide and the fourth polypeptide comprise any one antibody L chain selected from the following (c1) to (c10): (a1) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 1 (Q1-G4k); (a2) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 2 (Q31-z7); (a3) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 3 (Q64-z55); (a4) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 10 (Q64-z7); (a5) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 11 (Q85-G4k); (a6) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 12 (Q153-G4k); (a7) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 13 (Q354-z106); (a8) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 14 (Q360-G4k); (a9) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 15 (Q360-z118); (a10) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 16 (Q405-G4k); (a11) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 17 (Q458-z106); (a12) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 18 (Q460-z121); (a13) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 19 (Q499-z118); (a14) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 20 (Q499-z121); (b1) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 4 (J268-G4h); (b2) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 5 (J321-G4h); (b3) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 6 (J326-z107); (b4) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 7 (J344-z107); (b5) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 21 (J232-G4h); (b6) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 22 (J259-z107); (b7) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 23 (J300-z107); (b8) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 24 (J327-z107); (b9) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 25 (J327-z119); (b10) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 26 (J339-z119); (b11) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 27 (J346-z107); (b12) an antibody H chain consisting of the amino acid sequence of SEQ ID NO: 170 (J142-G4h); (c1) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 8 (L2-k); (c2) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 9 (L45-k); (c3) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 28 (L248-k); (c4) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 29 (L324-k); (c5) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 30 (L334-k); (c6) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 31 (L377-k); (c7) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 32 (L404-k); (c8) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 33 (L406-k); (c9) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 34 (L408-k); and (c10) an antibody L chain consisting of the amino acid sequence of SEQ ID NO: 171 (L180-k). [0291] 30. The combination, antibody or use according to embodiment 28, wherein the antibody is a bispecific antibody of any one of the following (a) to (u): (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 9; (b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 9; (c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 8; (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the first polypeptide is an H chain consisting of the amino acid sequence of SEQ ID NO: 3, 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 are a commonly shared L chain of SEQ ID NO: 9; (e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 29; (k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 33; (1) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 28; (n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 34; (o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein 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 a commonly shared L chain of SEQ ID NO: 30; (p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 30; (q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 31; (r) a bispecific antibody (Q499-z118/J346-z107/L248-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 28; (s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 32; (t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 31; and (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein 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 are a commonly shared L chain of SEQ ID NO: 171. [0292] 31. The combination, antibody or use according to any one or the preceding embodiments, wherein the FIX is administered in an amount of 10 U-200 U FIX/kg body weight in a patient with hemophilia A treated with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0293] 32. The combination, antibody or use according to embodiment 31, wherein additionally the FX is administered in an amount of 10 U-200 U FX/kg body weight. [0294] 33. The combination, antibody or use according to embodiment 31, wherein additionally the FII is administered in an amount of 10 U-200 U FII/kg body weight. [0295] 34. The combination, antibody or use according to embodiment 31, wherein additionally the FII and FX are administered in an amount of 10 U-200 U FII/kg body weight and 10 U-200 U FX/kg body weight. [0296] 35. The combination, antibody or use according to any one or the preceding embodiments, wherein prothrombin complex (PCC) is administered in amount of 10 U-200 U PCC/kg body weight in a patient with hemophilia A treated with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X. [0297] 36. The combination, antibody or use according to any one of the preceding embodiments, wherein said