Bispecific antibodies binding to coagulation factor ix and coagulation factor x

Abstract

To provide a method for new and effective treatment which solves a defect in treatment of hemophilia A by FVIII.SOLUTION: The present disclosure provides antibodies that selectively bind to specific forms of clotting factors, in particular, antibodies that specifically bind to activated factor IX (FIXa), wherein the anti-FIXa antibody or an antigen binding portion thereof preferentially binds to FIXa in the presence of FIXa and factor IX zymogen (FIXz), and antibodies that specifically bind to factor X zymogen (FXz), and the anti-FXz antibody or antigen binding portion thereof preferentially binds to FXz in the presence of FXz and activated factor X (FXa). Also provided are bispecific molecules (e.g., antibodies) comprising, e.g., an anti-FIXa antibody or antigen binding portion thereof and / or an anti-FXz antibody or antigen binding portion thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] References to Prior Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 425,921, filed November 23, 2016; U.S. Provisional Patent Application No. 62 / 452,809, filed January 31, 2017; U.S. Provisional Patent Application No. 62 / 529,805, filed July 7, 2017; and U.S. Provisional Patent Application No. 62 / 587,284, filed November 16, 2017, which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronically Filed Sequence Listing via EFS-WEB The content of the electronically submitted sequence listing filed with this application (Name: 4159.485PC04_Sequence_listing_ST25.txt; Size: 1,053,370 bytes; and Creation Date: November 21, 2017) is hereby incorporated by reference in its entirety.

[0003] This application relates, inter alia, to antibodies that preferentially bind to activated factor IX or factor X zymogen, and to bispecific molecules that include both specificities mimicking activated factor VIII cofactor.

Background Art

[0004] Hemophilia A is a severe X - linked recessive disorder caused by mutations in the factor VIII (FVIII) gene. FVIII is involved in the intrinsic pathway of blood coagulation, and the lack of FVIII results in poor or no blood clotting. FVIII deficiency, also known as hemophilia A, is one of the most common bleeding disorders, affecting approximately 1 in 10,000 males (Non - Patent Document 1). Hemophilia A has three grades of severity defined by plasma levels of factor FVIII of less than or equal to 1% (“severe”), 2 - 5% (“moderate”), and 6 - 30% (“mild”) (Non - Patent Document 2). In the severe form of the disorder, the first bleeding is usually seen at 5 - 6 months of age, while in the moderate form, this first bleeding is delayed until about 1 - 2 years of age. Bleeding can be seen spontaneously or after minimal trauma. Approximately half of all patients with hemophilia A are classified as having the severe form of the disease. These patients experience severe bleeding episodes starting in childhood and, later in life, frequent episodes of spontaneous or excessive bleeding. Bleeding commonly occurs in joints and muscles, and without appropriate treatment, recurrent bleeding can lead to irreversible hemarthropathy (Non - Patent Document 3).

[0005] An important goal in the treatment of hemophilia A is to maintain plasma levels of FVIII at ≥1%, which reduces the bleeding risk. To achieve this, recombinant or plasma - derived FVIII is frequently administered intravenously as a prophylactic treatment. However, the current standard treatment for hemophilia A is difficult and has several drawbacks, imposing a significant physical and mental burden on patients and their families.

Prior Art Documents

Non - Patent Documents

[0006]

Non - Patent Document 1

Non - Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] The most common adverse effect in FVIII treatment is the generation of alloantibodies against FVIII that act as FVIII inhibitors. As many as 30% of severely affected patients generate such alloantibodies, and once the generation occurs, the effectiveness of using FVIII to treat ongoing bleeding is limited (Kempton and White (2009) Blood 113(1): 11 - 7). In such cases, alternative methods that avoid the drug are used to control bleeding. However, these drugs usually have a shorter half-life and are not always effective. Furthermore, due to the short plasma half-life (with an average of about 12 hours in adults and even shorter in children), it is necessary to administer FVIII frequently. Such a regimen can be difficult, especially in young children. Since available treatments are associated with complications and side effects, there is no single treatment that optimally and effectively treats hemophilia. Therefore, the need for a new and effective treatment to solve the drawbacks of treating hemophilia A with FVIII remains unmet.

Means for Solving the Problems

[0008] The present disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to activated factor IX (FIXa) (the "anti-FIXa antibody or antigen-binding portion thereof"), wherein the anti-FIXa antibody or antigen-binding portion thereof preferentially binds to FIXa in the presence of FIXa and factor IX prothrombin (FIXz). In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to FIXa with a binding affinity that is higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXz. The present disclosure also provides an isolated anti-FIXa antibody or antigen-binding portion thereof that binds to FIXa with a binding affinity that is higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXz. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof has a K of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nM or less as determined by a biolayer interferometry (BLI) assay Dand binds to FIXa. In some embodiments, FIXa is free FIXa, FIXa in the tenase complex, or FIXa covalently linked to EGR-CMK (FIXa-SM). In some embodiments, FIXz comprises inactivated Factor IX (FIXn). In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and 3C. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and 3C. In some embodiments, such reference antibodies are selected from BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, BIIB-9-433, and any combination thereof. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof preferentially binds to FIXa-SM compared to free FIXa or FIXz and / or binds to FIXa-SM with a binding affinity higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for free FIXa or FIXz. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3A. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3A. In some embodiments, such reference antibodies are selected from BIIB-9-484, BIIB-9-440, BIIB-9-460, and any combination thereof. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof is FIXa-SM or Binds to free FIXa with a binding affinity that is preferential to free FIXa compared to FIXz and / or higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXa-SM or FIXz. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in Figure 3B. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figure 3B. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds preferentially to free FIXa or FIXa-SM compared to FXz and / or with a binding affinity higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXz, and binds to free FIXa or FIXa-SM. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in Figure 3C. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figure 3C. In some embodiments, such reference antibodies are selected from BIIB-9-882, BIIB-9-433, and combinations thereof. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3s in Figures 3A, 3B, and 3C or VH CDR3s having one or two mutations. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises ARDX 1 X 2 X 3 X 4 X 5 X 6 YYX 7 Contains MDV (SEQ ID NO: 753), and X 1 is V or G, and X 2 is G or V, and X 3 is G or R, and X 4 is Y or V, and X 5 is A or S, and X 6 is G or D, and X 7is G or does not exist. In some embodiments, CDR3 comprises ARDVGGYAGYYGMDV (SEQ ID NO: 905, BIIB-9-484, 1335, 1336), ARDISTDGESSLYYYMDV (SEQ ID NO: 901, BIIB-9-460), ARGPTDSSGYLDMDV (SEQ ID NO: 1186, BIIB-9-882) or ARDGPRVSDYY MDV (SEQ ID NO: 912, BIIB-9-619). In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises CDR1, CDR2 and CDR3, and CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIGS. 3A, 3B and 3C or a VH CDR1 having one or two mutations. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2 and CDR3, and CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIGS. 3A, 3B and 3C or a VH CDR2 having one or two mutations. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2 and CDR3, and CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIGS. 3A, 3B and 3C or a VL CDR1 having one or two mutations. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2 and CDR3, and CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIGS. 3A, 3B and 3C or a VL CDR2 having one or two mutations. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2 and CDR3, and CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIGS. 3A, 3B and 3C or a VL CDR3 having one or two mutations.

[0009] The present disclosure also provides an isolated anti-FIXa antibody or antigen-binding portion thereof that specifically binds to FIXa and includes VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 include VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, CDR2, and CDR3 of FIGS. 3A, 3B, and 3C, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VH CDR1, CDR2 and CDR3 sequences, and / or VL CDR1, CDR2, and CDR3 sequences (BIIB-9-484) that include SEQ ID NOs: 950, 995, and 1040, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VH CDR1, CDR2, and CDR3 sequences that include SEQ ID NOs: 809, SEQ ID NO: 854, and SEQ ID NO: 899, respectively, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (BIIB-9-440) that include SEQ ID NOs: 944, SEQ ID NO: 989, and SEQ ID NO: 1034, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VH CDR1, CDR2, and CDR3 sequences that include SEQ ID NOs: 1102, SEQ ID NO: 1144, and SEQ ID NO: 1186, respectively, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (BIIB-9-882) that include SEQ ID NOs: 1228, SEQ ID NO: 1270, and SEQ ID NO: 1312, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VH CDR1, CDR2, and CDR3 sequences that include SEQ ID NOs: 811, SEQ ID NO: 856, and SEQ ID NO: 901, respectively, and / or VL CDR1, VL It comprises CDR2 and VL CDR3 sequences (BIIB-9-460). In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 1108, SEQ ID NO: 1150, and SEQ ID NO: 1192, respectively, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (BIIB-9-433) comprising SEQ ID NO: 1234, SEQ ID NO: 1276, and SEQ ID NO: 1318, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 822, SEQ ID NO: 867, and SEQ ID NO: 912, respectively, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (BIIB-9-619) comprising SEQ ID NO: 957, SEQ ID NO: 1002, and SEQ ID NO: 1047, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, respectively The CDR1, CDR2, and CDR3 sequences, and / or the VL CDR1, VL CDR2, and VL CDR3 sequences each containing SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, or (ii) the anti-FIXa antibody or antigen-binding portion thereof contains the VH CDR1, CDR2, and CDR3 sequences each containing SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, and / or the VL CDR1, VL CDR2, and VL CDR3 sequences each containing SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040 (BIIB-9-1335 and BIIB-9-1336). The anti-FIXa antibody or antigen-binding portion thereof contains VH and VL, and VH contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181 and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof contains VH and VL, and VL contains SEQ ID NO: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293An amino acid sequence selected from the group consisting of 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises a VH and a VL, wherein the VH is derived from a germline sequence of VH1-46.0, VH1-46.4, VH1-46.5, VH1-46.7, VH1-46.9, VH1-69.9, VH3-07.0, VH3-21.0, VH3-21.2, VH3-23.0, VH3-23.1, VH4-31.0, VH4-34.0, VH4-39.0, VH4-39.2, VH4-39.3, VH4-39.5, VH4-39.6, VH4-39.8, VH4-59.6, VH4-0B.4, or VH4-0B.6. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises a VH and a VL, wherein the VL is derived from a germline sequence of VK1-05.0, VK1-05.6, VK1-05.9, VK1-05.21, VK1-12.0, VK1-12.3, VK1-33.0, VK1-33.1, VK1-33.2, VK1-33.8, VK1-33.10, VK1-39.0, VK1-39.6, VK2-28.0, VK2-28.1, VK3-11.0, VK3-11.2, VK3-11.6, VK3-11.10, VK3-11.14, VK3-15.0, VK3-15.6, VK3-15.8, VK3-15.11, VK3-15.20, VK3-15.26, VK3-20.0, VK3-20.4, VK3-20.5, VK3-20.8, or VK4-01.0.In one aspect, the anti-FIXa antibody or antigen-binding portion thereof comprises VH and VL, wherein (a1) VH and VL each comprise SEQ ID NO: 31 and 221 (BIIB-9-484); (a2) VH and VL each comprise SEQ ID NO: 19 and 209 (BIIB-9-440); (a3) VH and VL each comprise SEQ ID NO: 115 and 301 (BIIB-9-882); (a4) VH and VL each comprise SEQ ID NO: 23 and 213 (BIIB-9-460); (a5) VH and VL each comprise SEQ ID NO: 127 and 313 (BIIB-9-433); (a6) VH and VL each comprise SEQ ID NO: 45 and 235 (BIIB-9-619); (a7) VH and VL each comprise SEQ ID NO: 185 and 371 (BIIB-9-578); (a8) VH and VL each comprise SEQ ID NO: 87 and 221 (BIIB-9-1335); or (a9) VH and VL each comprise SEQ ID NO: 89 and 221 (BIIB-9-1336).

[0010] The present disclosure also provides an isolated antibody or antigen-binding portion thereof that specifically binds to FIXz (the "anti-FIXz antibody or antigen-binding portion thereof"), wherein the anti-FIXz antibody or antigen-binding portion thereof preferentially binds to FIXz in the presence of free FIXa or FIXa-SM and / or the anti-FIXz antibody or antigen-binding portion thereof binds to FIXz with a binding affinity that is higher than its binding affinity for free FIXa or FIXa-SM. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3D. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3D. In some embodiments, such a reference antibody is BIIB-9-578. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in FIG. 3D or a VH CDR3 having one or two mutations. In some embodiments, CDR3 comprises ARDKYQDYSFDI (SEQ ID NO: 1355, BIIB-9-578). In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CD Comprising R3, CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIG. 3D or a VH CDR1 having one or two mutations. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIG. 3D or a VH CDR2 having one or two mutations. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIG. 3D or a VL CDR1 having one or two mutations. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIG. 3D or a VL CDR2 having one or two mutations. In some embodiments, the anti-FIXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIG. 3D or a VL CDR3 having one or two mutations. In some embodiments, the anti-FIX antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof. In some embodiments, the anti-FIX antibody is an IgG4 antibody. In some embodiments, the anti-FIX antibody comprises an effectorless IgG4 Fc. In some embodiments, the anti-FIX antibody or antigen-binding portion thereof comprises a heavy chain constant region. In some embodiments, the anti-FIX antibody is a human antibody, an engineered antibody, or a humanized antibody. In some embodiments, the anti-FIX antigen-binding portion comprises Fab, Fab’, F(ab’)2, Fv, or single-chain Fv (scFv).

[0011] The present disclosure also provides a bispecific molecule comprising an anti-FIX antibody or an antigen-binding portion thereof disclosed herein, which is linked to a molecule having a second binding specificity. Similarly, provided are the heavy and / or light chain variable regions of the anti-FIX antibody or an antigen-binding portion thereof disclosed herein, or a nucleic acid encoding a bispecific molecule comprising the anti-FIX antibody or an antigen-binding portion thereof disclosed herein. Similarly, provided is an expression vector comprising a nucleic acid molecule encoding disclosed herein. Similarly, provided are cells transformed with the expression vector disclosed herein. The present disclosure also provides an immunoconjugate comprising any antibody or an antigen-binding portion thereof disclosed herein or a bispecific molecule disclosed herein, which is linked to a drug. The present disclosure also provides a composition comprising (i) an antibody or an antigen-binding portion thereof disclosed herein, a bispecific molecule disclosed herein, or an immunoconjugate disclosed herein, and (ii) a carrier. Similarly, provided is a kit comprising (i) an antibody or an antigen-binding portion thereof disclosed herein, a bispecific molecule disclosed herein, or an immunoconjugate disclosed herein, and (ii) instructions for use.

[0012] The present disclosure also provides a method for manufacturing an anti-FIX antibody or an antigen-binding portion thereof, comprising the steps of expressing the antibody or an antigen-binding portion thereof in a cell, and isolating the antibody or an antigen-binding portion thereof from the cell. Similarly, provided is a method for measuring the level of activated FIX in a subject in need thereof, comprising the steps of contacting a sample obtained from the subject with an anti-FIXa antibody or an antigen-binding portion thereof disclosed herein under suitable conditions, and measuring the binding of the anti-FIXa antibody or an antigen-binding portion thereof in the sample to FIXa. In some embodiments, the sample is blood or serum.

[0013] The present disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to factor X zymogen (FXz) (the "anti-FXz antibody or antigen-binding portion thereof"), wherein the anti-FXz antibody or antigen-binding portion thereof preferentially binds to FXz in the presence of FXz and activated factor X (FXa). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof binds to FXz with a binding affinity that is higher than the binding affinity of the antibody or antigen-binding portion thereof for FXa. Similarly, there is provided an isolated anti-FXz antibody or antigen-binding portion thereof that binds to FXz with a binding affinity that is higher than the binding affinity of the antibody or antigen-binding portion thereof for FXa. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof has a K of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less as measured by BLI Dand binds to FXz. In some embodiments, FXa is free FXa or FXa (FIXa-SM) covalently linked to EGR-CMK. In some embodiments, FXz includes inactivated Factor X (FXn). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIGS. 12A and 12B. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIGS. 12A and 12B. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of BIIB-12-915, BIIB-12-917, BIIB-12-932, and any combination thereof. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof includes CDR1, CDR2 and CDR3, and CDR3 includes a VH CDR3 selected from the group consisting of the VH CDR3s in FIGS. 12A and 12 or VH CDR3s having one or two mutations. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof includes CDR1, CDR2 and CDR3, and CDR3 includes ARX 1 X 2 X 3 RX 4 X 5 X 6 X 7 FDX 8 (SEQ ID NO: 766), and X 1 is G or L, and X 2 is R or G, and X 3 is F or Y, and X 4 is P or G, and X 5 is R or A, and X 6 is G or S, and X 7 is R or A, and X 8is Y or I. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises ARGRFRPRGRFDY (SEQ ID NO: 1575, BIIB-12-917), ARLGYRGASAFDI (SEQ ID NO: 1589, BIIB-12-932), or ARVGGGYANP (SEQ ID NO: 1573, BIIB-12-915). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIGS. 12A and 12B or a VH CDR1 having one or two mutations. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIGS. 12A and 12B or a VH CDR2 having one or two mutations. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIGS. 12A and 12B or a VL CDR1 having one or two mutations. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIGS. 12A and 12B or a VL CDR2 having one or two mutations. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIGS. 12A and 12B or a VL CDR3 having one or two mutations.

[0014] The present disclosure also provides an isolated antibody or antigen-binding portion thereof that specifically binds to FXz and comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 are the VH CDR1 in FIGS. 12A and 12B, respectively. , CDR2, and CDR3, and VL CDR1, CDR2, and CDR3. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences, each comprising SEQ ID NO: 1393, 1483, or 1573, and / or VL CDR1, VL CDR2, and VL CDR3 sequences, each comprising SEQ ID NO: 1663, 1753, and 1843 (BIIB-12-915). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences, each comprising SEQ ID NO: 1395, 1485, or 1575, and / or VL CDR1, VL CDR2, and VL CDR3 sequences, each comprising SEQ ID NO: 1665, 1755, and 1845 (BIIB-12-917). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences, each comprising SEQ ID NO: 1409, 1499, or 1589, and / or VL CDR1, VL It comprises CDR2 and VL CDR3 sequences (BIIB-12-932). In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises VH and VL, and VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, and 555, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises a VH and a VL, wherein the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 565, 567, 569, 571, 573, 575, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, and 743, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises a VH and a VL, wherein the VH is derived from a germline sequence of VH1-18.0, VH1-18.1, VH1-18.8, VH1-46.0, VH1-46.4, VH1-46.5, VH1-46.6, VH1-46.7, VH1-46.8, VH1-46.9, VH3-21.0, VH3-23.0, VH3-23.2, VH3-23.6, VH3-30.0, VH4-31.5, VH4-39.0, VH4-39.5, VH4-0B.4, or VH5-51.1. In some embodiments, the anti-FXz antibody or antigen-binding portion thereof comprises a VH and a VL, wherein the VL is VK1-05.6, VK1-05.12, VK1-12.0, VK1-12.4, VK1-12.7, VK1-12.10, VK1-12.15, VK1-39.0, VK1-39.3, VK1-39.15, VK2-28.0, VK2-28.1, VK2-28.5, VK3-11.0, VK3-11.2, VK3-11.6, VK3-11. .14, VK3-15.0, VK3-15.8, VK3-15.10, VK3-20.0, VK3-20.1, VK3-20.4, VK3-20.5, VK4-01.0, VK4-01.4, VK4-01.20. In some embodiments, the anti-FX antibody or antigen-binding portion thereof comprises VH and VL, and (b1) VH and VL each comprise SEQ ID NO: 423 and 611 (BIIB-12-915); (b2) VH and VL each comprise SEQ ID NO: 427 and 615 (BIIB-12-917); or (b3) VH and VL each comprise SEQ ID NO: 455 and 643 (BIIB-12-932).

[0015] The present disclosure also provides an isolated antibody or antigen-binding portion thereof that specifically binds to activated Factor X (FXa) (the "anti-FXa antibody or antigen-binding portion thereof"), wherein the anti-FXa antibody or antigen-binding portion thereof preferentially binds to FXa in the presence of FXz and FXa, and / or binds to FXa with a binding affinity higher than the binding affinity of the antibody or antigen-binding portion thereof for FXz. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 12C. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 12C. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of BIIB-12-925. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in FIG. 12C or a VH CDR3 having one or two mutations. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises CDR1, CDR2, and CDR3, and CDR3 comprises AKGPRYYWYSWYFDL (SEQ ID NO: 1919, BIIB-12-925). In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIG. 12C or a VH CDR1 having one or two mutations. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIG. 12C or a VH CDR2 having one or two mutations. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIG. 12C or a VH CDR1 having one or two mutations.In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIG. 12C or a VL CDR2 having one or two mutations. In some embodiments, the anti-FXa antibody or antigen-binding portion thereof comprises VL CDR1, CDR2, and CDR3, and CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIG. 12C or a VL CDR3 having one or two mutations.

[0016] The present disclosure also provides an isolated antibody or antigen-binding portion thereof that specifically binds to FXa and comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 comprise the VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 of FIG. 12C, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (BIIB-12-925) comprising SEQ ID NO: 1911, 1915, or 1919, and / or SEQ ID NO: 1923, 1927, or 1931, respectively. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH and VL, and VH and VL Each contains SEQ ID NOs: 559 and 747 (BIIB-12-925). In some embodiments, the anti-FX antibody or antigen-binding portion thereof is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof. In some embodiments, the anti-FX antibody or antigen-binding portion thereof is an IgG4 antibody. In some embodiments, the anti-FX antibody or antigen-binding portion thereof comprises an effectorless IgG4 Fc. In some embodiments, the anti-FX antibody or antigen-binding portion thereof comprises a heavy chain constant region. In some embodiments, the anti-FX antibody is a human antibody, engineered antibody, or humanized antibody. In some embodiments, the antigen-binding portion thereof comprises a Fab, Fab’, F(ab’)2, Fv, or single-chain Fv (scFv). The disclosure also provides a bispecific molecule comprising an anti-FX antibody disclosed herein linked to a molecule having a second binding specificity. Similarly, provided are nucleic acids encoding the heavy and / or light chain variable regions of an anti-FX antibody or antigen-binding portion thereof disclosed herein, or a bispecific molecule comprising an anti-FX antibody or antigen-binding portion thereof disclosed herein. Similarly, provided is an expression vector comprising the nucleic acid molecule. Similarly, provided is a cell transformed with the expression vector. Similarly, provided is an immunoconjugate comprising an antibody or antigen-binding portion thereof, or a bispecific molecule, conjugated to a drug. Similarly, provided is a composition comprising (i) an antibody or antigen-binding portion thereof, or a bispecific molecule, or an immunoconjugate, and (ii) a carrier. Similarly, provided is a kit comprising (i) an antibody or antigen-binding portion thereof, or a bispecific molecule, or an immunoconjugate, and (ii) instructions for use. Similarly, provided is a method for producing an anti-FX antibody or antigen-binding portion thereof, the method comprising expressing the antibody or antigen-binding portion thereof in a cell and isolating the antibody or antigen-binding portion thereof from the cell.

