BISPECIFIC ANTIBODIES BINDING TO COAGULATION FACTOR IX AND COAGULATION FACTOR X

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name: 4159.485PC04_Sequence_listing_ST25.txt; Size: 1,053,370 bytes; and Date of Creation: Nov. 21, 2017) filed with the application is incorporated herein by reference in its entirety.

BACKGROUND
Field

This application pertains to, among other things, antibodies that preferentially bind to activated coagulation factor IX or coagulation factor X zymogen, as well as bispecific molecules comprising both specificities that mimic the activated factor VIII cofactor.

Background

Hemophilia A is a severe X-chromosome-linked recessive disorder caused by mutations in the factor VIII (FVIII) gene. FVIII is involved in the intrinsic pathway of blood coagulation, and FVIII deficiency leads to blood either coagulating poorly, or barely at all. FVIII deficiency, alternatively known as hemophilia A, is one of the most common hemorrhagic disorders, and affects one in about 10,000 males (Stonebraker et al. (2012) Haemophilia 18(3):e91-4). Hemophilia A has three grades of severity defined by factor FVIII plasma levels of 1% or less (“severe”), 2 to 5% (“moderate”), and 6 to 30% (“mild”) (White et al. (2001) Thromb. Haemost. 85:560). In severe forms of the disorder, the first bleeds typically appear at 5 to 6 months of age, whereas the first bleeds are delayed until about 1 to 2 years of age in the moderate form. A bleed can appear spontaneously, or following minimum trauma. Approximately half of all patients with hemophilia A are classified as having the severe form of the disease. These patients experience severe bleeding starting in early childhood, and frequent episodes of spontaneous or excessive bleeding later in life. Bleeding commonly occurs into joints and muscles, and without appropriate treatment, recurrent bleeding can lead to irreversible hemoarthropathy (Manco-Johnson et al. (2007) N. Engl. J. Med. 357(6):535-44).

An important goal of hemophilia A treatment is maintenance of FVIII plasma levels ≥1%, which reduces bleeding risk. To achieve this, intravenous recombinant or plasma-derived FVIII is administered frequently as prophylactic therapy. However, this current standard of treatment of hemophilia A is difficult, has several drawbacks, and incurs a considerable physical and mental burden on patients and their families.

The most common hindrance in FVIII treatment is the production of alloantibodies against FVIII, which act as FVIII inhibitors. As many as 30% of severely affected patients develop such alloantibodies, and once development has occurred, the effective use of FVIII for treating on-going bleeds is restricted (Kempton & White (2009) Blood 113(1):11-7). In such cases, alternative bypassing agents are used to control bleeding. However, these agents typically have shorter half-lives and are not always effective. Furthermore, frequent administration of FVIII is required due to its short plasma half-life (an average of about 12 hours in adults, and even shorter in children). Such a regimen can be difficult, particularly in young children. Since available treatments are associated with complications and side effects, there is no single treatment that optimally and effectively treats hemophilia. Thus, there remains an unmet need for new and effective treatments that resolve the drawbacks of treating hemophilia A with FVIII.

BRIEF SUMMARY

The present disclosure provides an isolated antibody, or an antigen binding portion thereof, that specifically binds to activated factor IX (FIXa) (“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). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof binds to FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz. The disclosure also provides an isolated anti-FIXa antibody, or antigen binding portion thereof, which binds to FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof binds to FIXa with a K_Dof 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 Bio-Layer Interferometry (BLI) assay. In some aspects, the FIXa is free FIXa, FIXa in a tenase complex, or FIXa covalently linked to EGR-CMK (FIXa-SM). In some aspects, the FIXz comprises non-activatable Factor IX (FIXn). In some aspects, 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, FIG. 3B, and FIG. 3C. In some aspects, 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, FIG. 3B, and FIG. 3C. In some aspects, such reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, BIIB-9-433, and any combination thereof. In some aspects, 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 a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to free FIXa or FIXz. In some aspects, 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 aspects, 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 aspects, such reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-460, and any combination thereof. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof preferentially binds to free FIXa compared to FIXa-SM or FIXz and/or binds to free FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXa-SM or FIXz. In some aspects, 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 some aspects, 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 aspects, the anti-FIXa antibody, or antigen binding portion thereof preferentially binds to free FIXa or FIXa-SM compared to FIXz and/or binds to free FIXa or FIXa-SM with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz. In some aspects, 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 some aspects, 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. In some aspects, such reference antibody is selected from BIIB-9-882, BIIB-9-433, and the combination thereof. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR3 with one or two mutations. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises ARDX₁X₂X₃X₄X₅X₆YYX₇MDV (SEQ ID NO:753), wherein X₁is V or G, X₂is G or V, X₃is G or R, X₄is Y or V, X₅is A or S, X₆is G or D, X₇is G or none. In some aspects, the 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 aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR1 with one or two mutations. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR2 with one or two mutations. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR1 with one or two mutations. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR2 with one or two mutations. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR3 with one or two mutations.