multispecific antibody and FIX are co-administered simultaneously. [0298] 37. The combination, antibody or use according to any one of the preceding embodiments, wherein said multispecific antibody and FIX are co-administered sequentially. [0299] 38. A method of treating a patient suffering from hemophilia A, the method comprising administering effective amounts of a) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, an b) a coagulation factor IX, to the patient in need of such treatment. [0300] 39. A method of treating a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, the method comprising administering effective amounts of a) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, an b) a coagulation factor IX, to the patient in need of such treatment. [0301] 40. The method according to embodiment 39, wherein the patient suffers from a congenital or acquired deficiency of coagulation factor VIII. [0302] 41. The method according to embodiment 40, wherein the deficiency is acquired by antibodies, other inhibitors, consumption or dilution. [0303] 42. A method of [0304] a) in increasing the thrombin generation; [0305] b) in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0306] c) accelerating of the thrombin generation/formation; [0307] d) in increasing and accelerating the thrombin generation/formation; [0308] e) in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0309] f) in enhancing blood coagulation; [0310] g) in enhancing fibrin clot formation; and/or [0311] h) preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like, [0312] in a patient suffering from bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like, (e.g. suffering from a a) deficiency or b) malfunction of coagulation factor VIII, e.g. from hemophilia A), [0313] the method comprising administering effective amounts of a) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, an b) a (non-activated) coagulation factor IX, to the patient. [0314] 43. The method according to any one of the preceding embodiments [0315] a) wherein there exists an increased bleeding risk, [0316] b) during surgery or other invasive procedures, and/or [0317] c) after vascular injury. [0318] 44. The method according to any one of the preceding embodiments, wherein in addition a) a coagulation factor II or b) a coagulation factor X, c) coagulation factors II and X; or d) coagulation factors II, X and VII are used in the combination. [0319] 45. The method according to any one of the preceding embodiments, wherein coagulation factors IX is comprised in a prothrombin complex concentrates (PCC). [0320] 46. The method according to embodiment 45, wherein the prothrombin complex concentrates comprises FIX, FII, and FX. [0321] 47. The method according to embodiment 45, wherein the prothrombin complex concentrates comprises FIX, FII, FX and FVII. [0322] 48. The method according to any one of the preceding embodiments, wherein the antibody is bispecific and the first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX comprises a H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H chain CDRs of Q499)), respectively, and a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively. and the second antigen-binding site of comprises an H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain CDRs of J327), respectively a L chain CDR1, 2, and 3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain CDR of L404), respectively. [0323] 49. The method according to any one of the preceding embodiments, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0324] 50. The method according to any one of the preceding embodiments, wherein said multispecific antibody and FIX are co-administered simultaneously. [0325] 51. The method according to any one of the preceding embodiments, wherein said multispecific antibody and FIX are co-administered sequentially.
In the following some preferred embodiments of the invention are listed: [0326] 1. A multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, for use in the treatment of hemophilia A, wherein the antibody is used in combination with a (non-activated) coagulation factor IX. [0327] 2. A (non-activated) coagulation factor IX for use in the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0328] 3. A multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the antibody is used in combination with a (non-activated) coagulation factor IX. [0329] 4. A (non-activated) coagulation factor IX for use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0330] 5. A combination of [0331] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, and [0332] ii) a (non-activated) coagulation factor IX, [0333] for the use in the treatment of hemophilia A. [0334] 6. A combination of [0335] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, and [0336] ii) a (non-activated) coagulation factor IX [0337] for the use in the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII. [0338] 7. Use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, for the manufacture of a medicament for the treatment of hemophilia A, [0339] wherein the treatment is in combination with a coagulation factor IX. [0340] 8. Use of a (non-activated) coagulation factor IX for the manufacture of a medicament for the treatment of hemophilia A, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0341] 9. Use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the treatment is in combination with a (non-activated) coagulation