[0017] The present disclosure also provides a method for measuring thrombogen FX (FXz) in a subject in need thereof, the method comprising contacting an anti-FX antibody or an antigen-binding portion thereof disclosed herein with a sample obtained from the subject under suitable conditions, and measuring the binding of the anti-FX antibody or an antigen-binding portion thereof in the sample to FXz. In some embodiments, the sample is blood or serum derived from the subject. The present disclosure also provides a bispecific molecule comprising (i) an anti-FIX antibody or an antigen-binding portion thereof disclosed herein, and (ii) an anti-FX antibody or an antigen-binding portion thereof disclosed herein. In some embodiments, the bispecific molecule cross-competes with a reference bispecific antibody, the reference bispecific antibody comprising the VH and VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIGS. 3A, 3B, 3C, and 3D, and the VH and VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIGS. 12A, 12B, and 12C. In some embodiments, the bispecific molecule binds to the same epitope as a reference bispecific antibody, the reference bispecific antibody comprising the VH and VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIGS. 3A, 3B, 3C, and 3D, and the VH and VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIGS. 12A, 12B, and 12C.In some aspects of the bispecific molecules disclosed herein, (i) the anti-FIX antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, and VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 of the anti-FIX (BIIB-9) antibody in FIGS. 16A, 16B, 16C, and 16D; and (ii) the anti-FX antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, and VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 of the anti-FX (BIIB-12) antibody in FIGS. 16A, 16B, 16C, and 16D. In some aspects of the bispecific molecules disclosed herein. , (a) The anti-FIX antibody or an antigen-binding portion thereof comprises (a1) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 815, 860, or 905, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 950, 995, or 1040 (BIIB-9-484); (a2) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 822, 867, and 912, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 957, 1002, and 1047 (BIIB-9-619); (a3) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1347, 1351, and 1355, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1359, 1363, and 1367 (BIIB-9-578); (a4) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 843, 888, and 933, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 978, 1023, and 1068 (BIIB-9-1335); or (a5) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 844, 889, and 934, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 979, 1024, and 1069 (BIIB-9-1336); (b) The anti-FX antibody or an antigen-binding portion thereof comprises (b1) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1393, 1483, and 1573, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1663, 1753, and 1843 (BIIB-12-915); (b2) VH CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1395, 1485, and 1575, and / or VL CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 1665, 1755, and 1845 (BIIB-12-917);(b3) VH CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1911, 1915, and 1919, respectively, and / or VL CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1923, 1927, and 1931, respectively (BIIB-12-925); (b4) VH CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1409, 1499, and 1589, respectively, and / or VL CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1679, 1769, and 1859, respectively (BIIB-12-932); or (b5) VH CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1433, 1523, and 1613, respectively, and / or VL CDR1, CDR2, and CDR3 sequences, each containing SEQ ID NO: 1703, 1793, and 1883, respectively (BIIB-12-1306). In some embodiments of the bispecific molecules disclosed herein, (a) the anti-FIX antibody or antigen-binding portion thereof comprises (a1) VH and LV, each containing SEQ ID NO: 31 and 221, respectively (BIIB-9-484); (a2) VH and VL, each containing SEQ ID NO: 45 and 235, respectively (BIIB-9-619); (a3) VH and VL, each containing SEQ ID NO: 185 and 371, respectively (BIIB-9-578); (a4) VH and VL, each containing SEQ ID NO: 87 and 221, respectively (BIIB-9-1335); or (a5) VH and VL, each containing SEQ ID NO: 89 and 221, respectively (BIIB-9-1336); (b) the anti-FX antibody or antigen-binding portion thereof comprises (b1) VH and LV, each containing SEQ ID NO: 423 and 611, respectively (BIIB-12-915); (b2) VH and VL, each containing SEQ ID NO: 427 and 615, respectively (BIIB-12-917); (b3) VH and VL, each containing SEQ ID NO: 559 and 747, respectively (BIIB-12-925); (b4) VH and VL, each containing SEQ ID NO: 455 and 643, respectively (BIIB-12-932);or (b5) each contains a VH and a VL (BIIB-12-1306) containing SEQ ID NOs: 503 and 691. In some embodiments of the bispecific molecules disclosed herein, (i) the anti-FIX antibody or antigen-binding portion thereof each contains a VH and a LV (BIIB-9-484) containing SEQ ID NOs: 31 and 221; and the anti-FX antibody or antigen-binding portion thereof each contains a VH and a VL (BIIB-12-915) containing SEQ ID NOs: 423 and 611; also; (ii) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-484) comprising SEQ ID NOs: 31 and 221, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-917) comprising SEQ ID NOs: 427 and 615, respectively; or (iii) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-484) comprising SEQ ID NOs: 31 and 221, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-925) comprising SEQ ID NOs: 559 and 747, respectively; or (iv) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-484) comprising SEQ ID NOs: 31 and 221, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-932) comprising SEQ ID NOs: 455 and 643, respectively; or (v) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-578) comprising SEQ ID NOs: 185 and 371, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-915) comprising SEQ ID NOs: 423 and 611, respectively; or (vi) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-578) comprising SEQ ID NOs: 185 and 371, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-917) comprising SEQ ID NOs: 427 and 615, respectively; or (vii) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-619) comprising SEQ ID NOs: 45 and 235, respectively; the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-917) comprising SEQ ID NOs: 427 and 615, respectively; or (viii) the anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-619) comprising SEQ ID NOs: 45 and 235, respectively; and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-925) comprising SEQ ID NOs: 559 and 747, respectively.In some embodiments, the bispecific molecule functionally mimics activated factor VIII (FVIIIa) cofactor in at least one FVIIIa activity assay. In some embodiments, the FVIIIa activity assay is selected from a chromogenic FXa generation assay, a one-stage clotting assay, or a combination thereof. In some embodiments, the FVIIIa activity is at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the activity normally achieved by FVIII in the same assay. In some embodiments, the bispecific molecule can generate thrombin from prothrombin, fibrin from fibrinogen, and / or fibrin clots in vitro or in vivo. In some embodiments, the bispecific molecule binds simultaneously to both FIXa and FX as determined by BLI. In some embodiments, the bispecific molecule is of the IgG isotype. In some embodiments, the IgG isotype is of the IgG1 subclass. In some embodiments, the IgG isotype is of the IgG4 subclass. In some embodiments, the bispecific molecule is of the bispecific IgG format and is selected from the group consisting of the antibodies in Table 2. In some embodiments, the bispecific molecule is of the bispecific heterodimer format. In some embodiments, the bispecific molecule comprises two different heavy chains and two different light chains. In some embodiments, the bispecific molecule comprises two identical light chains and two different heavy chains. In some embodiments, the bispecific molecule is capable of controlling or reducing the incidence of bleeding episodes in a subject having hemophilia. In some embodiments, the bispecific molecule can maintain homeostasis in a subject having hemophilia. In some embodiments, the bispecific molecule can effect prophylaxis of a defined procedure in a subject having hemophilia. In some embodiments, the subject has developed or is expected to develop neutralizing antibodies against factor VIII.

[0018] The present disclosure also provides an immunoconjugate comprising a bispecific molecule disclosed herein conjugated to an agent, such as a therapeutic agent. Similarly, there is provided a composition comprising (i) a bispecific molecule disclosed herein or an immunoconjugate comprising a bispecific molecule, and (ii) a carrier. Similarly, there is provided a kit comprising (i) a bispecific molecule disclosed herein or an immunoconjugate comprising a bispecific molecule, and (ii) instructions for use. Similarly, there is provided a nucleic acid sequence encoding a bispecific molecule disclosed herein. Similarly, there is provided a vector comprising the nucleic acid, and a host cell comprising the vector. In some embodiments, the host cell is a prokaryotic cell, a eukaryotic cell, a protist cell, an animal cell, a plant cell, a fungal cell, a yeast cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell, a HEK cell, or a COS cell. Similarly, there is provided a method of generating a bispecific molecule disclosed herein, the method comprising culturing a host cell disclosed herein under conditions that allow for expression of the bispecific molecule. In some embodiments, the method of generating a bispecific molecule disclosed herein further comprises using conditions that enhance heterodimerization. A composition comprising (i) a bispecific molecule or an immunoconjugate comprising a bispecific molecule, and (ii) a carrier is provided. Similarly, a kit comprising (i) a bispecific molecule or an immunoconjugate comprising a bispecific molecule, and (ii) instructions for use is provided. Similarly, a nucleic acid sequence encoding a bispecific molecule is provided. Similarly, a vector comprising the nucleic acid, and a host cell comprising the vector are provided. In some embodiments, the host cell is a prokaryotic cell, a eukaryotic cell, a protist cell, an animal cell, a plant cell, a fungal cell, a yeast cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell, a HEK cell, or a COS cell. Similarly, a method of generating a bispecific molecule is provided, the method comprising culturing a host cell under conditions that allow for expression of the bispecific molecule. In some embodiments, the method of generating a bispecific molecule further comprises using conditions that enhance heterodimerization.

[0019] The present disclosure also provides a method of promoting FX activation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific molecule disclosed herein, or an immunoconjugate, composition, nucleic acid, vector, or host cell disclosed herein that comprises or encodes a bispecific molecule disclosed herein.

[0020] The present disclosure also provides a method of reducing the frequency or extent of bleeding episodes in a subject in need thereof, the method comprising administering to the subject an effective amount of a bispecific molecule disclosed herein, or an immunoconjugate, composition, nucleic acid, vector or host cell disclosed herein that comprises or encodes a bispecific molecule disclosed herein. In some embodiments, the subject generates or has a tendency to generate an inhibitor to factor VIII (“FVIII”). In some embodiments, the inhibitor to FVIII is a neutralizing antibody to FVIII. In some embodiments, the bleeding episodes are the result of hemarthrosis, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, intraoral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fractures, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, or any combination thereof.

[0021] The present disclosure also provides a method of treating a blood coagulation disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of a bispecific molecule disclosed herein, or an immunoconjugate, composition, nucleic acid, vector or host cell disclosed herein that comprises or encodes a bispecific molecule disclosed herein. In some embodiments, the blood coagulation disorder is hemophilia A or hemophilia B. In some embodiments, the subject is a human subject. In some embodiments, the subject is undergoing or has undergone FVIII replacement therapy. In some embodiments, the bispecific molecule is administered in combination with a hemophilia treatment. In some embodiments, the hemophilia treatment is FVIII replacement therapy. In some embodiments, the bispecific molecule, immunoconjugate, composition, nucleic acid, vector or host cell is administered before, during or after administration of the hemophilia treatment. In some embodiments, the bispecific molecule, immunoconjugate, composition, nucleic acid, vector or host cell is administered intravenously or subcutaneously. In some embodiments, administration of the bispecific molecule, immunoconjugate, composition, nucleic acid, vector or host cell results in a decrease in the occurrence of bleeding episodes, the frequency of spontaneous bleeding episodes or acute bleeding. In some embodiments, administration of the bispecific molecule, immunoconjugate, composition, nucleic acid, vector or host cell results in a 5%, 10%, 20%, 30% or 50% decrease in the annual bleeding rate.

[0022] The present disclosure also provides an anti-FIXa antibody or antigen-binding portion thereof that binds to the same epitope as BIIB-9-1336. Similarly, an anti-FIXa antibody or antigen-binding portion thereof that binds to an epitope that overlaps with the BIIB-9-1336 epitope is provided.

[0023] The present disclosure also provides an anti-FIXa antibody or an antigen-binding portion thereof that binds to an epitope region comprising at least one amino acid located between (i) positions 91 and 101, (ii) 125 and 128, (iii) 165 and 179, or (iv) 232 and 241 of the chymotrypsinogen numbering in the heavy chain sequence of FIXa. Similarly, provided is an anti-FIXa antibody or an antigen-binding portion thereof that binds to an epitope comprising at least one of the amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. In some embodiments, the epitope comprises the amino acid residues N93, R165, N178, and R233 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. In other embodiments, the epitope comprises the amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. In some embodiments, the epitope does not comprise at least one of the amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. In some embodiments, the epitope does not comprise the amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. In some embodiments, the anti-FIXa antibody or an antigen-binding portion thereof binds to at least one amino acid residue (SEQ ID NO: 756) in the light chain of FIXa. In some embodiments, the amino acid residue (SEQ ID NO: 756) in the light chain of FIXa is K100. In some embodiments, the epitope overlaps with the binding site of FVIIIa to FIXa. In some embodiments, the anti-FIXa antibody or an antigen-binding portion thereof cross-competes with FVIIIa for binding to FIXa.In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof blocks the binding of FVIIIa to FIXa.

[0024] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein (i) VH CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1 in Table 7 or VH CDR1 having one or two mutations; and / or (ii) VH CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2 in Table 7 or VH CDR2 having one or two mutations; and / or (iii) VH CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3 in Table 7 or VH CDR3 having one or two mutations; and / or (iv) VL CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1 in Table 7 or VL CDR1 having one or two mutations; and / or (v) VL CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2 in Table 7 or VL CDR2 having one or two mutations; and / or (vi) VL CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3 in Table 7 or VL CDR3 having one or two mutations.

[0025] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, and VH CDR3 comprises the amino acid sequence ARDX 1 GGYAGYYGMDV (SEQ ID NO: 2196), wherein X 1 is L or V. In some embodiments, (i) VH CDR1 comprises the amino acid sequence FTFX 1 SX 2 X 3 MX 4 (SEQ ID NO: 2194), wherein X1 is S, G or E, and X 2 is Y or F, and X 3 is S, E, G or D, and X 4 is N, V, A or T; and / or (ii) the VH CDR2 is the amino acid sequence X 5 ISX 6 X 7 X 8 X 9 X 10 contains IYYADSVKG (SEQ ID NO: 2195), and X 5 is S, A, Y or G, and X 6 is S or A, and X 7 is S, A or G, and X 8 is S, G or D, and X 9 is S, T or G, and X 10 is Y or T.

[0026] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VL CDR1, CDR2, and CDR3, and the VL CDR3 comprises the amino acid sequence QQYANFPYT (SEQ ID NO: 2168). In some embodiments, (i) the VL CDR1 comprises the amino acid sequence QASQDIANYLN (SEQ ID NO: 2116); and / or (ii) the VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO: 2142).

[0027] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises (i) VH CDR1, CDR2, and CDR3, wherein VH CDR1 is selected from SEQ ID NOs: 2038-2047, VH CDR2 is selected from SEQ ID NOs: 2064-2073, and VH CDR3 is selected from SEQ ID NOs: 2090-2099, and / or (ii) VL CDR1, CDR2, and CDR3, wherein VL CDR1 is selected from SEQ ID NOs: 2116-2125, VL CDR2 is selected from SEQ ID NOs: 2142-2151, and VL CDR3 is selected from SEQ ID NOs: 2168-2177.

[0028] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR3 comprises the amino acid sequence X 1 RDVX 2 GYAGX 3 YGMDV (SEQ ID NO: 2198), and X 1 is A or V, and X 2 is G or S, and X 3 is Y or F. In some embodiments, (i) VH CDR1 comprises the amino acid sequence FTFGSYDMN (SEQ ID NO: 2048); and / or (ii) VH CDR2 comprises the amino acid sequence SISX 1 X 2 X 3 SYIX 4 YAX 5 SVKG (SEQ ID NO: 2197), and X 1 is S or D, and X 2 is G or S, and X 3 is E or A, and X 4 is Y or A, and X 5 is E or D.

[0029] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VL CDR3 comprises the amino acid sequence X 1 QYAX 2 FPYT (SEQ ID NO: 2201), and X 1 is Q or S, and X 2 is N or R. In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VL CDR1, CDR2, and CDR3, and (i) VL CDR1 comprises the amino acid sequence X 1 AX 2 X 3 X 4 IX 5 X 6 YLN (SEQ ID NO: 2199), and X 1 is Q, G, or E, and X 2 is S or N, and X 3 is Q or E, and X 4 is D or Y, and X 5 is A or S, and X 6 is N or D; and / or (ii) VL CDR2 comprises the amino acid sequence DAX 7 NLX 8 X 9 (SEQ ID NO: 2200), and X 7 is S or A, and X 8 is E, H, or Q, and X 9 is T or Y.

[0030] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises (i) VH CDR1, CDR2, and CDR3, wherein The VH CDR1 is selected from SEQ ID NOs: 2048 to 2052, the VH CDR2 is selected from SEQ ID NOs: 2074 to 2078, the VH CDR3 is selected from SEQ ID NOs: 2100 to 2104, and the VH CDR1, CDR2 and CDR3, and / or (ii) the VL CDR1, CDR2 and CDR3, wherein the VL CDR1 is selected from SEQ ID NOs: 2126 to 2130, the VL CDR2 is selected from SEQ ID NOs: 2152 to 2156, and the VL CDR3 is selected from SEQ ID NOs: 2178 to 2182, and the VL CDR1, CDR2 and CDR3 are included.

[0031] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein the VH CDR3 has the amino acid sequence ARDGPX 1 X 2 X 3 and includes DYYMDV (SEQ ID NO: 2204), where X 1 is R or Q, and X 2 is V, D, L or E, and X 3 is S or V. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2 and CDR3, and (i) the VH CDR1 has the amino acid sequence YTFX 1 X 2 YX 3 and includes MH (SEQ ID NO: 2202), where X 1 is T or H, and X 2 is S, G or H, and X 3 is Y or P; and / or (ii) the VH CDR2 has the amino acid sequence X 4 INPSX 5 GX 6 TX 7 and includes YAQKFQG (SEQ ID NO: 2203), where X 4 is I or S, and X 5 is G or R, and X 6 is S or R, and X 7 is S or E.

[0032] In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VL CDR3 comprises the amino acid sequence QQRDNWPFT (SEQ ID NO: 2116). In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise VL CDR1, CDR2, and CDR3, wherein (i) VL CDR1 comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO: 2116); and / or (ii) VL CDR2 comprises the amino acid sequence DASNRAT (SEQ ID NO: 2116).

[0033] In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise (i) VH CDR1, CDR2, and CDR3, wherein VH CDR1 is selected from SEQ ID NOs: 2053-2057, VH CDR2 is selected from SEQ ID NOs: 2079-2083, and VH CDR3 is selected from SEQ ID NOs: 2105-2109, and / or (ii) VL CDR1, CDR2, and CDR3, wherein VL CDR1 is selected from SEQ ID NOs: 2131-2135, VL CDR2 is selected from SEQ ID NOs: 2157-2161, and VL CDR3 is selected from SEQ ID NOs: 2183-2187.

[0034] In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR3 comprises the amino acid sequence ARDKYQDYSX 1 DI (SEQ ID NO: 2207), wherein X 1is F or V. In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VH CDR1, CDR2, and CDR3, and (i) VL CDR1 comprises the amino acid sequence GSIX 1 SX 2 X 3 YX 4 WX 5 (SEQ ID NO: 2205), where X 1 is S or A, and X 2 is S, T, G, or V, and X 3 is S or A, and X 4 is Y or A, and X 5 is G, V, N, or S; and / or (ii) VH CDR2 comprises the amino acid sequence X 6 IX 7 X 8 X 9 GX 10 TX 11 YNPSLK S (SEQ ID NO: 2206), where X 6 is S or Y, and X 7 is S, Y, R, T, or Q, and X 8 is Y, G, P, or A, and X 9 is S or Q, and X 10 is S or, and X 11 is Y or Q.

[0035] In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VL CDR1, CDR2, and CDR3, and VL CDR3 comprises the amino acid sequence QQANFLPFT (SEQ ID NO: 2188). In some embodiments, an anti-FIXa antibody or antigen-binding portion thereof disclosed herein that specifically binds to FIXa comprises VL CDR1, CDR2, and CDR3, and (i) VL CDR1 comprises the amino acid sequence RASQGIDSWLA (SEQ ID NO: 2136); and / or (ii) VL CDR2 comprises the amino acid sequence AASSLQS (SEQ ID NO: 2162).

[0036] In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise (i) VH CDR1, CDR2, and CDR3, where VH CDR1 is selected from SEQ ID NOs: 2058 - 2063, VH CDR2 is selected from SEQ ID NOs: 2084 - 2089, and VH CDR3 is selected from SEQ ID NOs: 2110 - 2115, and / or (ii) VL CDR1, CDR2, and CDR3, where VL CDR1 is selected from SEQ ID NOs: 2136 - 2141, VL CDR2 is selected from SEQ ID NOs: 2162 - 2167, and VL CDR3 is selected from SEQ ID NOs: 2188 - 2193.

[0037] In some embodiments, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise VH and VL, where (i) VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1935, 1939, 1943, 1947, 1951, 1955, 1959, 1963, 1967, 1971, 1975, 1979, 1983, 1987, 1991, 1995, 1999, 2003, 2007, 2011, 2015, 2019, 2023, 2027, 2031, and 2035 and has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity thereto; and / or (ii) VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1937, 1941, 1945, 1949, 1953, 1957, 1961, 1965, 1969, 1973, 1977, 1981, 1985, 1989, 1993, 1997, 2001, 2005, 2009, 2013, 2017, 2021, 2025, 2029, 2033, and 2037 and has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity thereto.

[0038] In one aspect, the anti-FIXa antibodies or antigen-binding portions thereof disclosed herein that specifically bind to FIXa comprise VH and VL, and (a1) VH and VL each comprise SEQ ID NO: 1935 and 1937 (BIIB-9-3595); (a2) VH and VL each comprise SEQ ID NO: 1939 and 1941 (BIIB-9-3601); (a3) VH and VL each comprise SEQ ID NO: 1943 and 1945 (BIIB-9-3604); (a4) VH and VL each comprise SEQ ID NO: 1947 and 1949 (BIIB-9-3617); (a5) VH and VL each comprise SEQ ID NO: 1951 and 1953 (BIIB-9-3618); (a6) VH and VL each comprise SEQ ID NO: 1955 and 1957 (BIIB-9-3621); (a7) VH and VL each comprise SEQ ID NO: 1959 and 1961 (BIIB-9-3647); (a8) VH and VL each comprise SEQ ID NO: 1963 and 1965 (BIIB-9-3649); (a9) VH and VL each including SEQ ID NO: 1967 and 1969 (BIIB-9-3650); (a10) VH and VL each include SEQ ID NO: 1971 and 1973 (BIIB-9-3654); (a11) VH and VL each include SEQ ID NO: 1975 and 1977 (BIIB-9-3753); (a12) VH and VL each include SEQ ID NO: 1979 and 1981 (BIIB-9-3754); (a13) VH and VL each include SEQ ID NO: 1983 and 1985 (BIIB-9-3756); (a14) VH and VL each include SEQ ID NO: 1987 and 1989 (BIIB-9-3764); (a15) VH and VL each include SEQ ID NO: 1991 and 1993 (BIIB-9-3766); (a16) VH and VL each include SEQ ID NO: 1995 and 1997 (BIIB-9-3707); (a17) VH and VL each include SEQ ID NO: 1999 and 2001 (BIIB-9-3709); (a18) VH and VL each include SEQ ID NO: 2003 and 2005 (BIIB-9-3720); (a19) VH and VL each include SEQ ID NO: 2007 and 2009 (BIIB-9-3727); (a20) VH and VL each include SEQ ID NO: 2011 and 2013 (BIIB-9-3745); (a21) VH and VL each include SEQ ID NO: 2015 and 2017 (BIIB-9-3780); (a22) VH and VL each include SEQ ID NO: 2019 and 2021 (BIIB-9-3675); (a23) VH and VL each include SEQ ID NO: 2023 and 2025 (BIIB-9-3681); (a24) VH and VL each include SEQ ID NO: 2027 and 2029 (BIIB-9-3684); (a25) VH and VL each include SEQ ID NO: 2031 and 2033 (BIIB-9-3698); or (a26) VH and VL each include SEQ ID NO: 2035 and 2037 (BIIB-9-3704).

[0039] Embodiment Embodiment 1. An isolated antibody or antigen-binding portion thereof that specifically binds to activated Factor IX (FIXa) (the "anti-FIXa antibody or antigen-binding portion thereof"), wherein the anti-FIXa antibody or antigen-binding portion thereof preferentially binds to FIXa in the presence of FIXa and Factor IX zymogen (FIXz).

[0040] Embodiment 2. The anti-FIXa antibody or antigen-binding portion thereof according to Embodiment 1, which binds to FIXa with a binding affinity higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXz.

[0041] Embodiment 3. An isolated anti-FIXa antibody or antigen-binding portion thereof that binds to FIXa with a binding affinity higher than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof for FIXz.

[0042] Embodiment 4. When determined by a Biolayer Interference (BLI) assay, the K is about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less D for the anti-FIXa antibody or antigen-binding portion thereof according to any of the above embodiments that binds to FIXa.

[0043] Embodiment 5. The anti-FIXa antibody or antigen-binding portion thereof according to any of the above embodiments, wherein FIXa is free FIXa, FIXa in the tenase complex, or FIXa covalently linked to EGR-CMK (FIXa-SM).

[0044] Embodiment 6. The anti-FIXa antibody or antigen-binding portion thereof according to any of the above embodiments, wherein FIXz comprises inactivated Factor IX (FIXn).

[0045] Embodiment 7. The anti-FIXa antibody or antigen-binding portion thereof according to any of the above embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and 3C.

[0046] Embodiment 8. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the preceding embodiments that binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and 3C.

[0047] Embodiment 9. An anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 7 or 8, wherein the reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, BIIB-9-433, and any combination thereof.

[0048] Embodiment 10. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the preceding embodiments that binds preferentially to FIXa-SM compared to free FIXa or FIXz and / or binds to FIXa-SM with a binding affinity higher than the binding affinity of the anti-FIXa antibody or an antigen-binding portion thereof for free FIXa or FIXz.

[0049] Embodiment 11. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the preceding embodiments that cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3A.

[0050] Embodiment 12. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the preceding embodiments that binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3A.

[0051] Embodiment 13. An anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 11 or 12, wherein the reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-460, and any combination thereof.

[0052] Embodiment 14. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which binds preferentially to free FIXa compared to FIXa-SM or FIXz and / or binds to free FIXa with a binding affinity higher than the binding affinity of the anti-FIXa antibody or an antigen-binding portion thereof for FIXa-SM or FIXz.

[0053] Embodiment 15. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in Figure 3B.

[0054] Embodiment 16. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figure 3B.

[0055] Embodiment 17. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which binds preferentially to free FIXa or FIXa-SM compared to FIXz and / or binds to free FIXa or FIXa-SM with a binding affinity higher than the binding affinity of the anti-FIXa antibody or an antigen-binding portion thereof for FIXz.

[0056] Embodiment 18. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in Figure 3C.

[0057] Embodiment 19. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figure 3C.

[0058] Embodiment 20. The anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 18 or 19, wherein the reference antibody is selected from BIIB-9-882, BIIB-9-433, and any combination thereof. Embodiment 20. The anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 18 or 19, wherein the reference antibody is selected from BIIB-9-882, BIIB-9-433, and any combination thereof.

[0059] Embodiment 21. Any anti-FIXa antibody or antigen-binding portion thereof of the above embodiments, comprising CDR1, CDR2 and CDR3, wherein CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in FIGS. 3A, 3B and 3C or a VH CDR3 having one or two mutations.