The present disclosure also provides an isolated anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 comprise VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, CDR2, and CDR3 of FIG. 3A, FIG. 3B, and FIG. 3C, respectively. In some aspects, the anti-FIXa antibody or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 815, 860, and 905, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 950, 995, and 1040, respectively (BIIB-9-484). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 809, SEQ ID NO: 854, and SEQ ID NO: 899, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 944, SEQ ID NO: 989, and SEQ ID NO: 1034, respectively (BIIB-9-440). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 1102, SEQ ID NO: 1144, and SEQ ID NO: 1186, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 1228, SEQ ID NO: 1270, and SEQ ID NO: 1312, respectively (BIIB-9-882). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 811, SEQ ID NO: 856, and SEQ ID NO: 901, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 946, SEQ ID NO: 991, and SEQ ID NO: 1036, respectively (BIIB-9-460). In some aspects, 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 comprising SEQ ID NO: 1234, SEQ ID NO: 1276, and SEQ ID NO: 1318, respectively (BIIB-9-433). In some aspects, 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 comprising SEQ ID NO: 957, SEQ ID NO: 1002, and SEQ ID NO: 1047, respectively (BIIB-9-619). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively, respectively or (ii) the anti-FIXa antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively (BIIB-9-1335 and BIIB-9-1336). the anti-FIXa antibody, or antigen binding portion thereof comprises a VH and a VL, wherein the VH comprises an amino acid sequence which 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 the 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. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises a VH and a VL, wherein the VL comprises an amino acid sequence which 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 the 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. In some aspects, 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 aspects, 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 some aspects, the anti-FIXa antibody, or antigen binding portion thereof comprises a VH and a VL, wherein (a1) VH and VL comprise SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); (a2) VH and VL comprise SEQ ID NOs: 19 and 209, respectively (BIIB-9-440); (a3) VH and VL comprise SEQ ID NOs: 115 and 301, respectively (BIIB-9-882); (a4) VH and VL comprise SEQ ID NOs: 23 and 213, respectively (BIIB-9-460); (a5) VH and VL comprise SEQ ID NOs: 127 and 313, respectively (BIIB-9-433); (a6) VH and VL comprise SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); (a7) VH and VL comprise SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); (a8) VH and VL comprise SEQ ID NOs: 87 and 221, respectively (BIIB-9-1335); or, (a9) VH and VL comprise SEQ ID NOs: 89 and 221, respectively (BIIB-9-1336).

The present disclosure also provides an isolated antibody, or an antigen binding portion thereof, which specifically binds to FIXz (“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 a binding affinity of the anti-FIXz antibody or antigen binding portion thereof to free FIXa or FIXa-SM. In some aspects, 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 aspects, 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 aspects, such reference antibody is BIIB-9-578. In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 3D or the VH CDR3 with one or two mutations. In some aspects, the CDR3 comprises ARDKYQDYSFDI (SEQ ID NO: 1355, BIIB-9-578). In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 3D or the VH CDR1 with one or two mutations. In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 3D or the VH CDR2 with one or two mutations. In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 3D or the VL CDR1 with one or two mutations. In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 3D or the VL CDR2 with one or two mutations. In some aspects, the anti-FIXz antibody, or antigen binding portion thereof comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 3D or the VL CDR3 with one or two mutations. In some aspects, the anti-FIX antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4 or a variant thereof. In some aspects, the anti-FIX antibody is an IgG4 antibody. In some aspects, the anti-FIX antibody comprises an effectorless IgG4 Fc. In some aspects, the anti-FIX antibody, or antigen binding portion thereof comprises a heavy chain constant region. In some aspects, the anti-FIX antibody is a human antibody, an engineered antibody, or a humanized antibody. In some aspects, the anti-FIX antigen binding portion comprises an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv (scFv).