factor IX. [0342] 10. Use of a (non-activated) coagulation factor IX for the manufacture of a medicament for the treatment of a patient suffering from a a) deficiency or b) malfunction of coagulation factor VIII, wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0343] 11. A multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, [0344] a) for use in increasing the thrombin generation; [0345] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0346] c) for use in accelerating of the thrombin generation/formation; [0347] d) for use in increasing and accelerating the thrombin generation/formation; [0348] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0349] f) for use in enhancing blood coagulation; [0350] g) for use in enhancing fibrin clot formation; and/or [0351] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like; [0352] wherein the antibody is used in combination with a (non-activated) coagulation factor IX. [0353] 12. A (non-activated) coagulation factor IX [0354] a) for use in increasing the thrombin generation; [0355] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0356] c) for use in accelerating of the thrombin generation/formation; [0357] d) for use in increasing and accelerating the thrombin generation/formation; [0358] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0359] f) for use in enhancing blood coagulation; [0360] g) for use in enhancing fibrin clot formation; and/or [0361] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like; [0362] wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0363] 13. A combination of [0364] i) a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, and [0365] ii) a (non-activated) coagulation factor IX, [0366] a) for use in increasing the thrombin generation; [0367] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0368] c) for use in accelerating of the thrombin generation/formation; [0369] d) for use in increasing and accelerating the thrombin generation/formation; [0370] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0371] f) for use in enhancing blood coagulation; [0372] g) for use in enhancing fibrin clot formation; and/or [0373] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like. [0374] 14. Use of a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32, [0375] for the manufacture of a medicament [0376] a) for use in increasing the thrombin generation; [0377] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0378] c) for use in accelerating of the thrombin generation/formation; [0379] d) for use in increasing and accelerating the thrombin generation/formation; [0380] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0381] f) for use in enhancing blood coagulation; [0382] g) for use in enhancing fibrin clot formation; and/or [0383] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like;
[0384] wherein the medicament is used in combination with a (non-activated) coagulation factor IX.
[0385] 15. Use of a (non-activated) coagulation factor IX for the manufacture of a medicament [0386] a) for use in increasing the thrombin generation; [0387] b) for use in increasing the thrombin generation at the site of vascular injury/at the site of tissue factor release; [0388] c) for use in accelerating of the thrombin generation/formation; [0389] d) for use in increasing and accelerating the thrombin generation/formation; [0390] e) for use in accelerating the thrombin generation/formation at the site of vascular injury/at the site of tissue factor release; [0391] f) for use in enhancing blood coagulation; [0392] g) for use in enhancing fibrin clot formation; and/or [0393] h) for preventing and/or treating bleeding, diseases accompanying bleeding, diseases caused by bleeding, and the like; [0394] wherein the coagulation factor IX is used in combination with a multispecific antibody (that functionally substitutes for blood coagulation factor VIII) which comprises a first antigen-binding site that binds to coagulation factor IX and/or activated coagulation factor IX and a second antigen-binding site that binds to coagulation factor X, wherein the antibody is a bispecific antibody (Q499-z121/J327-z119/L404-k), comprising a) a H chain consisting of the amino acid sequence of SEQ ID NO: 20, b) a H chain consisting of the amino acid sequence of SEQ ID NO: 25, and c) a (commonly shared) L chain of consisting of the amino acid sequence of ID NO: 32. [0395] 16. The combination, antibody or use according to any one of embodiments 11 to 15 for use in increasing the thrombin generation; [0396] 17. The combination, antibody or use according to any one of embodiments 11 to 15 for use in accelerating of the thrombin generation. [0397] 18. The combination, antibody or use according to any one of embodiments 1 to 17, wherein said multispecific antibody and FIX are co-administered simultaneously. [0398] 19. The combination, antibody or use according to any one of embodiments 1 to 17, wherein said multispecific antibody and FIX are co-administered sequentially.
[0399] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Experimental Procedures:
Example 1
Thrombin Generation in FVIII Deficient Plasma
[0400] Plasma samples which were deficient in FVIII (FVIII deficient plasma, Siemens), (however containing normal levels of non activated FIX, FX and FII) were used as a model of a deficiency or malfunction of coagulation factor VIII (especially as model a hemophilia A patient samples).