[0060] Embodiment 22. Any anti-FIXa antibody or antigen-binding portion thereof of any one of Embodiments 1 to 21, comprising CDR1, CDR2 and CDR3, wherein CDR3 comprises ARDX 1 X 2 X 3 X 4 X 5 X 6 YYX 7 including MDV (SEQ ID NO: 753), wherein X 1 is V or G, and X 2 is G or V, and X 3 is G or R, and X 4 is Y or V, and X 5 is A or S, and X 6 is G or D, and X 7 is G or does not exist.

[0061] Embodiment 23. Any anti-FIXa antibody or antigen-binding portion thereof of Embodiment 22, wherein CDR3 comprises ARDVGGYAGYYGMDV (SEQ ID NO: 905, BIIB-9-484, 1335, 1336), ARDISTDGESSLYYYMDV (SEQ ID NO: 901, BIIB-9-460), ARGPTDSSGYLDMDV (SEQ ID NO: 1186, BIIB-9-882) or ARDGPRVSDYY MDV (SEQ ID NO: 912, BIIB-9-619).

[0062] Embodiment 24. Any anti-FIXa antibody or antigen-binding portion thereof of the above embodiments, comprising CDR1, CDR2 and CDR3, wherein CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIGS. 3A, 3B and 3C or a VH CDR1 having one or two mutations.

[0063] Embodiment 25. An anti-FIXa antibody or antigen-binding portion thereof according to any of the preceding embodiments, comprising VH CDR1, CDR2 and CDR3, wherein CDR2 is VH CDR2 as shown in FIGS. 3A, 3B and 3C or a VH having one or two mutations, and comprising a VH CDR2 selected from the group consisting of the CDR2s.

[0064] Embodiment 26. An anti-FIXa antibody or antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2 and CDR3, wherein CDR1 is VL CDR1 as shown in FIGS. 3A, 3B and 3C or a VL having one or two mutations, and comprising a VL CDR1 selected from the group consisting of the CDR1s.

[0065] Embodiment 27. An anti-FIXa antibody or antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2 and CDR3, wherein CDR2 is VL CDR2 as shown in FIGS. 3A, 3B and 3C or a VL having one or two mutations, and comprising a VL CDR2 selected from the group consisting of the CDR2s.

[0066] Embodiment 28. An anti-FIXa antibody or antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2 and CDR3, wherein CDR3 is VL CDR3 as shown in FIGS. 3A, 3B and 3C or a VL having one or two mutations, and comprising a VL CDR3 selected from the group consisting of the CDR3s.

[0067] Embodiment 29. An isolated anti-FIXa antibody or antigen-binding portion thereof that specifically binds to FIXa, comprising VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein VH CDR1, CDR2 and CDR3, and ​​​​An anti-FIXa antibody or an antigen-binding portion thereof, wherein the VL CDR1, CDR2, and CDR3 include the VH CDR1, VH CDR2, and VH CDR3, and the VL CDR1, CDR2, and CDR3 of FIGS. 3A, 3B, and 3C, respectively.

[0068] Embodiment 30. The anti-FIXa antibody or an antigen-binding portion thereof of Embodiment 29, including VH CDR1, CDR2, and CDR3 sequences each including SEQ ID NO: 815, 860, and 905, and / or VL CDR1, CDR2, and CDR3 sequences each including SEQ ID NO: 950, 995, and 1040 (BIIB-9-484).

[0069] Embodiment 31. (a1) The antibody includes VH CDR1, CDR2, and CDR3 sequences each including SEQ ID NO: 809, SEQ ID NO: 854, and SEQ ID NO: 899, and / or VL CDR1, VL CDR2, and VL CDR3 sequences each including SEQ ID NO: 944, SEQ ID NO: 989, and SEQ ID NO: 1034 (BIIB-9-440); (a2) The antibody includes VH CDR1, CDR2, and CDR3 sequences each including SEQ ID NO: 1102, SEQ ID NO: 1144, and SEQ ID NO: 1186, and / or VL CDR1, VL CDR2, and VL CDR3 sequences each including SEQ ID NO: 1228, SEQ ID NO: 1270, and SEQ ID NO: 1312 (BIIB-9-882); (a3) The antibody includes VH CDR1, CDR2, and CDR3 sequences each including SEQ ID NO: 811, SEQ ID NO: 856, and SEQ ID NO: 901, and / or VL CDR1, VL CDR2, and VL CDR3 sequences each including SEQ ID NO: 946, SEQ ID NO: 991, and SEQ ID NO: 1036 (BIIB-9-460); or (a4) The anti-FIXa antibody of embodiment 29 or an antigen-binding portion thereof, wherein the antibody comprises a VH CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 1108, SEQ ID NO: 1150, and SEQ ID NO: 1192, respectively, and / or a VL CDR1, VL CDR2, and VL CDR3 sequence comprising SEQ ID NO: 1234, SEQ ID NO: 1276, and SEQ ID NO: 1318, respectively (BIIB-9-433).

[0070] Embodiment 32. The anti-FIXa antibody of embodiment 29 or an antigen-binding portion thereof, wherein the antibody comprises a VH CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 822, SEQ ID NO: 867, and SEQ ID NO: 912, respectively, and / or a VL CDR1, VL CDR2, and VL CDR3 sequence comprising SEQ ID NO: 957, SEQ ID NO: 1002, and SEQ ID NO: 1047, respectively (BIIB-9-619).

[0071] Embodiment 33. (i) The anti-FIXa antibody of embodiment 29 or an antigen-binding portion thereof, wherein the antibody comprises a VH CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, respectively, and / or a VL CDR1, VL CDR2, and VL CDR3 sequence comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively, or (ii) the antibody comprises a VH CDR1, CDR2, and CDR3 sequence comprising SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, respectively, and / or a VL CDR1, VL CDR2, and VL CDR3 sequence comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively (BIIB-9-1335 and BIIB-9-1336).

[0072] Embodiment 34. An antibody or antigen-binding portion thereof according to any of the embodiments described above, comprising VH and VL, wherein VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 , 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181, and having an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.

[0073] Embodiment 35. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, comprising VH and VL, wherein VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical thereto.

[0074] Embodiment 36. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, comprising VH and VL, wherein VH is derived from a germline sequence of VH1-46.0, VH1-46.4, VH1-46.5, VH1-46.7, VH1-46.9, VH1-69.9, VH3-07.0, VH3-21.0, VH3-21.2, VH3-23.0, VH3-23.1, VH4-31.0, VH4-34.0, VH4-39.0, VH4-39.2, VH4-39.3, VH4-39.5, VH4-39.6, VH4-39.8, VH4-59.6, VH4-0B.4 or VH4-0B.6.

[0075] Embodiment 37. An anti-FIXa antibody or an antigen-binding portion thereof according to any of the above embodiments, comprising VH and VL, wherein VL is derived from a germline sequence of VK1-05.0, VK1-05.6, VK1-05.9, VK1-05.21, VK1-12.0, VK1-12.3, VK1-33.0, VK1-33.1, VK1-33.2, VK1-33.8, VK1-33.10, VK1-39.0, VK1-39.6, VK2-28.0, VK2-28.1, VK3-11.0, VK3-11.2, VK3-11.6, VK3-11.10, VK3-11.14, VK3-15.0, VK3-15.6, VK3-15.8, VK3-15.11, VK3-15.20, VK3-15.26, VK3-20.0, VK3-20.4, VK3-20.5, VK3-20.8, or VK4-01.0.

[0076] Embodiment 38. Comprising VH and VL, (a1) VH and VL each comprise SEQ ID NO: 31 and 221 (BIIB-9-484); (a2) VH and VL each comprise SEQ ID NO: 19 and 209 (BIIB-9-440); (a3) VH and VL each comprise SEQ ID NO: 115 and 301 (BIIB-9-882); (a4) VH and VL each comprise SEQ ID NO: 23 and 213 (BIIB-9-460); (a5) VH and VL each comprise SEQ ID NO: 127 and 313 (BIIB-9-4 33); (a6) VH and VL each comprise SEQ ID NO: 45 and 235 (BIIB-9-619); (a7) VH and VL each comprise SEQ ID NO: 185 and 371 (BIIB-9-578); (a8) VH and VL each comprise SEQ ID NO: 87 and 221 (BIIB-9-1335); or (a9) VH and VL each comprise SEQ ID NO: 89 and 221 (BIIB-9-1336) Any anti-FIXa antibody or antigen-binding portion thereof of the foregoing embodiments.

[0077] Embodiment 39. An isolated antibody or antigen-binding portion thereof that specifically binds to FIXz (the "anti-FIXz antibody or antigen-binding portion thereof"), wherein the anti-FIXz antibody or antigen-binding portion thereof preferentially binds to FIXz in the presence of free FIXa or FIXa-SM and / or the anti-FIXz antibody or antigen-binding portion thereof binds to FIXz with a binding affinity higher than its binding affinity for free FIXa or FIXa-SM.

[0078] Embodiment 40. Any anti-FIXz antibody or antigen-binding portion thereof of the foregoing embodiments that cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3D.

[0079] Embodiment 41. Any anti-FIXz antibody or antigen-binding portion thereof of the foregoing embodiments that binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3D.

[0080] Embodiment 42. The anti-FIXz antibody or antigen-binding portion thereof of Embodiment 40 or 41, wherein the reference antibody is BIIB-9-578.

[0081] Embodiment 43. Any anti-FIXz antibody or antigen-binding portion thereof of the foregoing embodiments, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in FIG. 3D or a VH CDR3 having one or two mutations.

[0082] Embodiment 44. The anti-FIXz antibody or antigen-binding portion thereof of Embodiment 43, wherein CDR3 comprises ARDKYQDYSFDI (SEQ ID NO: 1355, BIIB-9-578).

[0083] Embodiment 45. An anti-FIXz antibody or an antigen-binding portion thereof according to any of the preceding embodiments, comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIG. 3D or a VH CDR1 having one or two mutations.

[0084] Embodiment 46. An anti-FIXz antibody or an antigen-binding portion thereof according to any of the preceding embodiments, comprising VH CDR1, CDR2, and CDR3, wherein CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIG. 3D or a VH CDR2 having one or two mutations.

[0085] Embodiment 47. An anti-FIXz antibody or an antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2, and CDR3, wherein CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIG. 3D or a VL CDR1 having one or two mutations.

[0086] Embodiment 48. An anti-FIXz antibody or an antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2, and CDR3, wherein CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIG. 3D or a VL CDR2 having one or two mutations.

[0087] Embodiment 49. An anti-FIXz antibody or an antigen-binding portion thereof according to any of the preceding embodiments, comprising VL CDR1, CDR2, and CDR3, wherein CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIG. 3D or a VL CDR3 having one or two mutations.

[0088] Embodiment 50. An anti-FIX antibody or an antigen-binding portion thereof according to any one of the preceding embodiments and Embodiments 169 to 204, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof.

[0089] Embodiment 51. The anti-FIX antibody or antigen-binding portion thereof according to Embodiment 50, wherein the antibody is an IgG4 antibody.

[0090] Embodiment 52. The anti-FIX antibody or antigen-binding portion thereof according to Embodiment 50, wherein the antibody comprises an effectorless IgG4 Fc.

[0091] Embodiment 53. The anti-FIX antibody or antigen-binding portion thereof according to any one of the above embodiments and Embodiments 169 to 204, which comprises a heavy chain constant region.

[0092] Embodiment 54. The anti-FIX antibody according to any one of the above embodiments and Embodiments 169 to 204, wherein the antibody is a human antibody, an engineered antibody or a humanized antibody.

[0093] Embodiment 55. The anti-FIX antigen-binding portion according to any one of the above embodiments and Embodiments 169 to 204, wherein the antigen-binding portion comprises Fab, Fab’, F(ab’)2, Fv or single-chain Fv (scFv).

[0094] Embodiment 56. A bispecific molecule comprising the anti-FIX antibody or antigen-binding portion thereof according to any one of the above embodiments and Embodiments 169 to 204, which is linked to a molecule having a second binding specificity.

[0095] Embodiment 57. A nucleic acid encoding the heavy chain and / or light chain variable region of the anti-FIX antibody or antigen-binding portion thereof according to any one of Embodiments 1 to 55 and Embodiments 169 to 204, or the bispecific molecule according to Embodiment 56.

[0096] Embodiment 58. An expression vector comprising the nucleic acid molecule according to Embodiment 57.

[0097] Embodiment 59. A cell transformed with the expression vector according to Embodiment 58.

[0098] Embodiment 60. An immunoconjugate comprising an antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 55 and Embodiments 169 to 204, or the bispecific molecule of Embodiment 56, which is conjugated to a drug.

[0099] Embodiment 61. A composition comprising an antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 55 and Embodiments 169 to 204, the bispecific molecule of Embodiment 56, or the immunoconjugate of Embodiment 60, and a carrier.

[0100] Embodiment 62. A kit comprising an antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 55 and Embodiments 169 to 204, the bispecific molecule of Embodiment 56, or the immunoconjugate of Embodiment 60, and instructions for use.

[0101] Embodiment 63. A method for producing an anti-FIX antibody or an antigen-binding portion thereof, comprising the steps of expressing the antibody or an antigen-binding portion thereof in the cells of Embodiment 59, and isolating the antibody or an antigen-binding portion thereof from the cells.

[0102] Embodiment 64. A method for measuring the level of activated FIX in a subject in need thereof, comprising contacting a sample obtained from the subject with an anti-FIXa antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 55 and Embodiments 169 to 204 under suitable conditions, and measuring the binding of the anti-FIXa antibody or an antigen-binding portion thereof in the sample to FIXa.

[0103] Embodiment 65. The method of Embodiment 64, wherein the sample is blood or serum.

[0104] Embodiment 66. An isolated antibody or antigen-binding portion thereof that specifically binds to factor X zymogen (FXz) (the "anti-FXz antibody or antigen-binding portion thereof"), wherein the anti-FXz antibody or antigen-binding portion thereof preferentially binds to FXz in the presence of FXz and activated factor X (FXa).

[0105] Embodiment 67. The anti-FXz antibody or antigen-binding portion thereof according to Embodiment 66, which binds to FXz with a binding affinity higher than the binding affinity of the antibody or antigen-binding portion thereof for FXa.

[0106] Embodiment 68. An isolated anti-FXz antibody or antigen-binding portion thereof that binds to FXz with a binding affinity higher than the binding affinity of the antibody or antigen-binding portion thereof for FXa.

[0107] Embodiment 69. When measured by BLI, the K is about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less. D The anti-FXz antibody or antigen-binding portion thereof according to any one of Embodiments 66 to 68, which binds to FXz.

[0108] Embodiment 70. The anti-FXz antibody or antigen-binding portion thereof according to any one of Embodiments 66 to 69, wherein FXa is free FXa or FXa (FIXa-SM) covalently linked to EGR-CMK.

[0109] Embodiment 71. The anti-FXz antibody or antigen-binding portion thereof according to any one of Embodiments 66 to 70, wherein FXz contains inactive factor X (FXn).

[0110] Embodiment 72. The anti-FXz antibody or antigen-binding portion thereof according to any one of Embodiments 66 to 71, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIGS. 12A and 12B.

[0111] Embodiment 73. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 72 that binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIGS. 12A and 12B.

[0112] Embodiment 74. An anti-FXz antibody or an antigen-binding portion thereof according to Embodiment 73 that binds to the same epitope as a reference antibody selected from the group consisting of BIIB-12-915, BIIB-12-917, BIIB-12-932, and any combination thereof.

[0113] Embodiment 75. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 74, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3s in FIGS. 12A and 12B or a VH CDR3 having one or two mutations.

[0114] Embodiment 76. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 75, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises 1 X 2 X 3 RX 4 X 5 X 6 X 7 FDX 8 (SEQ ID NO: 766), wherein X 1 is G or L, X 2 is R or G, X 3 is F or Y, X 4 is P or G, X 5 is R or A, X 6 is G or S, X 7 is R or A, X 8 is Y or I.

[0115] Embodiment 77. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 76, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises ARGRFRPRGRFDY (SEQ ID NO: 1575, BIIB-12-917), ARLGYRGASAFDI (SEQ ID NO: 1589, BIIB-12-932), or ARVGGGYANP (SEQ ID NO: 1573, BIIB-12-915).

[0116] Embodiment 78. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 77, comprising VH CDR1, CDR2, and CDR3, wherein CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIGS. 12A and 12B or a VH CDR1 having one or two mutations.

[0117] Embodiment 79. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 78, comprising VH CDR1, CDR2, and CDR3, wherein CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIGS. 12A and 12B or a VH CDR2 having one or two mutations.

[0118] Embodiment 80. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 79, comprising VL CDR1, CDR2, and CDR3, wherein CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIGS. 12A and 12B or a VL CDR1 having one or two mutations.

[0119] Embodiment 81. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 80, comprising VL CDR1, CDR2, and CDR3, wherein CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIGS. 12A and 12B or a VL CDR2 having one or two mutations.

[0120] Embodiment 82. An anti-FXz antibody or antigen-binding portion thereof according to any one of Embodiments 66 to 81, comprising VL CDR1, CDR2 and CDR3, wherein CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIGS. 12A and 12B or a VL CDR3 having one or two mutations.

[0121] Embodiment 83. An isolated antibody or antigen-binding portion thereof that specifically binds to FXz, comprising VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3 are the VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3 in FIGS. 12A and 12B, respectively, of an isolated antibody or antigen-binding portion thereof.

[0122] Embodiment 84. The anti-FXz antibody or antigen-binding portion thereof according to Embodiment 83, wherein the antibody comprises VH CDR1, CDR2 and CDR3 sequences each comprising SEQ ID NO: 1393, 1483 or 1573, and / or VL CDR1, VL CDR2 and VL CDR3 sequences each comprising SEQ ID NO: 1663, 1753 and 1843 (BIIB-12-915).

[0123] Embodiment 85. The anti-FXz antibody or antigen-binding portion thereof according to Embodiment 83, wherein the antibody comprises VH CDR1, CDR2 and CDR3 sequences each comprising SEQ ID NO: 1395, 1485 or 1575, and / or VL CDR1, VL CDR2 and VL CDR3 sequences each comprising SEQ ID NO: 1665, 1755 and 1845 (BIIB-12-917).

[0124] Embodiment 86. An anti-FXz antibody or an antigen-binding portion thereof according to Embodiment 83, wherein the antibodies each comprise VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 1409, 1499, or 1589, and / or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 1679, 1769, and 1859, respectively (BIIB-12-932).

[0125] Embodiment 87. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 86, comprising VH and VL, wherein VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, and 555, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.

[0126] Embodiment 88. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 87, comprising VH and VL, wherein VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 565, 567, 569, 571, 573, 575, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, and 743, and an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.

[0127] Embodiment 89. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 88, comprising VH and VL, wherein VH is derived from a germline sequence of VH1-18.0, VH1-18. 1, VH1-18.8, VH1-46.0, VH1-46.4, VH1-46.5, VH1-46.6, VH1-46.7, VH1-46.8, VH1-46.9, VH3-21.0, VH3-23.0, VH3-23.2, VH3-23.6, VH3-30.0, VH4-31.5, VH4-39.0, VH4-39.5, VH4-0B.4 or VH5-51.1.

[0128] Embodiment 90. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 89, comprising VH and VL, wherein VL is derived from a germline sequence among VK1-05.6, VK1-05.12, VK1-12.0, VK1-12.4, VK1-12.7, VK1-12.10, VK1-12.15, VK1-39.0, VK1-39.3, VK1-39.15, VK2-28.0, VK2-28.1, VK2-28.5, VK3-11.0, VK3-11.2, VK3-11.6, VK3-11.14, VK3-15.0, VK3-15.8, VK3-15.10, VK3-20.0, VK3-20.1, VK3-20.4, VK3-20.5, VK4-01.0, VK4-01.4, VK4-01.20.

[0129] Embodiment 91. An anti-FXz antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 90, comprising VH and VL, (b1) wherein VH and VL each comprise SEQ ID NO: 423 and 611 (BIIB-12-915); (b2) wherein VH and VL each comprise SEQ ID NO: 427 and 615 (BIIB-12-917); or (b3) wherein VH and VL each comprise SEQ ID NO: 455 and 643 (BIIB-12-932).

[0130] Embodiment 92. An isolated antibody or an antigen-binding portion thereof that specifically binds to activated factor X (FXa) (the "anti-FXa antibody or an antigen-binding portion thereof"), wherein the anti-FXa antibody or an antigen-binding portion thereof preferentially binds to FXa in the presence of FXz and FXa, and / or binds to FXa with a binding affinity higher than the binding affinity of the antibody or an antigen-binding portion thereof for FXz.

[0131] Embodiment 93. The anti-FXa antibody or an antigen-binding portion thereof according to Embodiment 92, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 12C.

[0132] An anti-FXa antibody or an antigen-binding portion thereof according to Embodiment 92 or 93 that binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 12C.

[0133] Embodiment 95. An anti-FXa antibody or an antigen-binding portion thereof according to Embodiment 94 that binds to the same epitope as a reference antibody selected from the group consisting of BIIB-12-925.

[0134] Embodiment 96. An anti-FXa antibody or an antigen-binding portion thereof according to any one of Embodiments 92 to 95, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in FIG. 12C or a VH CDR3 having one or two mutations.

[0135] Embodiment 97. An anti-FXa antibody or an antigen-binding portion thereof according to any one of Embodiments 92 to 96, comprising CDR1, CDR2, and CDR3, wherein CDR3 comprises AKGPRYYWYSWYFDL (SEQ ID NO: 1919, BIIB-12-925).

[0136] Embodiment 98. An anti-FXa antibody or an antigen-binding portion thereof according to any one of Embodiments 92 to 97, comprising VH CDR1, CDR2, and CDR3, wherein CDR1 comprises a VH CDR1 selected from the group consisting of the VH CDR1 in FIG. 12C or a VH CDR1 having one or two mutations.

[0137] Embodiment 99. An anti-FXa antibody or an antigen-binding portion thereof according to any one of Embodiments 92 to 98, comprising VH CDR1, CDR2, and CDR3, wherein CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in FIG. 12C or a VH CDR2 having one or two mutations.

[0138] Embodiment 100. An anti-FXa antibody or antigen-binding portion thereof according to any one of Embodiments 92 to 99, comprising VL CDR1, CDR2 and CDR3, wherein CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in FIG. 12C or a VL CDR1 having one or two mutations.

[0139] Embodiment 101. An anti-FXa antibody or antigen-binding portion thereof according to any one of Embodiments 92 to 100, comprising VL CDR1, CDR2 and CDR3, wherein CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in FIG. 12C or a VL CDR2 having one or two mutations.

[0140] Embodiment 102. An anti-FXa antibody or antigen-binding portion thereof according to any one of Embodiments 92 to 101, comprising VL CDR1, CDR2 and CDR3, wherein CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in FIG. 12C or a VL CDR3 having one or two mutations.

[0141] Embodiment 103. An isolated antibody or antigen-binding portion thereof that specifically binds to FXa, comprising VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3 comprise the VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3 in FIG. 12C, respectively, of the isolated antibody or antigen-binding portion thereof.

[0142] Embodiment 104. The anti-FXa or antigen-binding portion thereof according to Embodiment 103, wherein the antibody comprises VH CDR1, CDR2 and CDR3 sequences each comprising SEQ ID NO: 1911, 1915 or 1919, and / or VL CDR1, VL CDR2 and VL CDR3 sequences (BIIB-12-925) each comprising SEQ ID NO: 1923, 1927 or 1931.

[0143] Embodiment 105. An anti-FXa antibody or an antigen-binding portion thereof according to any one of Embodiments 92 to 104, comprising VH and VL, wherein VH and VL each comprise SEQ ID NO: 559 and 747 (BIIB-12-925).

[0144] Embodiment 106. An anti-FX antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 105, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0145] Embodiment 107. The anti-FX antibody or an antigen-binding portion thereof according to Embodiment 106, wherein the antibody is an IgG4 antibody.

[0146] Embodiment 108. The anti-FX antibody or an antigen-binding portion thereof according to Embodiment 106, wherein the antibody comprises an effectorless IgG4 Fc. 6.

[0147] Embodiment 109. An anti-FX antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 108, comprising a heavy chain constant region.

[0148] Embodiment 110. An anti-FX antibody according to any one of Embodiments 66 to 109, wherein the antibody is a human antibody, an engineered antibody, or a humanized antibody.

[0149] Embodiment 111. An anti-FX antigen-binding portion according to any one of Embodiments 66 to 110, wherein the antigen-binding portion comprises Fab, Fab’, F(ab’)2, Fv, or single-chain Fv (scFv).

[0150] Embodiment 112. A bispecific molecule comprising an anti-FX antibody according to any one of Embodiments 66 to 111, linked to a molecule having a second binding specificity.

[0151] Embodiment 113. A nucleic acid encoding the heavy chain and / or light chain variable region of an antibody or an antigen-binding portion thereof according to any one of Embodiments 66 to 111, or the bispecific molecule according to Embodiment 112.