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

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

The present disclosure provides an isolated antibody, or an antigen binding portion thereof, that specifically binds to factor X zymogen (FXz) (“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 aspects, the anti-FXz antibody, or antigen binding portion thereof binds to FXz with a binding affinity higher than a binding affinity of the antibody or antigen binding portion thereof to FXa. Also provided is an isolated anti-FXz antibody, or antigen binding portion thereof, which binds to FXz with a binding affinity higher than a binding affinity of the antibody or antigen binding portion thereof to FXa. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, binds to FXz with a K_Dof 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. In some aspects, the FXa is free FXa or FXa covalently linked to EGR-CMK (FIXa-SM). In some aspects, the FXz comprises non-activatable Factor X (FXn). In some aspects, the anti-FXz antibody, or antigen binding portion thereof cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 12A and FIG. 12B. In some aspects, 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 FIG. 12A and FIG. 12B. In some aspects, 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 aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 12A and FIG. 12B or the VH CDR3 with one or two mutations. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises ARX₁X₂X₃RX₄X₅X₆X₇FDX₈(SEQ ID NO: 766), wherein X₁is G or L, X₂is R or G, X₃is F or Y, X₄is P or G, X₅is R or A, X₆is G or S, X₇is R or A, and X₈is Y or I. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises CDR1, CDR2, and CDR3, wherein the 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 aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 12A and FIG. 12B or the VH CDR1 with one or two mutations. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 12A and FIG. 12B or the VH CDR2 with one or two mutations. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 12A and FIG. 12B or the VL CDR1 with one or two mutations. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 12A and FIG. 12B or the VL CDR2 with one or two mutations. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 12A and FIG. 12B or the VL CDR3 with one or two mutations.

The present disclosure also provides an isolated antibody, or antigen binding portion thereof, which specifically binds to FXz, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 comprise VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 of FIG. 12A and FIG. 12B, respectively. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1393, 1483, or 1573, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1663, 1753, or 1843, respectively (BIIB-12-915). In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1395, 1485, or 1575, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1665, 1755, or 1845, respectively (BIIB-12-917). In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1409, 1499, or 1589, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1679, 1769, or 1859, respectively (BIIB-12-932). In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH and VL, wherein the VH comprises an amino acid sequence which 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 the 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. In some aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises VH and VL, wherein the VL comprises an amino acid sequence which 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 the 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. In some aspects, 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 aspects, the anti-FXz antibody, or antigen binding portion thereof, comprises a VH and a VL, wherein the VL is derived from a germline sequence 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. In some aspects, the anti-FX antibody, or antigen binding portion thereof, comprises VH and VL, wherein (b1) VH and VL comprise SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); (b2) VH and VL comprise SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or (b3) VH and VL comprise SEQ ID NOs: 455 and 643, respectively (BIIB-12-932).

The present disclosure also provides an isolated antibody, or an antigen binding portion thereof, that specifically binds to activated factor X (FXa) (“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 a binding affinity of the antibody or antigen binding portion thereof to FXz. In some aspects, 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 aspects, 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 aspects, 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 aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 12C or the VH CDR3 with one or two mutations. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises AKGPRYYWYSWYFDL (SEQ ID NO: 1919, BIIB-12-925). In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 12C or the VH CDR1 with one or two mutations. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 12C or the VH CDR2 with one or two mutations. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 12C or the VL CDR1 with one or two mutations. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 12C or the VL CDR2 with one or two mutations. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 12C or the VL CDR3 with one or two mutations.

The present disclosure also provides an isolated antibody, or antigen binding portion thereof, which specifically binds to FXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 comprise VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 of FIG. 12C, respectively. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1911, 1915, or 1919, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1923, 1927, or 1931, respectively (BIIB-12-925). In some aspects, the anti-FXa antibody, or antigen binding portion thereof, comprises VH and VL, wherein the VH and VL comprise SEQ ID NOs: 559 and 747, respectively (BIIB-12-925). In some aspects, the anti-FX antibody, or antigen binding portion thereof, is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4 or a variant thereof. In some aspects, the anti-FX antibody, or antigen binding portion thereof, is an IgG4 antibody. In some aspects, the anti-FX antibody, or antigen binding portion thereof, comprises an effectorless IgG4 Fc. In some aspects, the anti-FX antibody, or antigen binding portion thereof, comprises a heavy chain constant region. In some aspects, the anti-FX antibody is a human antibody, an engineered antibody, or a humanized antibody. In some aspects, the antigen binding portion thereof comprises an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv (scFv). The present disclosure also provides a bispecific molecule comprising an anti-FX antibody disclosed herein linked to a molecule having a second binding specificity. Also provided is a nucleic acid encoding the heavy and/or light chain variable region of an anti-FX antibody disclosed herein, or antigen binding portion thereof, or a bispecific molecule comprising an anti-FX antibody or antigen binding portion thereof disclosed herein. Also provided is an expression vector comprising the nucleic acid molecule. Also provided is a cell transformed with the expression vector. Also provided is an immunoconjugate comprising the antibody, or antigen binding portion thereof, or the bispecific molecule, linked to an agent. Also provided is a composition comprising (i) the antibody, or antigen binding portion thereof, or the bispecific molecule, or the immunoconjugate, and (ii) a carrier. Also provided is a kit comprising (i) the antibody, or antigen binding portion thereof, or the bispecific molecule, or the immunoconjugate, and (ii) instructions for use. Also provided is a method of preparing an anti-FX antibody, or antigen binding portion thereof, comprising expressing the antibody, or antigen binding portion thereof, in a cell and isolating the antibody, or antigen binding portion thereof, from the cell.