[0401] The bispecific antibody that binds to factor IX and binds to factor X (Q499-z121/J327-z119/L404-k) as described in US 2013/0330345 (comprising the amino acid sequences of sequences SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 32) and herein below abbreviated as Bsab FIX/FX, was spiked into the plasma samples in concentrations of 25, 50, 75 or 100 μg/ml resembling clinically applied concentrations of Bsab FIX/FX. The Bsab FIX/FX (Q499-z121/J327-z119/L404-k) is described in detail in US 2013/0330345 and comprises a first polypeptide comprising a first antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX and a third polypeptide comprising a third antigen-binding site that binds to blood coagulation factor IX and/or activated blood coagulation factor IX, as well as a second polypeptide comprising a second antigen-binding site that binds to blood coagulation factor X and a fourth polypeptide comprising a fourth antigen-binding site that binds to blood coagulation factor X wherein the first polypeptide is an H chain comprising the amino acid sequence of SEQ ID NO: 20, the second polypeptide is an H chain comprising the amino acid sequence of SEQ ID NO: 25, and the third polypeptide and the fourth polypeptide are a commonly shared L chain comprising the amino acid sequence of SEQ ID NO: 32.
[0402] In addition recombinant FIX (Benefix®) ((non-activated) FIX) was added in vitro in part of the experiments. Alternately a commercially available prothrombin complex concentrate (PCC) comprising FIX (in non-activated form) was added in vitro in a concentration of 1 U/ml (Octaplex®). Octaplex is a pooled plasma coagulation factor concentrate containing coagulation factors II (220-760 IU), VII (180-480 IU), IX (500 IU) and X (360-600 IU). It also contains protein C, protein S, albumin, heparin and sodium citrate. As PCCs contain small amounts of heparin, which may interfere with in vitro evaluation of their activity, the PCC was incubated for 15 minutes in Heparinase solution (Hepzyme, Siemens), an enzyme, which degrades heparin in vitro.
[0403] As a control recombinant FVIII (Advate, Baxter) was added to the FVIII deficient plasma in a concentration of 100%.
[0404] The dilutions of Bsab factor IX/factor X were prepared in 4% gelatin solution (Gelafusal).
TABLE-US-00001 TABLE 1 tested combinations of substances. FVIII dp FVIII dp +100% FVIII FVIII dp +100 μg/ml Bsab FIX/FX FVIII dp +75 μg/ml Bsab FIX/FX FVIII dp +50 μg/ml Bsab FIX/FX FVIII dp +25 μg/ml Bsab FIX/FX FVIII dp +100% FVIII +100% FIX FVIII dp +100 μg/ml Bsab FIX/FX +100% FIX FVIII dp +75 μg/ml Bsab FIX/FX +100% FIX FVIII dp +50 μg/ml Bsab FIX/FX +100% FIX FVIII dp +25 μg/ml Bsab FIX/FX +100% FIX FVIII dp +100% FVIII +1U PCC/ml FVIII dp +100 μg/ml Bsab FIX/FX +1U PCC/ml FVIII dp +75 μg/ml Bsab FIX/FX +1U PCC/ml FVIII dp +25 μg/ml Bsab FIX/FX +1U PCC/ml FVIII dp = FVIII deficient plasma
[0405] Thrombin generation was continuously determined by means of a fluorogenic substrate following the activation of coagulation with a small amount of tissue factor (“PPP low reagent”, instrument and all reagents by Thrombinoscope, Netherlands).
Description of Thrombin Generation Method:
[0406] Thrombin generation can be measured in biological plasma using the Calibrated Automated Thrombogram (CAT) method from Thrombinoscope BV, Maastricht, The Netherlands. In brief, thrombin generation is triggered through the extrinsic pathway of coagulation by addition of 1 pM tissue factor (TF), phospholipids and calcium ions (Ca2+). A low affinity fluorogenic substrate is added for the realtime analysis of thrombin generation. Plasma samples are calibrated against known thrombin calibrator in order to correct for the substrate depletion, sample color and inner filter effect. The fluorescence is read with a Thermo Fluoroskan. From the fluorescence signal measured the thrombin activity is calculated. The curves expressed show the free thrombin activity (y-axis, in nM thrombin) over time (x-axis, in sec).