[0152] Embodiment 114. An expression vector comprising the nucleic acid molecule of Embodiment 113.

[0153] Embodiment 115. A cell transformed with the expression vector of Embodiment 114.

[0154] Embodiment 116. An immunoconjugate comprising any one antibody or antigen-binding portion thereof of Embodiments 66 to 111, or the bispecific molecule of Embodiment 112, and linked to a drug.

[0155] Embodiment 117. A composition comprising any one antibody or antigen-binding portion thereof of Embodiments 66 to 111, or the bispecific molecule of Embodiment 112, or the immunoconjugate of Embodiment 116, and a carrier.

[0156] Embodiment 118. A kit comprising any one antibody or antigen-binding portion thereof of Embodiments 66 to 111, or the bispecific molecule of Embodiment 112, or the immunoconjugate of Embodiment 116, and instructions for use.

[0157] Embodiment 119. A method for producing an anti-FX antibody or antigen-binding portion thereof, comprising the steps of expressing the antibody or antigen-binding portion thereof in the cell of Embodiment 115, and isolating the antibody or antigen-binding portion thereof from the cell.

[0158] Embodiment 120. A method for measuring prothrombin FX (FXz) in a subject in need thereof, comprising contacting any one anti-FX antibody or antigen-binding portion thereof of Embodiments 66 to 111 with a sample obtained from the subject under suitable conditions, and measuring the binding of the anti-FX antibody or antigen-binding portion thereof in the sample to FXz.

[0159] Embodiment 121. The method of Embodiment 120, wherein the sample is blood or serum from the subject.

[0160] Embodiment 122. Any one of the anti-FIX antibodies or antigen-binding portions thereof according to Embodiments 1 to 55 and Embodiments 169 to 204, and (ii) any one of the anti-FX antibodies or antigen-binding portions thereof according to Embodiments 66 to 111. A bispecific molecule comprising any one of the anti-FX antibodies or antigen-binding portions thereof.

[0161] Embodiment 123. A bispecific molecule according to Embodiment 122 that cross-competes with a reference bispecific antibody, wherein the reference bispecific antibody comprises the VH and VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIGS. 3A, 3B, 3C, and 3D, and the VH and VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIGS. 12A, 12B, and 12C.

[0162] Embodiment 124. A bispecific molecule according to Embodiment 122 or 123 that binds to the same epitope as a reference bispecific antibody, wherein the reference bispecific antibody comprises the VH and VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIGS. 3A, 3B, 3C, and 3D, and the VH and VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIGS. 12A, 12B, and 12C.

[0163] Embodiment 125. (i) The anti-FIX antibody or antigen-binding portion thereof comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, and VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 of the anti-FIX (BIIB-9) antibody in FIGS. 16A, 16B, 16C, and 16D; and (ii) The anti-FX antibody or antigen-binding portion thereof comprises VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, and VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3 are selected from the group consisting of VH CDR1, VH CDR2 and VH CDR3 and VL CDR1, VL CDR2 and VL CDR3 of the anti-FX (BIIB-12) antibody in FIGS. 16A, 16B, 16C and 16D, and is any one of the bispecific molecules of Embodiments 122 to 124.

[0164] Embodiment 126. (a) The anti-FIX antibody or antigen-binding portion thereof (a1) VH CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 815, 860 or 905, and / or VL CDR1, VL CDR2 and VL CDR3 sequences each containing SEQ ID NO: 950, 995 or 1040 (BIIB-9-484); (a2) VH CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 822, 867 and 912, and / or VL CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 957, 1002 and 1047 (BIIB-9-619); (a3) VH CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 1347, 1351 and 1355, and / or VL CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 1359, 1363 and 1367 (BIIB-9-578); (a4) VH CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 843, 888 and 933, and / or VL CDR1, CDR2 and CDR3 sequences each containing SEQ ID NO: 978, 1023 and 1068 (BIIB-9-1335); or (a5) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 844, 889, and 934, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 979, 1024, and 1069, respectively (BIIB-9-1336); (b) The anti-FX antibody or antigen-binding portion thereof (b1) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1393, 1483, and 1573, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1663, 1753, and 1843, respectively (BIIB-12-915); (b2) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1395, 1485, and 1575, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1665, 1755, and 1845, respectively (BIIB-12-917); (b3) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1911, 1915, and 1919, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1923, 1927, and 1931, respectively (BIIB-12-925); (b4) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1409, 1499, and 1589, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1679, 1769, and 1859, respectively (BIIB-12-932); or (b5) Each contains a VH CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1433, 1523, and 1613, respectively, and / or a VL CDR1, CDR2, and CDR3 sequence containing SEQ ID NOs: 1703, 1793, and 1883, respectively (BIIB-12-1306), a bispecific molecule of any one of Embodiments 122 to 124.

[0165] Embodiment 127. (a) The anti-FIX antibody or antigen-binding portion thereof (a1) VH and VL (BIIB-9-484) each containing SEQ ID NO: 31 and 221; (a2) VH and VL (BIIB-9-619) each containing SEQ ID NO: 45 and 235; (a3) VH and VL (BIIB-9-578) each containing SEQ ID NO: 185 and 371; (a4) VH and VL (BIIB-9-1335) each containing SEQ ID NO: 87 and 221; or (a5) containing VH and VL (BIIB-9-1336) each containing SEQ ID NO: 89 and 221; (b) The anti-FX antibody or antigen-binding portion thereof is (b1) VH and VL (BIIB-12-915) each containing SEQ ID NO: 423 and 611; (b2) VH and VL (BIIB-12-917) each containing SEQ ID NO: 427 and 615; (b3) VH and VL (BIIB-12-925) each containing SEQ ID NO: 559 and 747; (b4) VH and VL (BIIB-12-932) each containing SEQ ID NO: 455 and 643; or (b5) A bispecific molecule of any one of Embodiments 122 to 124, containing VH and VL (BIIB-12-1306) each containing SEQ ID NO: 503 and 691.

[0166] Embodiment 128. (i) The anti-FIX antibody or antigen-binding portion thereof contains VH and VL (BIIB-9-484) each containing SEQ ID NO: 31 and 221, and the anti-FX antibody or antigen-binding portion thereof contains VH and VL (BIIB-12-915) each containing SEQ ID NO: 423 and 611; (ii) The anti-FIX antibody or antigen-binding portion thereof contains VH and VL (BIIB-9-484) each containing SEQ ID NO: 31 and 221, and the anti-FX antibody or antigen-binding portion thereof contains VH and VL (BIIB-12-917) each containing SEQ ID NO: 427 and 615; (iii) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-484) comprising SEQ ID NOs: 31 and 221, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-925) comprising SEQ ID NOs: 559 and 747, respectively; (iv) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-484) comprising SEQ ID NOs: 31 and 221, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-932) comprising SEQ ID NOs: 455 and 643, respectively; (v) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-578) comprising SEQ ID NOs: 185 and 371, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-915) comprising SEQ ID NOs: 423 and 611, respectively; (vi) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-578) comprising SEQ ID NOs: 185 and 371, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-917) comprising SEQ ID NOs: 427 and 615, respectively; (vii) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-619) comprising SEQ ID NOs: 45 and 235, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-917) comprising SEQ ID NOs: 427 and 615, respectively; or (viii) The anti-FIX antibody or its antigen-binding portion comprises VH and VL (BIIB-9-619) comprising SEQ ID NOs: 45 and 235, respectively, and the anti-FX antibody or its antigen-binding portion comprises VH and VL (BIIB-12-925) comprising SEQ ID NOs: 559 and 747, respectively, a bispecific molecule of any one of Embodiments 122 to 127.

[0167] Embodiment 129. In at least one FVIIIa activity assay, any one of the bispecific molecules of Embodiments 122 to 128 that functionally mimics activated Factor VIII (FVIIIa) cofactor.

[0168] Embodiment 130. The bispecific molecule of Embodiment 129, wherein the FVIIIa activity assay is selected from a chromogenic FXa generation assay, a one-stage clotting assay, or a combination thereof.

[0169] Embodiment 131. In the same assay, the FVIIIa activity achieves at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% of the activity normally achieved by FVIII. The bispecific molecule of Embodiment 129 or 130.

[0170] Embodiment 132. Any one of the bispecific molecules of Embodiments 122 to 131 that can generate thrombin from prothrombin, fibrin from fibrinogen, and / or fibrin clots in vitro or in vivo.

[0171] Embodiment 133. The bispecific molecule according to any one of Embodiments 122 to 132 that binds simultaneously to both FIXa and FX as determined by BLI.

[0172] Embodiment 134. Any one of the bispecific molecules of Embodiments 122 to 133 that is of IgG isotype.

[0173] Embodiment 135. The bispecific molecule of Embodiment 134, wherein the IgG isotype is IgG1 subclass.

[0174] Embodiment 136. The bispecific molecule of Embodiment 134, wherein the IgG isotype is IgG4 subclass.

[0175] Embodiment 137. A bispecific molecule according to any one of Embodiments 122 to 136, which is a bispecific IgG format and is selected from the group consisting of the antibodies in Table 2.

[0176] Embodiment 138. The bispecific molecule of Embodiment 137, which is a bispecific heterodimer format.

[0177] Embodiment 139. A bispecific molecule according to any one of Embodiments 122 to 138, which comprises two different heavy chains and two different light chains.

[0178] Embodiment 140. A bispecific molecule according to any one of Embodiments 122 to 138, which comprises two identical light chains and two different heavy chains.

[0179] Embodiment 141. A bispecific molecule according to any one of Embodiments 122 to 140, wherein the bispecific molecule is capable of controlling or reducing the incidence of bleeding episodes in a subject having hemophilia.

[0180] Embodiment 142. A bispecific molecule according to any one of Embodiments 122 to 140, which is capable of maintaining homeostasis in a subject having hemophilia.

[0181] Embodiment 143. A bispecific molecule according to any one of Embodiments 122 to 140, which can achieve the prevention of a determined procedure in a subject having hemophilia.

[0182] Embodiment 144. A bispecific molecule according to any one of Embodiments 122 to 143, wherein the subject produces or is expected to produce a neutralizing antibody against Factor VIII.

[0183] Embodiment 145. An immunoconjugate comprising a bispecific molecule according to any one of Embodiments 122 to 144, which is conjugated to a drug.

[0184] Embodiment 146. A composition comprising any one of the bispecific molecules of Embodiments 122 to 144, or the immunoconjugate of Embodiment 145, and a carrier.

[0185] Embodiment 147. A kit comprising any one of the bispecific molecules of Embodiments 122 to 144, or the immunoconjugate of Embodiment 145, and instructions for use.

[0186] Embodiment 148. A nucleic acid sequence encoding any one of the bispecific molecules of Embodiments 122 to 144.

[0187] Embodiment 149. A vector comprising the nucleic acid according to Embodiment 148.

[0188] Embodiment 150. A host cell comprising the vector of Embodiment 149.

[0189] Embodiment 151. The host cell of Embodiment 150, wherein the host cell is a prokaryotic cell, a eukaryotic cell, a protist cell, an animal cell, a plant cell, a fungal cell, a yeast cell, an Sf9 cell, a mammalian cell, an avian cell, an insect cell, a CHO cell, a HEK cell or a COS cell.

[0190] Embodiment 152. A method for producing a bispecific molecule, comprising culturing the host cell of Embodiment 150 under conditions that allow expression of the bispecific molecule.

[0191] Embodiment 153. A method for producing any one of the bispecific molecules of Embodiments 122 to 144, further comprising conditions that enhance heterodimerization.

[0192] Embodiment 154. A method for promoting FX activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any one of the bispecific molecules of Embodiments 122 to 144, the immunoconjugate of Embodiment 145, the composition of Embodiment 146, the nucleic acid of Embodiment 148, the vector of Embodiment 149, or the host cell of Embodiment 150.

[0193] Embodiment 155. A method for reducing the frequency or degree of bleeding episodes in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the bispecific molecules of Embodiments 122 to 144, the immunoconjugate of Embodiment 145, the composition of Embodiment 146, the nucleic acid of Embodiment 148, the vector of Embodiment 149, or the host cell of Embodiment 150.

[0194] Embodiment 156. The method of Embodiment 155, wherein the subject has developed or has a tendency to develop an inhibitor against factor VIII ("FVIII").

[0195] Embodiment 157. The method of Embodiment 156, wherein the inhibitor against FVIII is a neutralizing antibody against FVIII.

[0196] Embodiment 158. The method of any one of Embodiments 155 to 157, wherein the bleeding episode is the result of arthropathy, muscle bleeding, oral bleeding, hemorrhage, bleeding into muscle, intraoral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intra-abdominal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the iliopsoas sheath, or any combination thereof.

[0197] Embodiment 159. A method for treating a blood coagulation disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the bispecific molecules of Embodiments 122 to 144, the immunoconjugate of Embodiment 145, the composition of Embodiment 146, the nucleic acid of Embodiment 148, the vector of Embodiment 149, or the host cell of Embodiment 150.

[0198] Embodiment 160. The method of Embodiment 159, wherein the blood coagulation disorder is hemophilia A or hemophilia B.

[0199] Embodiment 161. The method of any one of Embodiments 154 to 160, wherein the subject is a human subject.

[0200] Embodiment 162. The method according to any one of Embodiments 154 to 160, wherein the subject is undergoing or has undergone FVIII replacement therapy.

[0201] Embodiment 163. The method according to any one of Embodiments 154 to 162, wherein the bispecific molecule is administered in combination with a hemophilia treatment method.

[0202] Embodiment 164. The method of Embodiment 163, wherein the hemophilia treatment method is FVIII replacement therapy 。

[0203] Embodiment 165. The method according to Embodiment 163 or 164, wherein the bispecific molecule is administered before, during or after the administration of the hemophilia treatment method.

[0204] Embodiment 166. The method according to any one of Embodiments 154 to 165, wherein the bispecific molecule is administered intravenously or subcutaneously.

[0205] Embodiment 167. The method according to any one of Embodiments 154 to 166, wherein the administration of the bispecific molecule reduces the occurrence of bleeding episodes, the frequency of spontaneous bleeding episodes or acute bleeding.

[0206] Embodiment 168. The method of Embodiment 167, wherein the annual bleeding rate is reduced by 5%, 10%, 20%, 30% or 50% by the administration of the bispecific molecule.

[0207] Embodiment 169. An anti-FIXa antibody or an antigen-binding portion thereof that binds to the same epitope as BIIB-9-1336.

[0208] Embodiment 170. An anti-FIXa antibody or an antigen-binding portion thereof that binds to an epitope overlapping with the BIIB-9-1336 epitope.

[0209] Embodiment 171. An anti-FIXa antibody or an antigen-binding portion thereof that binds to an epitope region containing at least one amino acid located between positions (i) 91 and 101, (ii) 125 and 128, (iii) 165 and 179, or (iv) 232 and 241 according to chymotrypsinogen numbering in the heavy chain sequence of FIXa.

[0210] Embodiment 172. An anti-FIXa antibody or an antigen-binding portion thereof that binds to an epitope containing at least one of the amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 according to chymotrypsinogen numbering in the heavy chain sequence of FIXa.

[0211] Embodiment 173. The anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 171 or 172, wherein the epitope contains the amino acid residues N93, R165, N178, and R233 according to chymotrypsinogen numbering in the heavy chain sequence of FIXa.

[0212] Embodiment 174. The anti-FIXa antibody or an antigen-binding portion thereof according to Embodiments 171 to 173, wherein the epitope contains the amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 according to chymotrypsinogen numbering in the heavy chain sequence of FIXa.

[0213] Embodiment 175. The anti-FIXa antibody or an antigen-binding portion thereof according to Embodiments 171 to 174, wherein the epitope does not contain at least one of the amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 according to chymotrypsinogen numbering in the heavy chain sequence of FIXa.

[0214] Embodiment 176. An anti-FIXa antibody or an antigen-binding portion thereof according to Embodiments 171 to 175, wherein the epitope does not include amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the heavy chain sequence of FIXa according to chymotrypsinogen numbering. 5.

[0215] Embodiment 177. An anti-FIXa antibody or an antigen-binding portion thereof according to Embodiments 171 to 176, which binds to at least one amino acid residue (SEQ ID NO: 756) in the light chain of FIXa.

[0216] Embodiment 178. An anti-FIXa antibody or an antigen-binding portion thereof according to Embodiment 177, wherein the amino acid residue (SEQ ID NO: 756) in the light chain of FIXa is K100.

[0217] Embodiment 179. An anti-FIXa antibody or an antigen-binding portion thereof according to any one of Embodiments 169 to 178, wherein the epitope overlaps with the binding site of FVIIIa to FIXa.

[0218] Embodiment 180. An anti-FIXa antibody or an antigen-binding portion thereof according to any one of Embodiments 169 to 179, which cross-competes with FVIIIa for binding to FIXa.

[0219] Embodiment 181. An anti-FIXa antibody or an antigen-binding portion thereof according to any one of Embodiments 169 to 180, which blocks the binding of FVIIIa to FIXa.

[0220] Embodiment 182. Specifically binds to FIXa and includes VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, (i) VH CDR1 includes a VH CDR1 selected from the group consisting of the VH CDR1 in Table 7 or a VH CDR1 having one or two mutations; and / or (ii) The VH CDR2 comprises a VH CDR2 selected from the group consisting of the VH CDR2 in Table 7 or a VH CDR2 having one or two mutations; and / or (iii) The VH CDR3 comprises a VH CDR3 selected from the group consisting of the VH CDR3 in Table 7 or a VH CDR3 having one or two mutations; and / or (iv) The VL CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 in Table 7 or a VL CDR1 having one or two mutations; and / or (v) The VL CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 in Table 7 or a VL CDR2 having one or two mutations; and / or (vi) The VL CDR3 comprises a VL CDR3 selected from the group consisting of the VL CDR3 in Table 7 or a VL CDR3 having one or two mutations. An anti-FIXa antibody or an antigen-binding portion thereof according to any one of Embodiments 1 to 12.

[0221] Embodiment 183. An anti-FIXa antibody or an antigen-binding portion thereof that specifically binds to FIXa and comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 has the amino acid sequence ARDX 1 GGYAGYYGMDV (SEQ ID NO: 2196), and X 1 is L or V.

[0222] Embodiment 184. (i) The VH CDR1 has the amino acid sequence FTFX 1 SX 2 X 3 MX 4 (SEQ ID NO: 2194), and X 1 is S, G, or E, X 2 is Y or F, X 3 is S, E, G, or D, and X 4 is N, V, A, or T; and / or (ii) The VH CDR2 has the amino acid sequence X 5 ISX6 X 7 X 8 X 9 X 10 including IYYADSVKG (SEQ ID NO: 2195), and X 5 where is S, A, Y or G, and X 6 where is S or A, and X 7 where is S, A or G, and X 8 where is S, G or D, and X 9 where is S, T or G, and X 10 The isolated anti-FIXa antibody or antigen-binding portion thereof of Embodiment 183, where is Y or T.

[0223] Embodiment 185. An isolated anti-FIXa antibody or antigen-binding portion thereof from Embodiment 182, including VL CDR1, CDR2 and CDR3, and VL CDR3 includes the amino acid sequence QQYANFPYT (SEQ ID NO: 2168). An isolated anti-FIXa antibody or antigen-binding portion thereof of any one of 184.

[0224] Embodiment 186. An isolated anti-FIXa antibody or antigen-binding portion thereof of Embodiment 185, including VL CDR1, CDR2 and CDR3, (i) VL CDR1 includes the amino acid sequence QASQDIANYLN (SEQ ID NO: 2116); and / or (ii) VL CDR2 includes the amino acid sequence DASNLET (SEQ ID NO: 2142).

[0225] Embodiment 187. The anti-FIXa antibody or antigen-binding portion thereof of Embodiment 182, including VH CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 2038-2047, VH CDR2 and CDR3 selected from SEQ ID NOs: 2064-2073, and VH CDR3 selected from SEQ ID NOs: 2090-2099, and / or VL CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 2116-2125, VL CDR2 selected from SEQ ID NOs: 2142-2151, and VL CDR3 selected from SEQ ID NOs: 2168-2177.

[0226] Specifically binds to FIXa and includes VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VH CDR3 has the amino acid sequence X 1 RDVX 2 GYAGX 3 includes YGMDV (SEQ ID NO: 2198), and X 1 is A or V, and X 2 is G or S, and X 3 is Y or F, the isolated anti-FIXa antibody of Embodiment 182 or an antigen-binding portion thereof.

[0227] Embodiment 189. (i) VH CDR1 includes the amino acid sequence FTFGSYDMN (SEQ ID NO: 2048); and / or (ii) VH CDR2 has the amino acid sequence SISX 1 X 2 X 3 SYIX 4 YAX 5 includes SVKG (SEQ ID NO: 2197), and X 1 is S or D, and X 2 is G or S, and X 3 is E or A, and X 4 is Y or A, and X 5 is E or D, the isolated anti-FIXa antibody of Embodiment 188 or an antigen-binding portion thereof.

[0228] Specifically binds to FIXa and includes VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein VL CDR3 has the amino acid sequence X 1 QYAX 2 includes FPYT (SEQ ID NO: 2201), and X 1 is Q or S, and X 2 is N or R, the isolated anti-FIXa antibody of any one of Embodiments 182, 188, or 189 or an antigen-binding portion thereof.

[0229] Includes VL CDR1, CDR2, and CDR3, (i) The VL CDR1 contains the amino acid sequence X 1 AX 2 X 3 X 4 IX 5 X 6 and contains YLN (SEQ ID NO: 2199), where X 1 is Q, G or E, and X 2 is S or N, and X 3 is Q or E, and X 4 is D or Y, and X 5 is A or S, and X 6 is N or D; and / or (ii) The VL CDR2 has the amino acid sequence DAX 7 NLX 8 X 9 (SEQ ID NO: 2200), where X 7 is S or A, and X 8 is E, H or Q, and X 9 is T or Y, an isolated anti-FIXa antibody of embodiment 190 or an antigen-binding portion thereof.

[0230] Embodiment 192. A VH CDR1, CDR2 and CDR3 comprising a VH CDR1 selected from SEQ ID NOs: 2048-2052, a VH CDR2 selected from SEQ ID NOs: 2074-2078, and a VH CDR3 selected from SEQ ID NOs: 2100-2104, and / or a VL CDR1 selected from SEQ ID NOs: 2126-2130, a VL CDR2 selected from SEQ ID NOs: 2152-2156, and a VL CDR3 selected from SEQ ID NOs: 2178-2182 An anti-FIXa antibody of embodiment 182 or an antigen-binding portion thereof, comprising VL CDR1, CDR2 and CDR3.

[0231] Embodiment 193. Specifically binds to FIXa and comprises VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein the VH CDR3 has the amino acid sequence ARDGPX 1 X 2 X 3Comprising DYYMDV (SEQ ID NO: 2204), X 1 wherein X is R or Q, and X 2 wherein X is V, D, L or E, and X 3 wherein X is S or V, an isolated anti-FIXa antibody of embodiment 182 or an antigen-binding portion thereof.

[0232] Embodiment 194. Comprising VH CDR1, CDR2 and CDR3, (i) wherein VH CDR1 has the amino acid sequence YTFX 1 X 2 YX 3 comprising MH (SEQ ID NO: 2202), X 1 wherein X is T or H, and X 2 wherein X is S, G or H, and X 3 wherein X is Y or P; and / or (ii) wherein VH CDR2 has the amino acid sequence X 4 INPSX 5 GX 6 TX 7 comprising YAQKFQG (SEQ ID NO: 2203), X 4 wherein X is I or S, and X 5 wherein X is G or R, and X 6 wherein X is S or R, and X 7 wherein X is S or E, an isolated anti-FIXa antibody of embodiment 193 or an antigen-binding portion thereof.

[0233] Embodiment 195. An isolated anti-FIXa antibody or an antigen-binding portion thereof of any one of embodiments 182, 193 or 194 that specifically binds to FIXa and comprises VH CDR1, CDR2 and CDR3, and VL CDR1, CDR2 and CDR3, wherein VL CDR3 comprises the amino acid sequence QQRDNWPFT (SEQ ID NO: 2116).

[0234] Embodiment 196. Comprising VL CDR1, CDR2 and CDR3, (i) wherein VL CDR1 comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO: 2116); and / or (ii) The isolated anti-FIXa antibody or antigen-binding portion thereof of embodiment 195, wherein the VL CDR2 comprises the amino acid sequence DASNRAT (SEQ ID NO: 2116).

[0235] Embodiment 197. The anti-FIXa antibody or antigen-binding portion thereof of embodiment 182, comprising VH CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 2053-2057, VH CDR2 selected from SEQ ID NOs: 2079-2083, and VH CDR3 selected from SEQ ID NOs: 2105-2109, and / or VL CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 2131-2135, VL CDR2 selected from SEQ ID NOs: 2157-2161, and VL CDR3 selected from SEQ ID NOs: 2183-2187.

[0236] Embodiment 198. An isolated anti-FIXa antibody or antigen-binding portion thereof of embodiment 182 that specifically binds to FIXa and comprises VH CDR1, CDR2, and CDR3, and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDKYQDYSX 1 DI (SEQ ID NO: 2207), and X 1 is F or V.

[0237] Embodiment 199. Comprising VH CDR1, CDR2, and CDR3, (i) The VH CDR1 comprises the amino acid sequence GSIX 1 SX 2 X 3 YX 4 WX 5 (SEQ ID NO: 2205), and X 1 is S or A, X 2 is S, T, G, or V, X 3 is S or A, X 4 is Y or A, X 5 is G, V, N, or S; and / or (ii) The VH CDR2 comprises the amino acid sequence X 6 IX 7 X 8X 9 GX 10 TX 11 comprising YNPSLKS (SEQ ID NO: 2206), X 6 wherein X is S or Y, and X 7 wherein X is S, Y, R, T or Q, and X 8 wherein X is Y, G, P or A, and X 9 wherein X is S or Q, and X 10 wherein X is S or K, and X 11 An isolated anti-FIXa antibody or antigen-binding portion thereof of Embodiment 198, wherein X is Y or Q.

[0238] Embodiment 200. An isolated anti-FIXa antibody of Embodiment 182, 198 or 199, comprising VL CDR1, CDR2 and CDR3, wherein VL CDR3 comprises the amino acid sequence QQANFLPFT (SEQ ID NO: 2188).

[0239] Embodiment 201. An isolated anti-FIXa antibody or antigen-binding portion thereof of Embodiment 200, comprising VL CDR1, CDR2 and CDR3, (i) wherein VL CDR1 comprises the amino acid sequence RASQGIDSWLA (SEQ ID NO: 2136); and / or (ii) wherein VL CDR2 comprises the amino acid sequence AASSLQS (SEQ ID NO: 2162).

[0240] Embodiment 202. An anti-FIXa antibody or antigen-binding portion thereof of Embodiment 182, comprising VH CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 2058-2063 for VH CDR1, SEQ ID NOs: 2084-2089 for VH CDR2, and SEQ ID NOs: 2110-2115 for VH CDR3, and / or VL CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 2136-2141 for VL CDR1, SEQ ID NOs: 2162-2167 for VL CDR2, and SEQ ID NOs: 2188-2193 for VL CDR3.

[0241] Embodiment 203. Comprising VH and VL, (i) The VH comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1935, 1939, 1943, 1947, 1951, 1955, 1959, 1963, 1967, 1971, 1975, 1979, 1983, 1987, 1991, 1995, 1999, 2003, 2007, 2011, 2015, 2019, 2023, 2027, 2031 and 2035; and / or (ii) The VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1937, 1941, 1945, 1949, 1953, 1957, 1961, 1965, 1969, 1973, 1977, 1981, 1985, 1989, 1993, 1997, 2001, 2005, 2009, 2013, 2017, 2021, 2025, 2029, 2033 and 2037, an anti-FIXa antibody or an antigen-binding portion thereof of any one of embodiments 182 to 202.

[0242] Embodiment 204. Comprising VH and VL, (a1) VH and VL each comprise SEQ ID NOs: 1935 and 1937 (BIIB-9-3595); (a2) VH and VL each comprise SEQ ID NOs: 1939 and 1941 (BIIB-9-3601); (a3) VH and VL each comprise SEQ ID NOs: 1943 and 1945 (BIIB-9-3604); (a4) VH and VL each comprise SEQ ID NOs: 1947 and 1949 (BIIB-9-3617); (a5) VH and VL each comprise SEQ ID NOs: 1951 and 1953 (BIIB-9-3618); (a6) VH and VL each contain SEQ ID NO: 1955 and SEQ ID NO: 1957 (BIIB-9 -3621); (a7) VH and VL each contain SEQ ID NO: 1959 and SEQ ID NO: 1961 (BIIB-9-3647); (a8) VH and VL each contain SEQ ID NO: 1963 and SEQ ID NO: 1965 (BIIB-9-3649); (a9) VH and VL each contain SEQ ID NO: 1967 and SEQ ID NO: 1969 (BIIB-9-3650); (a10) VH and VL each contain SEQ ID NO: 1971 and SEQ ID NO: 1973 (BIIB-9-3654); (a11) VH and VL each contain SEQ ID NO: 1975 and SEQ ID NO: 1977 (BIIB-9-3753); (a12) VH and VL each contain SEQ ID NO: 1979 and SEQ ID NO: 1981 (BIIB-9-3754); (a13) VH and VL each contain SEQ ID NO: 1983 and SEQ ID NO: 1985 (BIIB-9-3756); (a14) VH and VL each contain SEQ ID NO: 1987 and SEQ ID NO: 1989 (BIIB-9-3764); (a15) VH and VL each contain SEQ ID NO: 1991 and SEQ ID NO: 1993 (BIIB-9-3766); (a16) VH and VL each contain SEQ ID NO: 1995 and SEQ ID NO: 1997 (BIIB-9-3707); (a17) VH and VL each contain SEQ ID NO: 1999 and SEQ ID NO: 2001 (BIIB-9-3709); (a18) VH and VL each contain SEQ ID NO: 2003 and SEQ ID NO: 2005 (BIIB-9-3720); (a19) VH and VL each contain SEQ ID NO: 2007 and SEQ ID NO: 2009 (BIIB-9-3727); (a20) VH and VL each contain SEQ ID NO: 2011 and SEQ ID NO: 2013 (BIIB-9-3745); (a21) VH and VL each contain SEQ ID NO: 2015 and 2017 (BIIB-9-3780); (a22) VH and VL each contain SEQ ID NO: 2019 and 2021 (BIIB-9-3675); (a23) VH and VL each contain SEQ ID NO: 2023 and 2025 (BIIB-9-3681); (a24) VH and VL each contain SEQ ID NO: 2027 and 2029 (BIIB-9-3684); (a25) VH and VL each contain SEQ ID NO: 2031 and 2033 (BIIB-9-3698); or (a26) An anti-FIXa antibody or an antigen-binding portion thereof of any one of embodiments 182 to 203, wherein VH and VL each contain SEQ ID NO: 2035 and 2037 (BIIB-9-3704).

Brief Description of the Drawings

[0243]

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Mode for Carrying Out the Invention

[0244] The present disclosure provides antibodies that preferentially bind to specific forms of coagulation factors. In particular, the present disclosure provides antibodies and antigen-binding portions thereof that specifically bind to FIX, for example, antibodies that preferentially bind to activated factor IX (FIXa) in the presence of FIX (e.g., FIXa and factor IX zymogen (FIXz)) and antigen-binding portions thereof. The present disclosure also provides antibodies and antigen-binding portions thereof that specifically and preferentially bind to FX (FX zymogen (FXz)) in the presence of FXz and FXa.

[0245] Similarly, provided are bispecific molecules (e.g., antibodies) comprising any one of the anti-FIX antibodies or antigen-binding portions thereof disclosed herein and any one of the anti-FX antibodies or antigen-binding portions thereof disclosed herein. These bispecific antibodies can bind to FIX and FX simultaneously and mimic the function of coagulation factor VIIIa.

[0246] The present disclosure also provides compositions, methods of making, methods of treatment and diagnosis, immunoconjugates, and kits, including, for example, binding molecules such as the antibodies disclosed herein (e.g., pharmaceutical or diagnostic compositions), nucleic acids and vectors encoding the binding molecules disclosed herein, cells comprising nucleic acids encoding the binding molecules disclosed herein.

[0247] The headings presented herein are not limitations of the various aspects or embodiments of the present disclosure, which can be defined in their entirety by reference to the present specification. Accordingly, the terms defined immediately below are fully defined by reference herein in their entirety. Before describing the present invention in detail, it is to be understood that the present invention is not limited to specific compositions or method steps, and thus can vary.

[0248] I. Definitions To make the present disclosure more readily understandable, certain terms are first defined. As used in this application, unless specifically stated otherwise herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0249] The present invention includes embodiments in which exactly one component of a group is present in, used in, or otherwise related to a given product or method. The present invention includes embodiments in which more than one or all of the components of that group are present in, used in, or otherwise related to a given product or method.

[0250] In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), and the terms “one or more” and “at least one” can be used interchangeably herein. In certain aspects, the term “a” or “an” means “single.” In other aspects, the term “a” or “an” includes “two or more” or “plural.”

[0251] Furthermore, “and / or” as used herein is considered to specifically disclose each of the two specified features or components, whether or not it also includes other things. Thus, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone) and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0252] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press present many common dictionaries of terms used in this disclosure to one of ordinary skill in the art.

[0253] When an aspect is described herein with the expression "comprising", other similar aspects are also presented with respect to "consisting of" and / or "consisting essentially of".

[0254] Units, prefixes, and symbols are expressed in their accepted form of the International System of Units (SI). Numerical ranges include the numbers defining the range. When a range of values is recited, each integer value and each fraction thereof intervening between the recited upper and lower limits of the range is specifically also disclosed, together with each subrange between such values. The upper and lower limits of any range may independently be included in or excluded from the range, and each range that includes one of the boundary values, neither of the boundary values, or both of the boundary values is also encompassed by the invention. It is understood that values that are substantially the same amount or the same amount as the recited values are also within the scope of the invention when the values are explicitly recited. When combinations are disclosed, each subcombination of the elements of the combination is also specifically disclosed and within the scope of the invention. Conversely, when different elements or groups of elements are disclosed separately, their combinations are also disclosed. When any element of the invention is disclosed as having a plurality of alternatives, examples of such inventions in which each alternative is included singly or in any combination with other alternatives are also disclosed by the invention; more than one element of the invention can have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0255] Nucleotides are described by their generally accepted single letter codes. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction. Nucleotides are described herein by their generally known one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0256] Amino acids are described herein by either their generally known three letter symbols or their one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in the amino to carboxy direction.

[0257] About: When the term "about" is used with respect to a numerical value throughout this specification and the claims, it represents an interval of precision with which those skilled in the art are familiar and would tolerate. Generally, such an interval of precision is ±10%. When ranges are indicated, the endpoints are included. Further, unless otherwise indicated, or clear from the context, or obvious to those skilled in the art from the context, values indicated as ranges are assumed to be any specific value or sub-range within the ranges specified for various embodiments of the invention, to the extent of one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0258] Administered in combination: As used herein, the terms "administered in combination", "combination administration" or "combination therapy" mean that two or more agents, e.g., a binding molecule disclosed herein and a second agent, are administered to a subject either simultaneously or within an interval such that the actions of each agent on the subject may overlap. In some embodiments, those agents are administered to each other within about 60, 30, 15, 10, 5 or 1 minute. In some embodiments, the administration of the agents is provided at an interval close enough together such that a combined (e.g., synergistic) effect is achieved.

[0259]

[0260] ​Affinity: The term "affinity" refers to the degree to which a binding molecule, such as an antibody, binds to an antigen such that the equilibrium of the antigen and the binding molecule shifts in the direction of the presence of the complex formed by their binding. Thus, when an antigen and a binding molecule are combined at relatively equal concentrations, a high-affinity binding molecule will bind to the available antigen such that the equilibrium shifts in the direction of a high concentration of the resulting complex. A binding molecule, such as an antibody, antigen-binding fragment, variant, or derivative thereof of the present disclosure, is also described or specified with respect to its binding affinity for an antigen. The affinity of a binding molecule, such as an antibody, for an antigen can be determined experimentally using any suitable method (e.g., Berzofsky et al., "Antibody-Antigen Interactions", In Fundamental Immunology, Paul, W.E., ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H.Freeman and Company: New York, N.Y. (1992); and see the methods described herein).

[0261] When measured under different conditions (e.g., salt concentration, pH), the measured affinity of a particular binding molecule-antibody interaction can vary. Thus, the measurement of affinity and other antigen-binding parameters (e.g., K D 、K a 、K d ) is preferably performed using a standard solution of the binding molecule and the antigen, as well as a standard buffer solution.

[0262] "High affinity" for a binding molecule, such as an antibody, means at least about 1×10 7 liters / mole, or at least about 1×10 8 liters / mole, or at least about 1×10 9 liters / mole, or at least about 1×10 10 liters / mole, or at least about 1×10 11 liters / mole, or at least about 1×1012 liters / mole, or at least about 1×10 13 liters / mole, or at least about 1×10 14 liters / mole, or a higher equilibrium binding constant (K aff ). "High affinity" binding may vary in the case of antibody isotypes.

[0263] K, which is the equilibrium dissociation constant, D is also a term used to describe antibody affinity and is the reciprocal of K aff . K D is obtained from the ratio of k a to k d (i.e., k d / k a ) and is expressed as molar concentration (M). The K D value of an antibody can be determined using methods well established in the art. Available methods for determining the K D of an antibody include biosensor systems such as biolayer interferometry (BLI) assays, surface plasmon resonance, the Biacore® system, or flow cytometry —, and Scatchard analysis. When K D is used, the term "high affinity" for an antibody refers to an equilibrium dissociation constant (K -7 ) of less than about 1×10 -8 M, or less than about 1×10 -9 M, or less than about 1×10 -10 M, or less than about 1×10 -11 M, or less than about 1×10 -12 M, or less than about 1×10 13 M, or less than about 1×10 -14 M, or less than that. D )

[0264] Amino acid substitution: The term "amino acid substitution" refers to replacing an amino acid residue present in a parent or reference sequence (e.g., a wild-type sequence) with another amino acid residue. The amino acid is substituted in the parent or reference sequence (e.g., a wild-type polypeptide sequence) by, for example, chemical peptide synthesis or by recombinant methods known in the art. Thus, when referring to "a substitution at position X", it means that the amino acid present at position X is replaced by an alternative amino acid residue. In some embodiments, the substitution pattern is described according to the schematic AnY, where A is the one-letter code corresponding to the amino acid that is naturally or originally present at the nth position, and Y is the amino acid residue to be substituted. In other embodiments, the substitution pattern is described according to the schematic An(YZ), where A is the one-letter code corresponding to the amino acid residue that replaces the amino acid that is naturally or originally present at the nth position, and Y and Z are alternative substitution amino acid residues that can replace A.

[0265] In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitutions) are made at the nucleic acid level, i.e., replacing an amino acid residue with an alternative amino acid residue is done by replacing the codon encoding the first amino acid with the codon encoding the second amino acid.

[0266] Affinity matured: The term "affinity matured" refers to a binding molecule, such as an antibody, that has undergone the process of affinity maturation, i.e., an increase in affinity for a target antigen. Thus, an affinity matured antibody is an antibody that has one or more alterations in one or more of its CDRs and that results in an improvement in the antibody's affinity for the antigen compared to the parental antibody that does not have such alterations. Exemplary affinity matured antibodies are expected to have an affinity at nanomolar concentrations or even picomolar concentrations for the target antigen.

[0267] In accordance with various embodiments of the invention disclosed herein, any one or more methods of manufacturing and / or using an affinity maturation library available in the art can be used to generate affinity matured antibodies. Exemplary affinity maturation methods include random mutagenesis, bacterial mutagenesis strain passage, site-directed mutagenesis, mutagenesis of multiple target sites, parsimonious mutagenesis, antibody shuffling, light chain shuffling, heavy chain shuffling, mutagenesis of CDR1 and / or CDR1, and methods of generating and using an affinity maturation library compatible with the methods and uses according to various embodiments of the invention disclosed herein include, for example, Prassler et al. (2009); Immunotherapy, Vol. 1(4), pp. 571-583; Sheedy et al. (2007), Biotechnol. Adv., Vol. 25(4), pp. 333-352; WO2012 / 009568; WO2009 / 036379; WO2010 / 105256; US2002 / 0177170; WO2003 / 074679, all of which are hereby incorporated by reference in their entirety. Marks et al. (1992), BioTechnology 10:779-783 describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by Barbas et al. (1994) Proc. Nat. Acad. Sci. USA 91:3809-3813; Schier et al. (1995) Gene 169:147-155; Yelton et al. (1995) J. Immunol. 155:1994-2004; Ja ckson et al. (1995) J. Immunol. 154(7):3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226:889-896. Selective mutagenesis at positions, contacts or hypervariable positions by amino acid residues that enhance activity is described in U.S. Patent No. 6,914,128.

[0268] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cows, primates or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal or a clone.

[0269] Antibody: The terms "antibody" and "immunoglobulin" (abbreviated as "Ig") are used interchangeably herein and refer to a molecule that specifically binds to a particular antigen or is immunologically reactive with a particular antigen and includes at least one immunoglobulin domain. The term includes whole antibodies and any antigen-binding portion, or single chains thereof, and combinations thereof (e.g., bispecific antibodies).

[0270] A typical antibody includes at least two heavy chains ("HC") and two light chains ("L") interconnected by disulfide bonds.

[0271] Each "heavy chain" consists of a "variable heavy region" (abbreviated as "VH" herein) and a "constant heavy region" (abbreviated as "CH" herein). The constant heavy region in an unmodified antibody consists of three constant domains, CH1, CH2 and CH3.

[0272] Each "light chain" consists of a "variable light region" (abbreviated as "VL" herein) and a "constant light region". The constant light region in an unmodified antibody consists of one constant domain, "CL". The VH and VL regions are further subdivided into regions of hypervariable regions called "complementary determining regions" ("CDR"), regions more conserved and regions dispersed, regions called "framework regions" ("FW").

[0273] VH and VL each consist of three CDRs and four FWs arranged in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4, from the amino terminus to the carboxy terminus. The present disclosure represents VH and VL sequences, as well as partial sequences corresponding to CDR1, CDR2, and CDR3. Thus, those skilled in the art will understand that the sequences of FW1, FW2, FW3, and FW4 are equally disclosed. For a particular VH, FW1 is the partial sequence between the N-terminus of VH and the N-terminus of VH-CDR1, FW2 is the partial sequence between the C-terminus of VH-CDR1 and the N-terminus of VH-CDR2, FW3 is the partial sequence between the C-terminus of VH-CDR2 and the N-terminus of VH-CDR3, and FW4 is the partial sequence between the C-terminus of VH-CDR3 and the C-terminus of VH. Similarly, for a particular VL, FW1 is the partial sequence between the N-terminus of VL and the N-terminus of VL-CDR1, FW2 is the partial sequence between the C-terminus of VL-CDR1 and the N-terminus of VL-CDR2, FW3 is the partial sequence between the C-terminus of VL-CDR2 and the N-terminus of VL-CDR3, and FW4 is the partial sequence between the C-terminus of VL-CDR3 and the C-terminus of VL.

[0274] The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies of the present disclosure include typical antibodies, scFvs, and combinations thereof, where, for example, the scFv is covalently linked (e.g., by a peptide bond or by a chemical linker) to the N-terminus of either the heavy chain and / or the light chain of a typical antibody, or is intercalated into the heavy chain and / or the light chain of a typical antibody. Combinations thereof, where, for example, the scFv is covalently linked (e.g., by a peptide bond or by a chemical linker) to the N-terminus of either the heavy chain and / or the light chain of a typical antibody, or is intercalated into the heavy chain and / or the light chain of a typical antibody.

[0275] As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab’, F(ab’)2 and Fv fragments), single-chain variable fragments (scFv), disulfide-stabilized scFv, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies and / or their antigen-binding portions, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antigenic determinants of antibodies, and any other modified immunoglobulin molecules containing antigen recognition sites, provided that the antibody exhibits the desired biological activity.

[0276] Antibodies can be of any of the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are named based on the identity of their heavy-chain constant domains, respectively called alpha, delta, epsilon, gamma, and mu. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be used naked or conjugated to other molecules such as therapeutic or diagnostic agents to form immunoconjugates.

[0277] There are at least two techniques for determining CDRs: (1) methods based on interspecies sequence variation (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th Edition, 1991, National Institutes of Health, Bethesda, Md., USA), and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273: 927-948). Furthermore, combinations of these two methods are sometimes used in the art to determine CDRs. Kabat's numbering system is generally used when referring to residues in the variable domains (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0278] The expressions "numbering of amino acid positions similar to Kabat", "Kabat position", and their grammatical variants refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain few or additional amino acids corresponding to shortening or insertion into the FW or CDR of the variable domain. For example, the heavy chain variable domain can contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, and residues inserted after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c according to kabat, etc.). See Table 1.

[0279] [Table 1]

[0280] The Kabat numbering of residues can be determined for an antibody given by alignment in the regions of sequence homology of the antibody's sequence by the "standard" Kabat numbering sequence. Chothia, instead, refers to the positions of loop structures (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme considers the insertions at H35A and H35B, and if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are a compromise between the Kabat CDRs and the Chothia loop structures and are used by Oxford Molecular's AbM antibody modeling software.

[0281] IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including the CDRs. See, for example, Lefranc, M.P. et al., Dev. Comp. Immunol. 27:55-77 (2003), which is incorporated herein by reference. The IMGT numbering system is based on the alignment of over 5,000 sequences, structural data, and the characterization of hypervariable loops, and allows for easy comparison of variable and CDR regions for all species. According to the IMGT numbering scheme, VH-CDR1 is present at positions 26-35, VH-CDR2 is present at positions 51-57, VH-CDR3 is present at positions 93-102, VL-CDR1 is present at positions 27-32, VL-CDR2 is present at positions 50-52, and VL-CDR3 is present at positions 89-97.

[0282] With respect to the positions of all heavy chain constant region amino acids discussed in the present invention, the numbering conforms to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, which describes the amino acid sequence of the myeloma protein EU, the first human IgG to be sequenced. The EU index of Edelman et al. is also described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrase "EU index described in Kabat" or "Kabat's EU index" and "position according to the EU index described in Kabat" and its grammatical variations refer to the residue numbering system based on the Edelman et al. human IgG1 EU antibody described in Kabat 1991.

[0283] The numbering system used for the variable domains (both heavy and light chains) and the light chain constant region amino acid sequences is that described in Kabat 1991.

[0284] As used herein, the Fc region includes a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminus to these domains. In the case of IgA and IgM, Fc can include the J chain. In the case of IgG, Fc includes the immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3), and the hinge between C gamma 1 (Cγ1) and C gamma 2 (Cγ2).

[0285] The boundaries of the Fc region can vary, but the Fc region of the human IgG heavy chain is typically defined as including the C226 or P230 residue at its carboxy terminus, in which case the numbering follows the EU index as described by Kabat. Fc can refer to such a region in isolation or to such a region with respect to an antibody, antibody fragment or Fc fusion protein.

[0286] Polymorphisms are observed at several different positions within the antibody constant region (e.g., Fc positions including, but not limited to, positions 270, 272, 312, 315, 356 and 358 when numbered according to the EU index described by Kabat), and thus there may be minor differences between the sequences presented and those in the prior art. The polymorphisms of human immunoglobulins are well characterized. Currently, 18 Gm allotypes are known: G1m(1, 2, 3, 17) or G1m(a, x, f, z), G2m(23) or G2m(n), G3m(5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m(b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5). See Lefranc et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pages 43-78 (1990); Lefranc et al. (1979), Hum. Genet.: 50, pages 199-211. The antibodies of the present invention can incorporate any allotype, isoallotype or haplotype of any immunoglobulin gene and are specifically included not to be limited to the allotype, isoallotype or haplotype of the sequences presented herein.

[0287] Antibody binding site: The term "antibody binding site" refers to a region in an antigen (e.g., FIXa or FXz) that includes a continuous or discontinuous site (i.e., epitope) to which a complementary antibody specifically binds. Thus, an antibody binding site is an antigen over an epitope and can include additional regions in the antigen that can determine properties such as binding affinity and / or stability, or can affect properties such as antigen enzyme activity or dimerization. Thus, even if two antibodies bind to the same epitope within an antigen, if the antibody molecule establishes individual intramolecular contacts with amino acids outside the epitope, such antibodies are considered to bind to individual antibody binding sites.

[0288] Antigen-binding portion: As used herein, the term "antigen-binding portion" can be used interchangeably with "antigen-binding fragment" and refers to a portion of an intact antibody that is capable of specific binding to the same epitope as the intact antibody. In particular, an antigen-binding portion refers to a part or portion of an intact antibody that includes one or more CDRs of the intact antibody. It is known in the art that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2 and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0289] Antigen-binding molecule: The terms "antigen-binding molecule" and "binding molecule" are used interchangeably in this disclosure and encompass antibodies as defined herein and other molecular entities that include at least one CDR of an antibody disclosed herein that is capable of binding to the same epitope. For example, the term includes scaffolds of fibronectin type III (monobodies), other scaffold systems (e.g., tenascin) to which one or more CDRs are grafted, and aptamer-based mimetic antibodies. In some embodiments, an antibody-binding molecule can be bispecific, i.e., a "bispecific binding molecule" or "bispecific molecule".

[0290] About: As used herein, the term "about" when applied to one or more target values refers to a value similar to the recited reference value. In certain embodiments, the term "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than the recited reference value), unless otherwise specified or otherwise clear from the context (such numbers excluding cases where the possible values exceed 100%) of the recited reference value.

[0291] Associated: As used herein in connection with a disease, the term "associated" means that the symptom, measurement, feature or situation in question is linked to the diagnosis, onset, presence or progression of that disease. Association may, but need not, imply a causal link to the disease.