Control Measurements: FVIII Deficient Plasma and FVIII Deficient Plasma+100% FVIII:
[0407] The analysis of FVIII deficient plasma alone revealed as expected a very weak thrombin generation. This shows the physiological reason of the bleeding disorder in hemophilia A patients.
[0408] In
Addition of Bsab FIX/FX:
[0409] Also the addition of Bsab FIX/FX leads to a significant increase of the thrombin generation to a 3.4 fold-4.4 fold thrombin generation of the sample lacking FVIII. The results are shown in
Comparison of the Activity of Bsab FIX/FX with the Activity of FVIII on the Thrombin Generation of a Plasma Sample Lacking FVIII:
[0410] Comparing the thrombin generation of samples with the supplementation of FVIII or Bsab FIX/FX, significantly more thrombin was formed using FVIII, and also that the time to peak thrombin generation was shorter with FVIII compared to Bsab FIX/FX. ((see
Addition of FIX to Bsab FIX/FX Treated FVIII Deficient Plasma:
[0411] The addition of FIX (100%) to FVIII deficient plasma treated with Bsab FIX/FX leads to a significant increase of thrombin generation (see
[0412] In the sample with 75 μg Bsab FIX/FX/ml this resulted to a doubling of the thrombin generation. In the sample with 50 μs Bsab FIX/FX/ml a 75% increase of thrombin generation was determined. In the sample with 25 μg Bsab FIX/FX/ml a 50% increase of thrombin generation was found (see Figure . . . . And.
[0413] In addition the time to peak was significantly shortened by the addition of FIX, i.e. thrombin generation was not only increased, but the thrombin generation was also accelerated.
[0414] As seen in
Addition of Prothrombin Complex Concentrate (PCC) to Bsab FIX/FX Treated FVIII Deficient Plasma:
[0415] The addition of PCC (1 U/ml) to FVIII deficient plasma treated with Bsab FIX/FX lead to a significant increase of thrombin generation.
[0416] In the sample with 75 μg Bsab FIX/FX/ml this resulted to a 94% increase of thrombin generation. In the sample with 25 μg RO5534262/ml a 90% increase of thrombin generation was found. In both cases the peak thrombin generation was similar for the Bsab FIX/FX treated samples with the addition of PCC compared to the FVIII supplemented sample. (see
TABLE-US-00002 TABLE 2 Results: Induced/increased thrombin generation by tested combinations of substances in FVIII deficient plasma. peak time to height peak (nM) (min) FVIII dp 34 14.0 FVIII dp +100% FVIII 238 5.7 FVIII dp +100 μg/ml Bsab FIX/FX 152 10.0 FVIII dp +75 μg/ml Bsab FIX/FX 124 11.0 FVIII dp +50 μg/ml Bsab FIX/FX 119 11.5 FVIII dp +25 μg/ml Bsab FIX/FX 117 12.0 FVIII dp +100 μg/ml Bsab FIX/FX +100% FIX 272 5.5 FVIII dp +75 μg/ml Bsab FIX/FX +100% FIX 247 6.3 FVIII dp +50 μg/ml Bsab FIX/FX +100% FIX 208 6.7 FVIII dp +25 μg/ml Bsab FIX/FX +100% FIX 176 7.5 FVIII dp +100 μg/ml Bsab FIX/FX +1U PCC/ml 361 10.7 FVIII dp +75 μg/ml Bsab FIX/FX +1U PCC/ml 240 11.0 FVIII dp +25 μg/ml Bsab FIX/FX +1U PCC/ml 223 12.0
[0417] Using 100 μg Bsab FIX/FX/ml and either 100% FIX or 1 U PCC/ml even a thrombin generation can be achieved which exceeds the thrombin generation found with 100% FVIII/ml. This may be desirable in case of large bleeding complications, e.g. following trauma or larger surgeries.
Summary on the Experimental Data:
[0418] The experimental data shows that the addition of either FIX or PCC to plasma samples treated with Bsab FIX/FX (Q499-z121/J327-z119/L404-k) leads to a significant increase of thrombin generation in the sample.