[0292] The terms "associated", "conjugated", "linked", "bound" and "constrained", when used with respect to two or more moieties, mean that those moieties are physically joined or linked to each other, either directly or by one or more additional moieties acting as cross-linking agents, to form a sufficiently stable structure such that the moieties are in a physically joined state under the conditions under which the structure is used, e.g., under physiological conditions. "Binding" need not be by strictly direct chemical covalent bonds. "Binding" may also imply sufficiently stable ionic or hydrogen bonds, or hybridization-based bonds, such that the "bound" entities maintain a physically joined state.

[0293] Binding affinity: "Binding affinity" generally refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the potential binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is the dissociation constant (K DThus, it can be generally represented. Affinity can be measured by general methods known in the art, including the methods described herein. Antibodies with low affinity generally tend to bind to antigens slowly and dissociate easily, while antibodies with high affinity generally tend to bind more quickly and maintain the binding for a longer time. Various methods for measuring binding affinity are known in the art, and any of these can be used for the purposes of the present disclosure.

[0294] The term "higher binding affinity" or "greater affinity", when applied to any of the antibodies of the present invention, refers to an increase in binding affinity (e.g., as measured by K D with respect to a reference antibody. In some embodiments, the reference antibody is the corresponding immature affinity antibody. In some embodiments, the reference antibody is another antibody having the same specificity (e.g., in the case of anti-FIXa disclosed herein, the reference antibody can be another anti-FIX or anti-FIXa antibody known in the art). In some embodiments, the increase in binding affinity is, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% higher than the binding affinity of the reference antibody for the same coagulation factor (e.g., FIX or FX), factor form (e.g., FIXa or FXz) or antigen-binding site (e.g., epitope). In some embodiments, the increase in binding affinity is, for example, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold or at least about 10-fold higher than the binding affinity of the reference antibody for the same coagulation factor (e.g., FIX or FX), factor form (e.g., FIXa or FXz) or antigen-binding site (e.g., epitope).

[0295] Binding: The term "binding" refers to the physical interaction between two molecules, e.g., an antibody and an antigen. (i) Binding specificity: The term "specificity" refers to the ability of a binding molecule, such as an antibody, to preferentially bind to one antigenic site (e.g., an epitope) over various antigenic sites, and does not necessarily imply high affinity. The terms "binding specificity" and "specificity" are used interchangeably and can refer to both (i) a particular part of a binding molecule, and (ii) the ability of a binding molecule to specifically bind to a particular epitope (see the definition of "specific binding" below). For example, in some embodiments, the bispecific antibodies disclosed herein include two binding specificities, i.e., a first binding specificity to, for example, FIXa, and a second binding specificity to, for example, FXz (in this context, "binding specificity", such as the specific region of binding of a bispecific antibody to a particular antigenic determinant, is equivalent to "binding domain"). (ii) Specific binding: A binding molecule, such as an antibody, "binds specifically" if there is an immunological reaction of specific interaction between the antigen and the binding molecule. The term "binds specifically" means that the antibody is generated to bind to the antigen via its variable region. The term "non-specific binding" means that the antibody is not generated to specifically bind to the antigen, but binds to the antigen to some extent via non-specific means. As an example, an antibody is expected to non-specifically bind to an Fc receptor via the Fc portion of the antibody molecule. As another example, certain antibodies may cross-react erroneously with antigens they are not generated against. (iii) Preferential binding: A binding molecule, such as an antibody, "binds preferentially" to an antigen if it binds more readily and / or for a longer period of time with greater affinity, avidity, than it binds to other substances. For example, an antibody that binds preferentially to a FIXa epitope is one that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other FIXa epitopes or non-FIXa epitopes. For example, if more than 50%, 60%, 70%, 80%, 90% or 95% of an anti-FIX antibody binds to FIXa in the presence of both FIXa and FIXz, the anti-FIX antibody binds preferentially to activated FIX over FIX prothrombin. For example, it will also be understood from a reading of this definition that an antibody (or portion or epitope) that binds preferentially to a first target may or may not bind preferentially to a second target. Thus, "preferential binding" does not necessarily require exclusive binding (it can include exclusive binding). Thus, in some embodiments, "preferential binding" can be "exclusive binding". To illustrate these concepts, if 50% of an anti-FIX binds specifically to FIX prothrombin and 50% specifically to FIXa, such binding is "non-selective" or "non-preferential". If less than 50% of the anti-FIX binds to FIX prothrombin and more than 50% binds to FXa, the anti-FIX "binds preferentially" to FIXa. If the anti-FIX does not bind to FIX prothrombin and binds only to FIXa, the anti-FIX "binds exclusively" to FIXa. If the anti-FIX does not bind to FIX prothrombin and binds only to FIXa, the anti-FIX "binds exclusively" to FIXa.

[0296] Biological sample: As used herein, the term "biological sample" refers to any sample obtained from a subject, cell line, tissue culture, or other source that potentially contains a molecule comprising an antigen specifically recognized by a binding molecule disclosed herein. In some embodiments, the biological sample is a blood sample or a sample derived from a blood sample (e.g., plasma). Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0297] Bispecific antibody: A “bispecific antibody” is a particular type of “bispecific molecule” or “bispecific binding molecule”. The term “bispecific antibody” means an antibody that can bind to at least two antigenic determinants (e.g., epitopes) via two different antigen-binding sites. In certain embodiments, a bispecific antibody can bind to two antigenic determinants (e.g., epitopes) simultaneously. In some embodiments, a bispecific antibody binds to one antigen (epitope) on one of its binding arms (a pair of heavy / light chains) and binds to a different antigen (or epitope) on its second binding arm (a different pair of heavy / light chains). In some embodiments, a bispecific antibody can have two separate antigen-binding arms (both in terms of specificity and CDR sequences), being monovalent for each antigen to which it binds. Bispecific antibodies include, for example, those generated by the quadroma technique (Milstein and Cuello (1983) Nature 305(5934):537 - 40), by chemical conjugation of two different monoclonal antibodies (Staerz et al. (1985) Nature 314(6012):628 - 31), or by knob-into-hole or similar techniques (Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(14):6444 - 6448) that introduce mutations into the Fc region.

[0298] A wide variety of recombinant bispecific antibody formats have been developed over the last decade, for example, by IgG antibody formats and by fusion of single-chain domains (see Kontermann RE, mAbs 4:2 (2012) 1 - 16). Bispecific antibodies in which the variable domains VL and VH, or the constant domains CL and CH1, are replaced with each other are described in WO2009080251 and WO2009080252.

[0299] A method known as "knob-into-hole" aims to force the pairing of two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. Bulky amino acids were replaced by amino acids with short side chains on one chain to create a "hole". Conversely, amino acids with large side chains were introduced into the other CH3 domain to create a "knob". By co-expressing these two heavy chains (and the two identical light chains that need to be appropriate for both heavy chains), a high yield of heterodimer formation ("knob-hole") was observed compared to homodimer formation ("hole-hole" or "knob-knob") (Ridgway JB, Presta LG, Carter P; and WO1996027011). The proportion of heterodimers can be further increased by remodeling the interaction surface of the two CH3 domains using phage display techniques and the introduction of disulfide bridges to stabilize the heterodimers (Merchant A.M et al., Nature Biotech 16 (1998) 677-681; Atwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). A novel approach to knob-into-hole technology is described, for example, in EP1870459A1, Xie, Z. et al., J Immunol Methods 286 (2005) 95-101, and refers to a format of bispecific antibodies using scFv in combination with knob-into-hole technology for the FC portion.

[0300] Using the modular structure of antibodies, more than 60 different bispecific antibody formats have been created. See Spiess et al. (2015) Molecular Immunology 67:95-106, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the bispecific antibody format is a CrossMab, DAF (dual action Fab) (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge pair, Fab-arm exchange, SEED body, Triomab, LUZ-Y (bispecific antibody containing a leucine zipper including heterodimerization of two HCs), Fcab, Kλ-body, orthogonal Fab, DVD-IgG (dual variable domain IgG), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zy body, DVI-IgG (four-in-one), nanobody, nanobody-HSA, BiTE (bispecific T cell engager), diabody, DART (dual affinity retargeting), TandAb (tandem antibody), sc diabody, sc diabody-CH3, triple body, mini antibody, minibody, TriBi mini antibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-ScFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HC Ab, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, ImmTAC, HSA body, sc diabody-HSA, tandem scFv-toxin, IgG-IgG, Cov-X-body, and scFv1-PEG-scFV2.

[0301] In some embodiments, the bispecific antibody is an asymmetric (e.g., heterodimeric) antibody comprising chain A and chain B, (i) Chain A contains the T336W mutation, and chain B contains the T366W, L368A, and Y407V mutations (knob-in-hole format); (ii) Chain A contains the F405L mutation, and chain B contains the K409R mutation (duobody format); (iii) Chain A contains the T350V, L351Y, F405A, and Y407V mutations, and chain B contains the T350V, T366L, K392L, and T394W mutations (azymetric format); (iv) Chain A contains the K409D and K392D mutations, and chain B contains the D399K and E356K mutations (charge pair format); (v) Chain A contains the D221E, P228E, and L368E mutations, and chain B contains the D221R, P228R, and K409R mutations (charge pair format); (vi) Chain A contains the S364H and F405A mutations, and chain B contains the Y349T and T394F mutations (HA-TF format); or (vii) Chain A contains an IgG / A chimera, and chain B also contains an IgG / A chimera (SEED body format).

[0302] In some embodiments, the bispecific antibody is a monospecific antibody engineered to be bispecific by adding either the amino terminus or the carboxy terminus of either the light chain or the heavy chain, which has an additional antigen-binding unit. Alternatives to these additional antigen-binding units include single domain antibodies (unpaired VL or VH), paired antibody variable domains (e.g., Fv or scFv) or engineered protein scaffolds. In some embodiments, the bispecific molecules of the invention include bispecific antibody fragments. A number of bispecific fragment forms lacking some or all of the bispecific antibody constant domains are known in the art. In some embodiments, the bispecific molecules of the invention are bispecific fusion proteins, such as ImmTAC (scFv linked to an affinity matured receptor). In other embodiments, the bispecific molecule is a bispecific antibody conjugate.

[0303] Bispecific molecule: See the definition of the above "antigen-binding molecule" / "binding molecule".

[0304] Chimeric antibody: The term "chimeric" antibody and its grammatical variations refer to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Usually, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) that has the desired specificity, and / or affinity, and / or ability, while the constant regions are homologous to the sequences in an antibody derived from another species (usually human) and avoid eliciting an immune response in that species.

[0305] Complementary determining region: The term "complementary determining region" or "CDR" refers to either the variable region of the H (heavy) or L (light) chain that contains an amino acid sequence capable of specifically binding to an antigen target. These CDR regions are responsible for the basic specificity of the antibody with respect to the structure of a particular antigen determinant. Such regions are also referred to as "hypervariable regions". See the definition of "antibody" above.

[0306] Conserved Amino Acid Substitutions: A "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, when an amino acid in a polypeptide is replaced by another amino acid derived from the same side chain family, this amino acid substitution is considered to be conservative. In another aspect, the amino acid chain is conservatively replaced by a structurally similar chain in which the order and / or composition of the side chain family members is different.

[0307] Non-conservative amino acid substitutions are those in which (i) a residue having an electropositive side chain (e.g., Arg, His, or Lys) is replaced by or with an electronegative side chain (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is replaced by or with a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) cysteine or proline is replaced by or with any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is replaced by or with one having a small side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0308] Other amino acid substitutions can be readily identified by those skilled in the art. For example, in the case of the amino acid alanine, the substitution is selected from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine. In the case of lysine, the substitution can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine or D-ornithine. Generally, substitutions in functionally important regions that are expected to induce changes in the properties of the isolated polypeptide are such that (i) polar residues, such as serine or threonine, are replaced by (or with) hydrophobic residues, such as leucine, isoleucine, phenylalanine or alanine; (ii) cysteine residues are replaced by (or with) any other residue; (iii) residues with electropositive side chains, such as lysine, arginine or histidine, are replaced by (or with) residues with electronegative side chains, such as glutamic acid or aspartic acid; or (iv) residues with bulky side chains, such as phenylalanine, are replaced by (or with) those without such side chains, such as glycine. The possibility that one of the above non-conservative substitutions can modify the functional properties of the protein is also related to the substitution position with respect to the functionally important region of the protein: thus, some non-conservative substitutions may have little or no effect on the biological properties. or D-ornithine. In general, substitutions in functionally important regions that are expected to induce changes in the properties of the isolated polypeptide are such that (i) polar residues, such as serine or threonine, are replaced by (or with) hydrophobic residues, such as leucine, isoleucine, phenylalanine or alanine; (ii) cysteine residues are replaced by (or with) any other residue; (iii) residues with electropositive side chains, such as lysine, arginine or histidine, are replaced by (or with) residues with electronegative side chains, such as glutamic acid or aspartic acid; or (iv) residues with bulky side chains, such as phenylalanine, are replaced by (or with) those without such side chains, such as glycine. The possibility that one of the above non-conservative substitutions can modify the functional properties of the protein is also related to the substitution position with respect to the functionally important region of the protein: thus, some non-conservative substitutions may have little or no effect on the biological properties.

[0309] Conserved: As used herein, the term "conserved" refers to a nucleotide or amino acid residue of a polynucleotide sequence or polypeptide sequence, respectively, at the same position in two or more sequences being compared, in which no modification has occurred. Comparatively, conserved nucleotides or amino acids are those that are conserved among more related sequences than the nucleotides or amino acids that appear elsewhere in the sequence.

[0310] In some embodiments, two or more sequences can be said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are approximately 70% identical, approximately 80% identical, approximately 90% identical, approximately 95%, approximately 98% or approximately 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are approximately 30% identical, approximately 40% identical, approximately 50% identical, approximately 60% identical, approximately 70% identical, approximately 80% identical, approximately 90% identical, approximately 95% identical, approximately 98% identical, or approximately 99% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to a portion, region or feature thereof.

[0311] Cross-competing: As used herein with respect to a binding molecule, e.g., an antibody, the term "competing" or "cross-competing" means that a first binding molecule, e.g., a first antibody or an antigen-binding portion thereof, is sufficiently similar to the binding of a second binding molecule, e.g., a second antibody or an antigen-binding portion thereof, to bind to an epitope such that the binding of the first binding molecule to its cognate epitope is detectably reduced in the presence of the second binding molecule compared to the binding of the first binding molecule in the absence of the second binding molecule. It may or may not be the case that the binding of the second binding molecule to that epitope is also detectably reduced in the presence of the first binding molecule. That is, the first binding molecule can inhibit the binding of the second binding molecule to its epitope without the second molecule inhibiting the binding of the first binding molecule to its epitope. However, if each binding molecule, regardless of whether it is to the same extent, to a greater extent or to a lesser extent, detectably inhibits the binding of other binding molecules having their cognate epitopes, this binding molecule is said to "cross-compete" with the binding of those individual epitopes to each other. Both competitive and cross-competitive binding molecules are encompassed by the present invention.

[0312] When binding molecules cross-compete, the binding molecules, e.g., antibodies, are said to "bind to the same epitope" or "contain the same binding site" or have "substantially the same binding" characteristics, such that only one antibody can bind to an epitope at a given point in time, i.e., one binding molecule inhibits or modulates the binding of another molecule.

[0313] Competition as used herein, when determined by competitive ELISA assay or, for example, by the ForteBio assay described in the Examples section, means a relative inhibition greater than at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%. In certain contexts, it may be desirable to set a higher threshold for relative inhibition as a diagnostic criterion for a suitable level of competition. Thus, for example, diagnostic criteria for competitive binding can be set such that a relative inhibition of at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or about 100% is detected before an antibody is considered to be sufficiently competitive.

[0314] Effective amount: As used herein, the term "effective amount" of an agent, such as a therapeutic agent like an antibody, is such an amount that is sufficient to produce a beneficial or desired result, e.g., a clinical result, and thus the "effective amount" depends on the circumstances to which it is applied. For example, in the context of administering a therapeutic agent to treat bleeding, an effective amount of the agent is an amount sufficient to reduce or decrease the occurrence of bleeding, e.g., as compared to the response obtained if the agent were not administered. The term "effective amount" can be used interchangeably with "effective dose", "therapeutically effective amount" or "therapeutically effective dose".

[0315] Effector function: The "effector function" of an antibody is the ability to bind to complement proteins that can assist in lysing a target antigen, such as a cellular pathogen, in a process called complement-dependent cytotoxicity (CDC). Another effector activity of the Fc region is to bind to Fc receptors (e.g., FcγR) on the surface of immune cells, so-called effector cells, which have the ability to trigger other immune effects. The effector function of an antibody can be avoided, for example, by using antibody fragments without an Fc region (e.g., Fab, F(ab’)2 or single-chain Fv (scFv)), by removing sugars linked to specific residues in the Fc region (non-glycosylated antibody), or by using an Fc region derived from an IgG4 antibody ("effectorless IgG4 Fc") instead of IgG1. It is well known that IgG4 antibodies are characterized by having lower levels of complement activation and antibody-dependent cytotoxicity than IgG1.

[0316] Engineered antibody: As used herein, embodiments of the invention are "engineered" if they are designed to have features or properties that vary from a starting point, wild-type or native molecule, whether structurally or chemically. In this regard, an "engineered antibody" is, for example, an antibody in which affinity, plasma half-life, etc. have been improved by substitution / mutation, and the format of these antibodies has been modified (e.g., by generating scFv or bispecific antibodies), or affinity maturation has been performed on the antibody.

[0317] Epitope: As used herein, the term "epitope" refers to an antigenic protein determinant (e.g., an amino acid subsequence of FIXa or FXz) capable of binding to a binding molecule, such as an antibody. Epitopes usually consist of the chemically active surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features and specific charge features. The portion of an antibody or binding molecule that recognizes an epitope is called a paratope. Epitopes of protein antigens can be classified into two categories, namely conformational epitopes and linear epitopes, based on their structure and interaction with paratopes. Conformational epitopes consist of discontinuous regions of the amino acid sequence of an antigen. These epitopes interact with paratopes based on the features and shape of the 3D surface of the antigen, or its tertiary structure. In contrast, linear epitopes interact with paratopes based on their primary structure. Linear epitopes are formed by a continuous sequence of amino acids derived from an antigen.

[0318] Expression vector: An "expression vector" is a polynucleotide that, when introduced into a suitable host, is transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell consisting of an expression vector that can function to produce a desired expression product. The antibodies according to the present invention (e.g., bispecific antibodies) are preferably produced by recombinant means. Such methods are widely known in the art and include the expression of proteins in prokaryotic and eukaryotic cells, including the subsequent isolation of the antibody polypeptide, and usually purification to pharmaceutically acceptable purity.

[0319] Germline sequences: As used herein, the term "germline sequence" refers to the sequence of an unrearranged immunoglobulin DNA sequence. Any suitable source of unrearranged immunoglobulin can be used. The term "germline" refers to the sequences of V, D, and J minigenes prior to exposure of the antibody to antigen. A rearranged "V region" describes a genetic element resulting from a rearrangement event between V, D, and J (in the case of the heavy chain) or V and J minigenes (in the case of the light chain). An "antibody V region" refers to the polypeptide region encoded by the V, D, and J elements. An "antibody V region" is encoded by the rearranged V, D, and J minigenes. The term "V(D)J recombination" refers to any method by which a V, D, or J minigene is rearranged to another V, D, or J minigene. The V region can be part of a full-length antibody, Fab, scFv, or any other derivative of an antibody (see the definition of antibody below). A "germline V region" refers to the sequence of rearranged V, D, and J minigenes prior to significant mutagenesis. A germline V region may have random insertions or deletions at the V-D, D-J, or V-J minigene junctions. A non-germline V region (or "mature" V region) is typically different from the germline sequence of the minigene by more than 5 residues (excluding deletions or insertions at the junction).

[0320] Homology: As used herein, the term "homology" refers to the overall homology between polymer molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In general, the term "homology" implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous are expected to have a common evolutionary ancestor. In the context of the present invention, the term homology encompasses both identity and similarity.

[0321] In some embodiments, polymer molecules are considered to be "homologous" to each other if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the monomers in the molecule are identical (exactly the same monomer) or similar (conservative substitution). The term "homologous" always refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0322] Human antibody: The term "human antibody" refers to an antibody produced by a human or an amino acid sequence corresponding to an antibody produced by a human and made using any of the known techniques in the art (e.g., recombinant expression in cultured cells or expression in transgenic animals). Thus, the term human antibody also includes an antibody (or engineered variant or derivative thereof) having an amino acid sequence corresponding to an antibody originally produced by a human but expressed in a non-human system (e.g., generated by chemical synthesis; recombinantly expressed in bacterial, mammalian or insect cells; or expressed in an animal subject). Thus, an antibody obtained from a human subject or from human cells (e.g., a hybridoma or a cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal, e.g., a mouse, is considered a human antibody. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies containing at least one human heavy and / or light chain polypeptide, such as an antibody containing a mouse light chain and a human heavy chain peptide.

[0323] ​Humanized antibody: The term "humanized antibody" refers to an antibody derived from a non-human (e.g., murine) immunoglobulin that has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, a humanized antibody is a human immunoglobulin in which the residues derived from the CDRs have been replaced by residues derived from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and capacity (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the FW residues of the human immunoglobulin are replaced by the corresponding residues in an antibody derived from a non-human species having the desired specificity, and / or affinity, and / or capacity.

[0324] A humanized antibody can be further modified by substitution of additional residues, either in the FW regions and / or within the replaced non-human residues, to define and optimize antibody specificity, and / or affinity, and / or capacity. Generally, a humanized antibody comprises substantially all of at least one, usually two or three, variable domains that contain all or substantially all of the CDR regions corresponding to the non-human immunoglobulin, while all or substantially all of the FW regions are those of a human immunoglobulin consensus sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), usually that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 or 5,639,641.

[0325] Identity: As used herein, the term "identity" refers to the overall conservation of monomers between polymer molecules, such as between polypeptide molecules or polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). For example, the term "identical" protein A without any additional modifiers being identical to protein B means that its sequence is 100% identical (100% sequence identity). For example, if two sequences are described as "70% identical", this is the same as describing them as having, for example, "70% sequence identity".

[0326] The percent identity of two polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps are introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences are ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. If the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percent identity between two sequences correlates with the number of identical positions the sequences share, taking into account the number of gaps that need to be introduced for the optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the identity rate between two sequences can be performed using arithmetic algorithms. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. When the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percent identity between two sequences correlates with the number of identical positions the sequences share, taking into account the number of gaps that need to be introduced for the optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the identity rate between two sequences can be performed using arithmetic algorithms. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0327] Software programs suitable for the alignment of both protein and nucleotide sequences are available from various sources. One suitable program for determining the sequence identity rate is bl2seq, which is part of the BLAST suite of programs available from the website of the National Center for Biotechnology Information BLAST (blast.ncbi.nlm.nih.gov). bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0328] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (Clustal W, Clustal X, or Clustal Omega), MUSCLE, etc.

[0329] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own sequence identity rate. It is noted that the values of the sequence identity rate are rounded to the second decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are truncated to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It is also noted that the length values are always integers.

[0330] In certain embodiments, the percent identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100×(Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) scored as identical matches in an alignment of the first and second sequences (aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence is higher than the percent identity of the second sequence to the first sequence.

[0331] One of ordinary skill in the art will appreciate that the generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence-sequence comparisons that are driven solely by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., protein crystal structures), functional data (e.g., positions of mutations), or phylogenetic data. Suitable programs for integrating heterogeneous data to generate multiple sequence alignments include T-Coffee, available at www.tcoffee.org and alternatively available from, e.g., EBI. It will also be appreciated that the final alignment used to calculate sequence identity percentages can be curated either automatically or manually.

[0332] Immunoconjugate: As used herein, the term “immunoconjugate” Refers to a compound comprising a binding molecule (e.g., anti-FIXa, anti-FXz or bispecific anti-FIXa / anti-FXz), and one or more moieties, e.g., a therapeutic or diagnostic moiety that is chemically conjugated to the binding molecule. Generally, an immunoconjugate is defined by the general formula: A-(L-M)n, where A is a binding molecule (e.g., an antibody), L is an optional linker, M is a non-homologous moiety that can be, for example, a therapeutic agent, a detectable label, etc., and n is an integer. The immunoconjugate can also be defined by the general formula in the reverse order. In some embodiments, the immunoconjugate is an "antibody-drug conjugate" ("ADC"). In the context of the present disclosure, the term "immunoconjugate" is not limited to chemically or enzymatically conjugated molecules. The term "immunoconjugate" as used in the present disclosure also includes genetic fusions.

[0333] Isolated: As used herein, the term "isolated" refers to a substance or entity (e.g., a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature) that is separated from at least a portion of the components to which it was bound (whether natural or in an experimental setting). Isolated substances (e.g., nucleotide sequences or protein sequences) can have various levels of purity with reference to the substances to which they were bound.

[0334] Isolated substances and / or entities are separated from other components to which they were initially bound by at least about 10%, at least about 15%, at least about 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least 95% or more.

[0335] In some embodiments, the isolated agent is at a purity of greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%.

[0336] As used herein, a substance is "pure" if it is substantially free of other components. The term "substantially isolated" means that the compound is substantially separated from the environment in which it is formed or detected. Partial separation can, for example, include a composition enriched in the compounds of the present disclosure. Substantial separation can include a composition containing the compound or salt thereof of the present disclosure at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight.

[0337] "Isolated", as disclosed herein, a polynucleotide (e.g., an antibody), vector, polypeptide, cell, or any composition is a polynucleotide (e.g., an antibody), vector, polypeptide, cell, or composition in a form not found in nature. Isolated polynucleotides, vectors, polypeptides or compositions include those that have been purified to the extent that they are no longer in their naturally occurring form. In some embodiments, the isolated polynucleotide, vector, polypeptide or composition is substantially pure.

[0338] Mimicking FVIIIa activity: The ability of a binding molecule disclosed herein to "mimic FVIIIa activity", i.e., the ability to mimic the activity of activated Factor VIII, can be measured by various methods known in the art. One such method is the present It is the chromogenic assay described in the Examples section of the specification. In one aspect, a binding molecule (e.g., a bispecific antibody) disclosed herein is said to "mimic FVIIIa activity" if the rate of observed FXa substrate cleavage is at least 3 standard deviations higher than the average basal rate in the absence of the added binding molecule (e.g., a bispecific antibody). Another exemplary method is the activated partial thromboplastin time (aPTT) assay. The term "activated partial thromboplastin time (APTT)" is derived from the original form of a test (devised in 1953) where only the phospholipid concentration of the test is controlled (as opposed to phospholipid and surface activator concentrations), and the name "partial thromboplastin" is applied to the time to a phospholipid preparation that accelerates clotting but does not correct for the prolonged clotting time of hemophilic plasma. Substantially, the term "partial" means that phospholipids are present but tissue factor is not. The aPTT is also known as the kaolin cephalin clotting time (KCCT) or kaolin plus partial thromboplastin time (PTTK). Other useful methods include a one-stage (OS) clotting assay modified from the above-described conventional aPTT assay. The one-stage clotting assay uses FVIII-deficient plasma and a diluted test sample and can be made quantitative for FVIII activity. See Example 4. In contrast, the aPTT assay uses sample plasma containing aPTT reagent and calcium and reports the clotting time.

[0339] Monoclonal antibody: The term "monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigen determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain a variety of antibodies directed against various antigen determinants. The term "monoclonal antibody" encompasses both intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv, etc.), single-chain variable fragments (scFv), fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies produced by any of a number of methods, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals (e.g., expression of human antibodies in transgenic mice).

[0340] Mutation: In the context of the present disclosure, the terms "mutation" and "amino acid substitution" (sometimes simply referred to as "substitution") as defined above are considered to be interchangeable. In some embodiments, the term "mutation" refers to any nucleotide deletion, insertion, or substitution in a nucleic acid encoding an antibody germline gene, such that the amino acid sequence of the resulting polypeptide is modified at one or more amino acid residues. In some embodiments, a mutation in a nucleic acid sequence disclosed herein results in an amino acid substitution. In other embodiments, a mutation of a codon in a nucleic acid sequence disclosed herein where the resulting codon is a synonymous codon does not result in an amino acid substitution. Thus, in some embodiments, a nucleic acid sequence disclosed herein can be codon-optimized by introducing one or more synonymous codon changes. Such codon optimization can, for example, (i) improve protein yield in recombinant protein expression, or (ii) improve stability, half-life, or other desired properties of an mRNA or DNA encoding a binding molecule disclosed herein, where such mRNA or DNA is administered to a subject in need thereof.

[0341] Patient: As used herein, "patient" refers to an individual who may seek treatment, requires treatment, demands treatment, is currently receiving treatment, will receive treatment in the future, or is under the supervision of a professional trained with respect to a particular disease or condition.

[0342] Pharmaceutical composition: The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of an active ingredient (e.g., a binding molecule disclosed herein such as an antibody) is effective, and which contains no additional constituent that would be unacceptable toxicity to the subject to which the composition is administered. Such compositions are sterile.

[0343] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" as used herein refers to those compounds, substances, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and which exhibit a reasonable benefit / risk ratio. In general, approval by a federal or state regulatory authority (or as listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias) for use in animals, and more particularly in humans, implies that such compounds, substances, compositions and / or dosage forms are pharmaceutically acceptable. Compounds, substances, compositions and / or dosage forms that are generally accepted as safe for therapeutic purposes are "therapeutically acceptable". Compounds, substances, compositions and / or dosage forms that are generally accepted as safe for diagnostic purposes are "diagnostically acceptable".

[0344] Pharmaceutically acceptable additives: The phrase "pharmaceutically acceptable additives" as used herein, when used in this specification, refers to any component other than the compounds described in this specification (for example, a vehicle capable of suspending or dissolving the active compound) that has the property of being substantially non-toxic and non-inflammatory in a patient. Additives can include, for example: anti-adhesion agents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), emollients, emulsifiers, fillers (excipients), film-forming agents or coating agents, flavoring agents, fragrances, flow promoters (fluidity enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweetening agents, and wetting water. Exemplary additives include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0345] Additives that are generally accepted as safe for therapeutic purposes are "therapeutically acceptable additives". Additives that are generally accepted as safe for diagnostic purposes are "diagnostically acceptable additives".

[0346] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" are derivatives of the disclosed compounds in which the parent compound is modified by converting any acidic or basic moieties present into their salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzenesulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate Salts include acetate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., and, without limitation, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and other non-toxic ammonium, quaternary ammonium, and amine cations. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting these compounds in the form of the free acid or free base in water, an organic solvent, or a mixture of the two with a stoichiometric amount of the appropriate base or acid; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is hereby incorporated by reference in its entirety.

[0347] Pharmaceutically acceptable solvate: As used herein, the term "pharmaceutically acceptable solvate" means a compound of the present invention in which molecules of a suitable solvent are incorporated into the crystal lattice. Suitable solvents are physiologically tolerated at the dosage administered. For example, solvates can be prepared by crystallization, recrystallization or precipitation from a solution containing an organic solvent, water or a mixture thereof. Examples of suitable solvents include ethanol, water (e.g., monohydrate, dihydrate and trihydrate), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate and the like. When water is the solvent, the solvate is called a "hydrate".

[0348] Pharmacokinetics: As used herein, "pharmacokinetics" refers to any one or more properties of a molecule or compound when referring to the determination of the fate of a substance administered to a living organism. Pharmacokinetics is classified into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is generally referred to as ADME, where: (A) Absorption is the process by which a substance enters the blood circulation; (D) Distribution is the dispersion or spread of a substance throughout the body's fluids and tissues; (M) Metabolism (or biotransformation) is the irreversible conversion of a parent compound to daughter metabolites; and (E) Excretion (or elimination) refers to the removal of a substance from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

[0349] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof or mixtures thereof. This term refers to the primary structure of a molecule. Thus Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). It also includes modified forms of polynucleotides, as well as their unmodified forms, for example, by alkylation and / or capping. More particularly, the term "polynucleotide" includes polydeoxyribonucleotides (including 2-deoxy-D-ribose), polyribonucleotides (including D-ribose) including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA"), and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that these polymers contain nucleobases in a conformation that permits base pairing and base stacking as found in DNA and RNA. In certain embodiments, the polynucleotide includes mRNA. In other embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class are replaced with unnatural nucleobases (e.g., all uridines in the polynucleotides disclosed herein are replaced with an unnatural nucleobase, such as 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., synthetic RNA or synthetic DNA) contains only natural nucleobases, i.e., only A, C, T, and U in the case of synthetic DNA, or only A, C, G, and U in the case of synthetic RNA.

[0350] One skilled in the art would understand that the T base in the codon map disclosed herein exists in DNA, while the T base is replaced by the U base in the corresponding RNA. For example, in DNA form, the codon-nucleotide sequences disclosed herein, such as vectors or templates for in vitro translation (IVT), have that T base which is transcribed as the U base in its corresponding translated mRNA. In this regard, the codon-optimized DNA sequence (including T) and its corresponding RNA sequence (including U) are considered the codon-optimized nucleotide sequences of the present invention. One skilled in the art would also understand that equivalent codon maps can be generated by one or more bases replaced by non-natural bases. Thus, for example, the TTC codon (DNA map) corresponds to the UUC codon (RNA map), and then this UUC codon is thought to correspond to the ΨΨC codon (RNA map where U is replaced by pseudouridine).

[0351] between the N3-H and C4-oxy of thymidine and the N1 and C6-NH of adenosine respectively 2 and between the C2-oxo, N3 and C4-NH of cytidine 2 and the C2-NH of guanosine respectively 2Under conditions that allow the formation of hydrogen bonds between N'-H and C6-oxy, standard A-T and G-C base pairs form. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such a modification results in a nucleoside base that no longer effectively forms a standard base pair with cytosine. However, modifying cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleoside that does not effectively form a base pair with guanosine but forms a base pair with isoguanosine (U.S. Patent No. 5,681,702 to Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine is prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and the references cited therein; 2'-deoxy-5-methyl-isocytidine is described by Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4 manufactured by the method of page 467 and the references cited therein; and isoguanine nucleotides can be manufactured using the methods described by Switzer et al., 1993, and Mantsch et al. (1993) Biochem. 14:5593 - 5601, or by the method described in U.S. Patent No. 5,780,610 to Collins et al. Other non - natural base pairs can be synthesized by the method described by Piccirilli et al. (1990) Nature 343:33 - 37 regarding the synthesis of 2,6 - diamino - pyrimidine and its complement (1 - methylpyrazolo - [4,3] pyrimidine - 5,7 - (4H,6H) - dione). Other such modified nucleotide units that form unique base pairs, such as those described by Leach et al. (1992) J. Am. Chem. Soc. 114:3675 - 3683 and Switzer et al., are known.

[0352] Polypeptide: The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can contain modified amino acids. The term also encompasses naturally - modified amino acid polymers, or amino acid polymers modified by any other operation or modification, such as disulfide - bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Similarly, polypeptides containing one or more analogs of amino acids (including non - natural amino acids such as homocysteine, ornithine, p - acetylphenylalanine, D - amino acids and creatine, etc.) and other modifications known in the art are included in this definition.

[0353] As used herein, this term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include the aforementioned gene products, natural polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs. A polypeptide can be a single polypeptide or a multimeric molecular complex such as a dimer, trimer, or tetramer. They can also include single-chain or multichain polypeptides. The most common disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids. In some embodiments, a "peptide" can be 50 amino acids in length or less, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0354] Prevention: As used herein, the term "preventing" refers to partially or completely delaying the onset of a disease, disorder, and / or condition; partially or completely delaying the onset of one or more of the symptoms, features, or clinical manifestations of a particular disease, disorder, and / or condition; partially or completely delaying the onset of one or more of the symptoms, features, or manifestations of a particular disease, disorder, and / or condition; partially or completely delaying the progression from a particular disease, disorder, and / or condition; and / or reducing the risk of developing the pathology associated with a disease, disorder, and / or condition.

[0355] Preventive: As used herein, "preventive" refers to a therapeutic process, or a process of action used to prevent the onset of a disease or condition, or to prevent or delay symptoms associated with a bleeding episode, such as those associated with hemophilia.

[0356] Prevention: As used herein, "prevention" refers to means employed to maintain health and to prevent or delay the onset of bleeding episodes or to prevent or delay symptoms associated with a disease or condition.

[0357] Recombinant: A "recombinant" polypeptide or protein refers to a polypeptide or protein produced by recombinant DNA methods. Polypeptides and proteins produced recombinantly and expressed in an engineered host cell are considered to be isolated for the purposes of the present invention in the same manner as a native or recombinant polypeptide that has been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be produced recombinantly using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be synthesized chemically.

[0358] Similarity: As used herein, the term "similarity" refers to the overall homology between polymeric molecules, such as polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. The calculation of the similarity rate between the interacting polymeric molecules can be performed in the same manner as the calculation of the identity rate, except that the similarity rate takes into account conservative substitutions, as understood in the art.

[0359] Subject: As used herein, the terms "subject", "individual", "animal", "patient", or "mammal" mean any subject, particularly a mammalian subject, whose diagnosis, prognosis, or treatment is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sports animals, pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as bears, cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters, and guinea pigs; etc. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In specific embodiments, the subject is a human patient or a cell thereof, whether in vivo, in vitro, or ex vivo, that is amenable to the methods described herein.

[0360] Substantially: As used herein, the term "substantially" refers to a quantitative state indicating the total or near total degree or extent of a desired feature or characteristic. One of ordinary skill in the biological arts will understand that biological and chemical phenomena, if any, are not completed and / or do not proceed to completion or achieve absolute results that are not or cannot be avoided. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0361] Substantially equivalent: As used herein, since it is related to the time difference between dosages, this term means plus / minus 2%.

[0362] Substantially simultaneously: As used herein, and in relation to multiple dosages, this term means within 2 seconds.

[0363] Affected: An individual who is "affected" by a disease, disorder, and / or condition has received a diagnosis of the disease, disorder, and / or condition or exhibits one or more of their symptoms.

[0364] Prone to being affected: An individual who is prone to being affected by a disease, disorder, and / or condition has not received a diagnosis of the disease, disorder, and / or condition and / or may not exhibit their symptoms, but has an inherent tendency to develop the disease or its symptoms. In some embodiments, an individual who is prone to being affected by a disease, disorder, and / or condition (e.g., cancer) can be characterized by one or more of the following: (1) genetic mutations associated with the development of the disease, disorder, and / or condition; (2) genetic polymorphisms associated with the development of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression level and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) habits and / or lifestyle associated with the development of the disease, disorder, and / or condition; (5) family history of the disease, disorder, and / or condition; (6) exposure to a microorganism associated with the development of the disease, disorder, and / or condition and / or infection by this microorganism. In some embodiments, an individual who is prone to being affected by a disease, disorder, and / or condition is expected to develop the disease, disorder, and / or condition. In some embodiments, an individual who is prone to being affected by a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0365] Therapeutic agent: The term "therapeutic agent" or "agent" refers to a molecular entity that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect. For example, in some embodiments, the bispecific antibodies disclosed herein can be therapeutic agents. In some embodiments, the agent is another molecule (e.g., a clotting factor, a complement factor, etc.) that is co-administered as part of a combination therapy with at least one of the antibodies disclosed herein.

[0366] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that, when administered to a subject suffering from or susceptible to an infectious disease, disease, disorder, and / or condition, is considered sufficient to treat the infectious disease, disease, disorder, and / or condition, to improve their symptoms, to diagnose them, to prevent them, and / or to delay their onset.

[0367] Therapeutically effective outcome: As used herein, the term "therapeutically effective outcome" means an outcome that is sufficient to treat an infectious disease, disease, disorder, and / or condition, to improve their symptoms, to diagnose them, to prevent them, and / or to delay their onset in a subject suffering from or susceptible to an infectious disease, disease, disorder, and / or condition.

[0368] Treat, treatment, therapy: As used herein, the term "treat" or "treatment" or "therapy" or grammatical variations thereof refers to the partial or complete alleviation, amelioration, improvement, reduction, delay in the onset, arrest in the progression, reduction in the severity, and / or decrease in the incidence of one or more symptoms or characteristics of a bleeding disorder, disorder, or condition, e.g., hemophilia. For example, "treating" a bleeding disorder can refer to preventing bleeding and / or reducing the frequency and / or severity of bleeding episodes, etc. Treatment can be carried out for the purpose of reducing the risk of developing the pathology associated with a disease, disorder, and / or condition, in a subject who does not exhibit signs of the disease, disorder, and / or condition, and / or in a subject who exhibits only early signs of the disease, disorder, and / or condition.

[0369] Vector: A "vector" is a nucleic acid molecule, particularly a self-replicating one, that transfers nucleic acid molecules inserted into host cells and / or between them. This term includes vectors that function primarily in inserting DNA or RNA into cells (e.g., chromosomal integration), vectors that function primarily in replicating DNA or RNA, and expression vectors that function in the transcription and / or translation of DNA or RNA. In some embodiments, administration and / or expression of nucleic acids (such as DNA or RNA like mRNA) encoding the binding molecules disclosed herein can be performed in vitro (e.g., during recombinant protein production), while , in other cases, it can be performed in vivo (e.g., administration of mRNA to a subject) or ex vivo (e.g., introduction of DNA or RNA into autologous or allogeneic cells for administration to a subject in need thereof). Similarly, vectors that provide more than one of the described functions are included.

[0370] II. Anti-FIX and Anti-FX Binding Molecules The present disclosure provides antibodies that bind to factor IX and factor X, and antigen-binding portions thereof. These antibodies are capable of preferentially binding to specific functional forms of these coagulation factors. For example, in some embodiments, the disclosed antibodies to FIX preferentially bind to activated FIX (FIXa), such as free FIXa or FIXa covalently linked to a mimetic substrate at the active site (FXa + EGR-CMK). In other embodiments, the disclosed antibodies preferentially bind to FIXa-SM over free FIXa or FIX prothrombin. In still other embodiments, the disclosed antibodies preferentially bind to free FIXa over FIXa-SM or FIX prothrombin. In contrast, in some embodiments, the disclosed antibodies to FX preferentially bind to FX prothrombin (FXz) over activated FX (FXa). This preferential binding is important for generating bispecific molecules that can specifically and simultaneously bind to FIXa and FXz, comprising an anti-FIXa portion and an anti-FXz portion. Factor VIII is Ca 2+In the presence of phospholipids, it is a cofactor for FIXa that forms a complex with FX which converts FX to activated FXa. Thus, the formation of an antibody-mediated complex between FIXa and FXz mimics the action of FVIIIa.

[0371] In yet other embodiments, some of the disclosed antibodies preferentially bind to FIX prothrombin over free FIXa or FIXa-SM (“anti-FIXz antibodies”). Thus, anti-FIXz antibodies can be used to generate bispecific molecules that include an anti-FIXz antibody and an anti-FX antibody (e.g., an anti-FXz antibody or an anti-FXa antibody).

[0372] In certain embodiments, some of the disclosed anti-FX antibodies preferentially bind to FXa over FXz (“anti-FXa antibodies”). Anti-FXa antibodies can be used to generate bispecific molecules that include an anti-FXa antibody and an anti-FIX antibody (e.g., an anti-FIXa antibody or an anti-FIXz antibody).

[0373] Thus, the formation of an antibody-mediated complex between FIX and FX can be used to bypass FVIII replacement therapy, particularly in subjects that have developed antibodies to FVIII or are at risk of developing antibodies to FVIII.

[0374] The present disclosure also provides bispecific binding molecules that bind to FX (FXz and / or FXa) and FIX (FIXz and / or FIXa). In one embodiment, the bispecific binding molecule can be a combination of any one of an anti-FIXa antibody or an anti-FIXz antibody and any one of an anti-FXa antibody or an anti-FXz antibody. In some embodiments, the bispecific binding molecule specifically binds to FXz, FIXz and FIXa, but has no detectable binding to FXa. In certain embodiments, the bispecific binding molecule binds to FIXz, FIXa and FXz with various binding affinities (e.g., K D )). In other embodiments, the bispecific binding molecule has a K D of less than 1 μM for each of FIXz, FIXa and FXz.to bind (e.g., 8 nM, 2 nM, or 20 nM each).

[0375] (a) Anti-FIXa binding molecule The present disclosure provides an anti-FIX binding molecule, e.g., an anti-FIX antibody that preferentially binds to activated FIX (FIXa) over FIX zymogen, or a molecule comprising an antigen-binding portion thereof .

[0376] Factor IX (FIX) is synthesized by stem cells as a zymogen precursor that requires extensive post-translational modification. The preprozymogen contains a prepeptide (hydrophobic signal peptide) at its amino terminus that transports the growing polypeptide into the lumen of the endoplasmic reticulum. This signal peptide is cleaved once inside the ER by signal peptidase. The propeptide functions as a recognition element for vitamin K-dependent carboxylase (γ-glutamyl carboxylase), which modifies 12 glutamic acid residues to γ-carboxyglutamyl (Gla) residues. These residues are required for binding to an anionic phospholipid surface by Ca2+-dependent binding.

[0377] The amino acid sequence of preproFIX zymogen is presented below (the signal sequence is underlined (1-28); the propeptide sequence (29-46) is shown in bold): [Chemical formula]

[0378] After cleavage of the signal peptide and propeptide, FIX is in the zymogen form. FIX zymogen thus circulates as a single-chain polypeptide of 415 amino acids. See Vysotchin et al., J. Biol. Chem. 268:8436 (1993). In one embodiment, FIX zymogen is amino acids 47-461 of SEQ ID NO: 764. In another embodiment, FIX zymogen is amino acids 47-461 of SEQ ID NO: 764, where amino acid residue 180 is alanine instead of arginine (i.e., non-activated FIX).

[0379] FIX zymogen is activated by FXIa or by the tissue factor / FVIIa complex. The first cleavage occurs at Arg191 (Arg145 in the mature FIX sequence) to produce inactive FIX-alpha. The second cleavage at Arg226 (Arg180 in the mature FIX sequence) removes the 35 amino acid FIX activation peptide to yield the catalytically active molecule FIXa-beta. This catalytically active FIXa, which does not bind FVIIIa, is also referred to herein as free FIXa. The resulting heterodimer is held together by a Cys178-Cys335 disulfide bridge. The serine protease contains the catalytic triad residues His267, Asp315, and Ser411. Upon cleavage at Arg226, Val227 can form an ionic bridge with Asp410, which is characteristic of the active serine protease. In one embodiment, free FIXa consists of amino acids 47-191 of SEQ ID NO: 764 and amino acids 227-461 of SEQ ID NO: 764, where amino acids 178 and 335 of SEQ ID NO: 764 form a disulfide bond.

[0380] However, the activity of free FIXa is known to be similar to that of FIX zymogen. Complex formation with the cofactor FVIIIa is an important second stage of activation, after which the potential Xase complex reaches an approximately 200,000-fold enhancement in activity that is highly specific for the physiological substrate FX and is restricted to the surface of activated platelets (van Dieije n et al., J Biol Chem. April 10, 1981; 256(7):3433-42). Activation of this macromolecule is assisted by low molecular weight agonists including Ca2+ (Mathur et al., Biol. Chem., 272 (1997), 23418-23426). A number of lines of evidence have shown that Ca2+ binding is associated with a conformational rearrangement (Bajaj et al., Proc. Natl. Acad. Sci. USA, 89 (1992), 152-156, Enfield and Thompson, Blood, 64 (1984), 821-831). This highly active FIXa in the tenase complex is mimicked by a pseudosubstrate (e.g., Glu-Gly-Arg-chloromethyl ketone (EGR-CMK)) by covalent binding to the active site of FIXa (also called FXa+EGR-CMK, FIXa-SM). Thus, FIXa-SM can be used as an important means to distinguish an antibody or an antibody-binding portion thereof that preferentially binds to highly active FIXa (in the tenase complex) compared to free FIXa.

[0381] Tripeptide chloromethyl ketones are generally accepted in this field as pseudosubstrates, the tripeptide sequence being the natural substrate sequence cleaved by a specific enzyme, and the CMK moiety being able to irreversibly trap this tripeptide at the active site by reacting with the serine at the active site. As a result, when the enzyme is bound to the pseudosubstrate, this should be the bound form of the substrate, i.e., the truly active conformation. See Brandsteter et al. (1995) Proc. Natl. Acad. Sci. USA 92(21):9796-80, and Hopfner et al. (1999) Structure 7(8):989-96.

[0382] The term "FIX zymogen" can be used interchangeably herein with "FIX z", "FIX precursor", "inactivated FIX", "non-activated FIX", or "non-activated FIX precursor". In one embodiment, FIX zymogen (FIX z) is a non-activated FIX precursor in which the activation peptide (e.g., the 35-amino acid activation peptide represented as amino acids 146-180 of SEQ ID NO: 764) (mature numbering) is not cleaved from the precursor. FIX zymogen can include any natural or engineered variant. A non-limiting example of FIX zymogen is shown in SEQ ID NO: 764. In another embodiment, FIX zymogen is non-activatable FIX (FIX n), which is engineered to be inactivated in the presence of factor XIa, an active plasma thromboplastin precursor. An example of non-activatable FIX can be FIX that results in an arginine-to-alanine mutation at position 180 (mature numbering), which blocks its activation and maintains factor IX in the zymogen form (FIX z). FIX zymogen can optionally contain a signal peptide and / or a propeptide.

[0383] The term "activated FIX" can be used interchangeably herein with "FIX a". In one embodiment, activated FIX is wild-type native FIX a (also referred to herein as "wild-type FIX a"). In another embodiment, FIX a includes non-native FIX a, such as FIX a conformational variants. For example, FIX a can be FIX a-SM, which is designed to have the same conformation as wild-type native FIX a bound to its substrate, FX. In certain embodiments, FIX a-SM is an activated FIX having a mimetic substrate covalently bound to the active site, which is intended to mimic the most active conformation of activated FIX.

[0384] FIX and FIXa can include FIX variants. In one embodiment, the FIX variants are cloned as described in U.S. Patent Nos. 4,770,999 and 7,700,734, and the cDNA encoding human factor IX is isolated, characterized, and cloned into an expression vector (e.g., Choo et al. , Nature 299:178-180 (1982); Fair et al., Blood 64:194-204 (1984); and Kurachi et al., Proc. Natl. Acad. Sci., U.S.A. 79:6461-6464 (1982)). One specific variant of FIX, the R338L FIX (Padua) variant characterized in 2009 by Simioni et al., contains a gain-of-function mutation, which correlates with a nearly 8-fold improvement in the activity of the Padua variant compared to native FIX. The FIX variant can also include any FIX polypeptide having one or more conservative amino acid substitutions that do not affect the FIX activity of the FIX polypeptide.

[0385] Accordingly, the present disclosure provides an antibody (e.g., an isolated antibody) or an antigen-binding portion thereof that specifically binds to activated factor IX (FIXa) (e.g., free FIXa or FIXa-SM), wherein the anti-FIXa antibody or an antigen-binding portion thereof preferentially binds to FIXa in the presence of FIXa and FIX prothrombin (an "anti-FIXa antibody or an antigen-binding portion thereof").

[0386] In some embodiments, the anti-FIXa antibody or an antigen-binding portion thereof binds to FIXa with a binding affinity higher than the binding affinity of the anti-FIXa antibody or an antigen-binding portion thereof for FIXz. In one embodiment, the binding affinity is represented as K D as.

[0387] The present disclosure also provides an isolated anti-FIXa antibody or an antigen-binding portion thereof that binds to FIXa with a binding affinity higher than that for FIXz of the anti-FIXa antibody or its antigen-binding portion. In some embodiments, the anti-FIXa antibody or its antigen-binding portion has a K D of about 100 nM or less (e.g., 1 nM to 100 nM or 0.1 nM to 100 nM), about 95 nM or less, about 90 nM or less, about 85 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 65 nM or less, about 60 nM or less, about 55 nM or less, about 50 nM or less, about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less as determined by a biolayer interferometry (BLI) assay, and binds to FIXa. In other embodiments, the anti-FIXa antibody or its antigen-binding portion has a K D of about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.2 nM or less, about 0.1 nM or less, or about 0.05 nM or less, and binds to FIXa. In yet other embodiments, the anti-FIXa antibody or its antigen-binding portion has a K D of 1 nM to 100 nM, 1 nM to 90 nM, 1 nM to 80 nM, 1 nM to 70 nM, 1 nM to 60 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, 1 nM to 10 nM, 0.1 nM to 100 nM, 0.1 nM to 90 nM, 0.1 nM to 80 nM, 0.1 nM to 70 nM, 0.1 nM to 60 nM, 0.1 nM to 50 nM, 0.1 nM to 40 nM, 0.1 nM to 30 nM, 0.1 nM to 20 nM, 0.1 nM to 10 nM, or 0.1 nM to 1 nM, and binds to FIXa.

[0388] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and / or 3C. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIGS. 3A, 3B, and / or 3C. In some embodiments, the reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, BIIB-9-433, and any combination thereof.

[0389] In a further embodiment, the anti-FIXa antibody or antigen-binding portion thereof can be further classified into the following three classes: Class I: an anti-FIXa antibody or antigen-binding portion thereof that preferentially binds to FIXa-SM over free FIXa or FIXz (the antibody in FIG. 3A); Class II: an anti-FIXa antibody or antigen-binding portion thereof that preferentially binds to free FIXa over FIXa-SM or FIXz (the antibody in FIG. 3B); and Class III: an anti-FIXa antibody or antigen-binding portion thereof that binds to free FIXa and FIXa-SM almost equivalently but does not bind significantly to FIXz (the antibody in FIG. 3C).

[0390] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3A. In other embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3A. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3B. In other embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3B. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3C. In other embodiments, the anti-FIXa antibody or antigen-binding portion thereof binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3C.

[0391] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and VH CDR3 is (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3A, or (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3A, except for one, two, or three amino acid substitutions and comprises.

[0392] In other embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and VH CDR3 is (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3B, or (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3B, except for one, two, or three amino acid substitutions and comprises.

[0393] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and VH CDR3 is (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3C, or (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of the VH CDR3 sequences in FIG. 3C, except for one, two, or three amino acid substitutions and comprises.

[0394] In some embodiments, the amino acid substitution is a conservative amino acid substitution. In other embodiments, the amino acid substitution is a reverse mutation.

[0395] In one aspect, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2 and VH CDR3, and the VH CDR3 sequence is ARDX 1 X 2 X 3 X 4 X 5 X 6 YYX 7 includes MDV (SEQ ID NO: 753), and X 1 is V or G, and X 2 is G or V, and X 3 is G or R, and X 4 is Y or V, and X 5 is A or S, and X 6 is G or D, and X 7 is G or does not exist.

[0396] One of ordinary skill in the art understands that when a position is described as "absent" or "not present" in a consensus sequence, the absence of such presence does not necessarily indicate a cleavage in the polypeptide chain. These terms merely reflect the occurrence of insertions and deletions in the amino acid chain observed in a multiple sequence alignment. Thus, when two sequences, one of which contains an amino acid insertion, are aligned to generate a consensus sequence, the sequence without the insertion is expected to have an "absent" ("none") amino acid at that position.

[0397] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR3 sequence comprises an amino acid sequence selected from ARDVGGYAGYYGMDV (SEQ ID NO: 905; VH CDR3 in the case of BIIB-9-484, BIIB-9-1335, and BIIB-9-1336), ARDISTDGESSLYYYMDV (SEQ ID NO: 901; BIIB-9-460), ARGPTDSSGYLDMDV (SEQ ID NO: 1186; BIIB-9-882), ARSPRHKVRGPNWFDP (SEQ ID NO: 899; BIIB-9-440), or ARDGPRVSDYYMDV (SEQ ID NO: 912; BIIB-9-619). In some embodiments, the VH CDR3 sequences disclosed herein can comprise one, two, or three amino acid substitutions.

[0398] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR1 sequence (i) is identical to a sequence selected from the group consisting of VH CDR1 sequences disclosed in FIG. 3A, or (ii) is identical to a sequence selected from the group consisting of VH CDR1 sequences disclosed in FIG. 3A, except for one, two, or three amino acid substitutions. The VH CDR1 sequence contains include.

[0399] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR1 sequence (i) is identical to a sequence selected from the group consisting of VH CDR1 sequences disclosed in FIG. 3B, or (ii) is identical to a sequence selected from the group consisting of VH CDR1 sequences disclosed in FIG. 3B, except for one, two, or three amino acid substitutions. The VH CDR1 sequence contains include.

[0400] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR1 sequence is (i) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3C, or (ii) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3C, excluding one, two, or three amino acid substitutions and includes.

[0401] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR2 sequence is (i) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3A, or (ii) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3A, excluding one, two, or three amino acid substitutions and includes.

[0402] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR2 sequence is (i) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3B, or (ii) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3B, excluding one, two, or three amino acid substitutions and includes.

[0403] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3, and the VH CDR2 sequence is (i) An VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3C, or (ii) An VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3C, except for one, two, or three amino acid substitutions and includes. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VL CDR1, VL CDR2, and VL CDR3, and the VL CDR1 sequence is

[0404] (i) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3A, or (ii) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3A, except for one, two, or three amino acid substitutions and includes. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VL CDR1, VL CDR2, and VL CDR3, and the VL CDR1 sequence is (i) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B, or

[0405] (ii) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B, except for one, two, or three amino acid substitutions (i) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B, or (ii) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B, except for one, two, or three amino acid substitutions and includes. In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof includes VL CDR1, VL CDR2, and VL CDR3, and the VL CDR1 sequence is

[0406] (i) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3C, or (ii) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3C, except for one, two, or three amino acid substitutions (ii) An VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3C, except for one, two, or three amino acid substitutions A VL CDR1 sequence identical to a sequence selected from the group consisting of CDR1 sequences is included.

[0407] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, VL CDR2, and VL CDR3, and the VL CDR2 sequence is (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3A, or (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3A, with the exception of one, two, or three amino acid substitutions is included.

[0408] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, VL CDR2, and VL CDR3, and the VL CDR2 sequence is (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3B, or (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3B, with the exception of one, two, or three amino acid substitutions is included.

[0409] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, VL CDR2, and VL CDR3, and the VL CDR2 sequence is (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3C, or (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in Figure 3C, with the exception of one, two, or three amino acid substitutions is included.

[0410] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VL CDR1, VL CDR2, and VL CDR3, and the VL CDR3 sequence is (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in Figure 3A, or (ii) the VL disclosed in FIG. 3A except for one, two or three amino acid substitutions a VL CDR3 sequence identical to a sequence selected from the group consisting of Includes.

[0411] In some embodiments, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2 and a VL CDR3, wherein the VL CDR3 sequence is (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in Figure 3B, or (ii) the VL disclosed in FIG. 3B except for one, two or three amino acid substitutions a VL CDR3 sequence identical to a sequence selected from the group consisting of Includes.

[0412] In some embodiments, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2 and a VL CDR3, wherein the VL CDR3 sequence is (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in Figure 3C, or (ii) the VL disclosed in FIG. 3C except for one, two or three amino acid substitutions a VL CDR3 sequence identical to a sequence selected from the group consisting of Includes.

[0413] The present disclosure also provides an isolated antibody or antigen-binding portion thereof that specifically binds to FIXa and includes VH CDR1, VH CDR2, and VH CDR3 as well as VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 as well as VL CDR1, VL CDR2, and VL CDR3 are the VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 of the antibodies: BIIB-9-605, BIIB-9-475, BIIB-9-477, BIIB-9-479, BIIB-9-480, BIIB-9-558, BIIB-9-414, BIIB-9-415, BIIB-9-425, BIIB-9-440, BIIB-9-452, BIIB-9-460, BIIB-9-461, BIIB-9-465, BIIB-9-564, BIIB-9-484, BIIB-9-469, BIIB-9-566, BIIB-9-567, BIIB-9-569, BIIB-9-588, BII B-9-611, BIIB-9-619, BIIB-9-626, BIIB-9-883, BIIB-9-419, BIIB-9-451, BIIB-9-473, BIIB-9-565, BIIB-9-573, BIIB-9-579, BIIB-9-581, BIIB-9-582, BIIB-9-585, BIIB-9-587, BIIB-9-590, BIIB-9-592, BIIB-9-606, BIIB-9-608 BIIB-9-616, BIIB-9-621, BIIB-9-622, BIIB-9-627, BIIB-9-1335, and BIIB-9-1336, and include the VH CDR1, VH CDR2, and VH CDR3 as well as VL CDR1, VL CDR2, and VL CDR3 of anti-FIXa antibodies selected from the group consisting of.

[0414] In some embodiments, the present disclosure includes an isolated antibody or antigen-binding portion thereof that specifically binds to FIXa, comprising VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 are the VH CDRs of an anti-FIXa antibody selected from the group consisting of antibodies: BIIB-9-408, BIIB-9-416, BIIB-9-629, or BIIB-9-885 in FIG. 3B comprising VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3.

[0415] In other embodiments, the present disclosure provides an isolated antibody or antigen-binding portion thereof that specifically binds to FIXa and includes VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3, and VL CDR1, VL CDR2, and VL CDR3 are the VH CDR1, VH CDR2, and VH CDR3 of the anti-FIXa antibodies selected from the group consisting of antibodies: BIIB-9-607, BIIB-9-471, BIIB-9-472, BIIB-9-439, BIIB-9-446, BIIB-9-568, BIIB-9-615, BIIB-9-628, BIIB-9-882, BIIB-9-884, BIIB-9-886, BIIB-9-887, BIIB-9-888, BIIB-9-889, BIIB-9-433, BIIB-9-445, BIIB-9-470, BIIB-9-625, BIIB-9-1264, BIIB-9-1265, BIIB-9-1266, BIIB-9-1267, BIIB-9-1268, BIIB-9-1269, BIIB-9-1270, BIIB-9-1271, BIIB-9-1272, BIIB-9-1273, BIIB-9-1274, BIIB-9-1275, BIIB-9-1276, BIIB-9-1277, BIIB-9-1278, BIIB-9-1279, BIIB-9-1280, BIIB-9-1281, BIIB-9-1282, BIIB-9-1283, BIIB-9-1284, BIIB-9-1285, BIIB-9-1286, and BIIB-9-1287 in FIG. 3C, and VL CDR1, VL include CDR2 and VL CDR3.

[0416] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of class I antibodies) comprising SEQ ID NOs: 800-844, 845-889, and 890-934, and / or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 935-979, 980-1024, and 1025-1069, respectively (VL CDRs of class I antibodies).

[0417] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of class II antibodies) comprising SEQ ID NOs: 1070-1073, 1074-1077, and 1078-1081, and / or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1082-1085, 1086

[0418] ~1089, and 1090-1093, respectively (VL CDRs of class II antibodies). In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of class III antibodies) comprising SEQ ID NOs: 1094-1135, 1136-1177, and 1178-1219, and / or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1220-1261, 1262-1303, and 1304-1345, respectively (VL CDRs of class III antibodies).

[0419] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-484 antibody) comprising SEQ ID NO: 815, SEQ ID NO: 860, and SEQ ID NO: 905, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-484 antibody) comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively.

[0420] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-1335 antibody) comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-1335 antibody) comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively.

[0421] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises, respectively, VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-1336 antibody) comprising SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-1336 antibody) comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively.

[0422] In other aspects, the anti-FIXa antibody or antigen-binding portion thereof cross-competes with, and / or binds to the same epitope as, antibodies BIIB-9-484, BIIB-9-1335, and BIIB-9-1336. In certain aspects, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3 as well as VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR3 comprises ARDVGGYAGYYGMDV (SEQ ID NO: 905, BIIB-9-484 VH CDR3), VH CDR2 comprises SISSX 1 X 2 SYIYYAX 3 comprises SVKG (SEQ ID NO: 754), and X 1 comprises S, G, or any conservative substitution, and X 2 comprises S, E, or any conservative substitution, and X 3 comprises D, E, or any conservative substitution, and VH CDR1 comprises FTFX 4 SYX 5 MX 6( comprises SEQ ID NO: 755), and X 4 comprises S, G, or any conservative substitution, and X 5 comprises D, S, or any conservative substitution, and X 6 comprises H, N, or any conservative substitution. The anti-FIXa antibody or antigen-binding portion thereof can comprise SEQ ID NO: 815 for VH CDR1, SEQ ID NO: 860 for VH CDR2, and SEQ ID NO: 905 for VH CDR3 (BIIB-9-484 VH CDR).

[0423] In some aspects, the anti-FIXa antibody or antigen-binding portion thereof (a1) VH CDR1, VH CDR2, and VH CDR3 sequences each comprising SEQ ID NO: 809, SEQ ID NO: 854, and SEQ ID NO: 899 (of the BIIB-9-440 antibody (VH CDRs), and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-440 antibody) each comprising SEQ ID NO: 944, SEQ ID NO: 989, and SEQ ID NO: 1034; (a2) VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-882 antibody) each comprising SEQ ID NO: 1102, SEQ ID NO: 1144, and SEQ ID NO: 1186, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-882 antibody) each comprising SEQ ID NO: 1228, SEQ ID NO: 1270, and SEQ ID NO: 1312; (a3) VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-460 antibody) each comprising SEQ ID NO: 811, SEQ ID NO: 856, and SEQ ID NO: 901, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-460 antibody) each comprising SEQ ID NO: 946, SEQ ID NO: 991, and SEQ ID NO: 1036; or (a4) VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-433 antibody) each comprising SEQ ID NO: 1108, SEQ ID NO: 1150, and SEQ ID NO: 1192, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-433 antibody) each comprising SEQ ID NO: 1234, SEQ ID NO: 1276, and SEQ ID NO: 1318; comprising.

[0424] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-619 antibody) each comprising SEQ ID NO: 822, SEQ ID NO: 867, and SEQ ID NO: 912, and / or VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-619 antibody) each comprising SEQ ID NO: 957, SEQ ID NO: 1002, and SEQ ID NO: 1047.

[0425] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof (i) the VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-1335 antibody) each comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, respectively, and / or the VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-1335 antibody) each comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively; or (ii) the VH CDR1, VH CDR2, and VH CDR3 sequences (VH CDRs of the BIIB-9-1336 antibody) each comprising SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, respectively, and / or the VL CDR1, VL CDR2, and VL CDR3 sequences (VL CDRs of the BIIB-9-1336 antibody) each comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040 is included.

[0426] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises a VH, and the VH is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181 (in the case of class I antibodies, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 2 5, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89; for class II antibodies, SEQ ID NOs: 91, 93, 95, and 97; and for class III antibodies, SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181), and an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.

[0427] In some embodiments, the anti-FIXa antibody or antigen-binding portion thereof comprises a VL, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367 (for class I antibodies, SEQ ID NOs: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, and 275; for class II antibodies, SEQ ID NOs: 277, 279, 281, and 283; for class III antibodies, SEQ ID NOs: 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367) and comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical.

[0428] In one aspect, the anti-FIXa antibody or antigen-binding portion thereof comprises VH and VL, and (i) the VH is set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181 (for class I antibodies, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89; for class II antibodies, SEQ ID NOs: 91, 93, 95, and 97; and for class III antibodies, SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, and 181) and comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical; (ii) VL is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367 (for class I antibodies, SEQ ID NOs: 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, and 275; for class II antibodies, SEQ ID NOs: 277, 279, 281, and 283; for class III antibodies, SEQ ID NOs: 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, and 367) and comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical.

[0429] In certain embodiments, the anti-FIXa antibody comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region from a particular germline light chain immunoglobulin gene. In some embodiments, the VH sequence of the anti-FIXa antibody can be derived from any one of the V, D, or J germline sequences, and / or the VL sequence of the anti-FIXa antibody can be derived from any one of the kappa or lambda germline sequences.

[0430] As demonstrated herein, human antibodies specific for FIXa are produced that comprise or are derived from heavy chain variable regions that are products of human germline genes. Accordingly, provided herein are isolated FIXa antibodies or antigen-binding portions thereof that comprise or are derived from heavy chain variable regions that are products of human VH germline genes selected from the group consisting of VH1-18, VH1-46, VH3-21, VH3-30, VH4-31, VH4-39, VH4-0B, VH5-51, and any combination thereof. In certain embodiments, the VH germline gene is selected from the group consisting of VH1-18.0, VH1-18.1, VH1-18.8, VH1-46.0, VH1-46.4, VH1-46.5, VH1-46.6, VH1-46.7, VH1-46.8, VH1-46.9, VH3-21.0, VH3-23.0, VH3-23.2, VH3-23.6, VH3-30.0, VH4-31. 5, VH4-39.0, VH4-39.5, VH4-0B.4, VH5-51.1, and any combination thereof.

[0431] In other embodiments, provided herein is an isolated FIXa antibody or antigen-binding portion thereof that is a product of, or derived from, a human VL germline gene selected from the group consisting of VK1-05, VK1-12, VK1-39, VK2-28, VK3-11, VK3-15, VK3-20, VK4-01, and any combination thereof. In certain embodiments, the VL germline gene is selected from the group consisting of VK1-05.6, VK1-05.12, VK1-12.0, VK1-12.4, VK1-12.7, VK1-12.10, VK1-12.15, VK1-39.0, VK1-39.3, VK1-39.15, VK2-28.0, VK2-28.1, VK2-28.5, VK3-11.0, VK3-11.2, VK3-11.6, VK3-11.14, VK3-15.0, VK3-15.8, VK3-15.10, VK3-20.0, VK3-20.1, VK3-20.4, VK3-20.5, VK4-01.0, VK4-01.4, VK4-01.20, and any combination thereof.

[0432] As shown in the figures, the antibodies described herein include those that are a product of, or derived from, one of the human germline VH genes listed above and include a heavy chain variable region, and also include those that are a product of, or derived from, one of the human germline VK genes listed above and also include a light chain variable region.

[0433] As used herein, a human antibody includes heavy and light chain variable regions that are “products of, or derived from” specific germline sequences when the variable regions of th...

Claims

1. An isolated antibody or antigen-binding portion thereof that specifically binds to activated factor IX (FIXa) (an "anti-FIXa antibody or antigen-binding portion thereof"), wherein the anti-FIXa antibody or antigen-binding portion thereof preferentially binds to FIXa in the presence of FIXa and factor IX zymogen (FIXz), or the anti-FIXa antibody or antigen-binding portion thereof binds to FIXa with a higher binding affinity than the binding affinity of the anti-FIXa antibody or antigen-binding portion thereof to FIXz.

2. 2. The anti-FIXa antibody or antigen-binding portion thereof of claim 1, which cross-competes with or binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figures 3A, 3B and 3C.

3. 3. The anti-FIXa antibody or antigen-binding portion thereof of claim 1 or 2, wherein the epitope comprises amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240 and K241 according to the chymotrypsinogen numbering of the sequence of the heavy chain of FIXa, or a combination thereof.

4. and / or (ii) the VH CDR2 comprises a VH CDR2 selected from the group consisting of a VH CDR2 of FIG. 3A, FIG. 3B, and FIG. 3C, a VH CDR2 of SEQ ID NO: 2084-2089, or ... and / or (iv) the VL CDR1 comprises a VL CDR1 selected from the group consisting of the VL CDR1 of Figure 3A, Figure 3B and Figure 3C, SEQ ID NOs: 2136-2141, or said VL CDR1 having one or two mutations; and / or (v) the VL CDR2 comprises a VL CDR2 selected from the group consisting of the VL CDR2 of Figure 3A, Figure 3B and Figure 3C, SEQ ID NOs: 2162-2167, or said VL CDR2 having one or two mutations; and / or (vi) the VL CDR3 comprises a VL CDR2 of Figure 3A, Figure 3B and Figure 3C, SEQ ID NOs: 2188-2193.

4. The anti-FIXa antibody or antigen-binding portion thereof of any one of claims 1 to 3, comprising a VL CDR3 selected from the group consisting of: a VL CDR3 having one or two mutations;

5. (a1) the VH and VL comprise SEQ ID NOs: 31 and 221 (BIIB-9-484); (a2) the VH and VL comprise SEQ ID NOs: 19 and 209 (BIIB-9-440); (a3) ​​the VH and VL comprise SEQ ID NOs: 115 and 301 (BIIB-9-882); (a4) the VH and VL comprise SEQ ID NOs: 23 and 213 (BIIB-9-460); (a5) the VH and VL comprise SEQ ID NOs: 127 and 313 (BIIB-9 (a5) the VH and VL comprise SEQ ID NOs: 47 and 48, respectively (BIIB-9-433); (a6) the VH and VL comprise SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); (a7) the VH and VL comprise SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); (a8) the VH and VL comprise SEQ ID NOs: 87 and 221, respectively (BIIB-9-1335); or (a9) the VH and VL comprise SEQ ID NOs: 89 and 221, respectively (BIIB-9-1336).

6. An isolated antibody or antigen-binding portion thereof that specifically binds to factor X zymogen (FXz) (an "anti-FXz antibody or antigen-binding portion thereof"), wherein the anti-FXz antibody or antigen-binding portion thereof preferentially binds to FXz in the presence of FXz and activated factor X (FXa).

7. 7. The anti-FXz antibody or antigen-binding portion thereof of claim 6, which cross-competes with or binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in Figures 12A and 12B.

8. The anti-FXz antibody or antigen-binding portion thereof of claim 6 or 7, comprising a VH and a VL, wherein (b1) the VH and VL comprise SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); (b2) the VH and VL comprise SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or (b3) the VH and VL comprise SEQ ID NOs: 455 and 643, respectively (BIIB-12-932).

9. An isolated antibody or antigen-binding portion thereof that specifically binds to activated factor X (FXa) (an "anti-FXa antibody or antigen-binding portion thereof"), wherein the anti-FXa antibody or antigen-binding portion thereof preferentially binds to FXa in the presence of FXz and FXa and / or binds to FXa with a binding affinity that is higher than the binding affinity of the antibody or antigen-binding portion thereof to FXz.

10. 10. An anti-FXa antibody or antigen-binding portion thereof as described in claim 9, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 12C or binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 12C.

11. 11. The anti-FXa antibody or antigen-binding portion thereof of claim 9 or 10, comprising a VH and a VL, the VH and VL comprising SEQ ID NOs: 559 and 747 (BIIB-12-925), respectively.

12. The anti-FIX antibody or antigen-binding portion thereof of any one of embodiments 1 to 5, and / or (ii) A bispecific molecule comprising an anti-FX antibody or antigen-binding portion thereof according to any one of claims 6 to 11.

13. A nucleic acid encoding the antibody according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 12, the nucleic acid according to claim 13, and a pharma- ceutically acceptable carrier.

15. An antibody according to any one of claims 1 to 12, a nucleic acid according to claim 13 or a pharmaceutical composition according to claim 14 for use in therapy.

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