The present disclosure also provides a method of measuring zymogen FX (FXz) in a subject in need thereof comprising contacting the anti-FX antibody, or 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 antigen binding portion thereof to FXz in the sample. In some aspects, the sample is blood or serum from the subject. The present disclosure also provides a bispecific molecule comprising (i) an anti-FIX antibody, or antigen binding portion thereof, disclosed herein, and (ii) an anti-FX antibody, or antigen binding portion thereof, disclosed herein. In some aspects, the bispecific molecule cross-competes with a reference bispecific antibody, wherein the reference bispecific antibody comprises a VH and a VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D, and a VH and a VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIG. 12A, FIG. 12B, and FIG. 12C. In some aspects, the bispecific molecule binds to the same epitope as a reference bispecific antibody, wherein the reference bispecific antibody comprises a VH and a VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D and a VH and a VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIG. 12A, FIG. 12B, and FIG. 12C. In some of 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, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1s, VH CDR2s, and VH CDR3s and VL CDR1s, VL CDR2s, and VL CDR3s of the anti-FIX (BIIB-9) antibodies in FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D; and, (ii) the anti-FX antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1s, VH CDR2s, and VH CDR3s and VL CDR1s, VL CDR2s, and VL CDR3s of the anti-FX (BIIB-12) antibodies in FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D. In some aspects of the bispecific molecule disclosed herein (a) the anti-FIX antibody, or antigen binding portion thereof, comprises (a1) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 815, 860, or 905, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 950, 995, or 1040, respectively (BIIB-9-484); (a2) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 822, 867, and 912, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 957, 1002, and 1047, respectively (BIIB-9-619); (a3) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1347, 1351, and 1355, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1359, 1363, and 1367, respectively (BIIB-9-578); (a4) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 843, 888, and 933, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 978, 1023, and 1068, respectively (BIIB-9-1335); or (a5) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 844, 889, and 934, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 979, 1024, and 1069, respectively (BIIB-9-1336); and (b) the anti-FX antibody, or antigen binding portion thereof, comprises (b1) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1393, 1483, and 1573, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1663, 1753, and 1843, respectively (BIIB-12-915); (b2) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1395, 1485, and 1575, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1665, 1755, and 1845, respectively (BIIB-12-917); (b3) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1911, 1915, and 1919, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1923, 1927, and 1931, respectively (BIIB-12-925); (b4) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1409, 1499, and 1589, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1679, 1769, and 1859, respectively (BIIB-12-932); or (b5) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1433, 1523, and 1613, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1703, 1793, and 1883, respectively (BIIB-12-1306). In some aspects of the bispecific molecule disclosed herein (a) the anti-FIX antibody, or antigen binding portion thereof, comprises (a1) a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); (a2) a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); (a3) a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); (a4) a VH and a VL comprising SEQ ID NOs: 87 and 221, respectively (BIIB-9-1335); or (a5) a VH and a VL comprising SEQ ID NOs: 89 and 221, respectively (BIIB-9-1336); and, (b) the anti-FX antibody, or antigen binding portion thereof, comprises (b1) a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); (b2) a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); (b3) a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925); (b4) a VH and VL comprising SEQ ID NOs: 455 and 643, respectively (BIIB-12-932); or, (b5) a VH and a VL comprising SEQ ID NOs: 503 and 691, respectively (BIIB-12-1306). In some aspects of the bispecific molecule disclosed herein (i) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); or (ii) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or (iii) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925); or (iv) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 455 and 643, respectively (BIIB-12-932); or, (v) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); or, (vi) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or, (vii) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or, (viii) the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925). In some aspects, the bispecific molecule functionally mimics activated factor VIII (FVIIIa) cofactor in at least one FVIIIa activity assay. In some aspects, the FVIIIa activity assay is selected from a chromogenic FXa generation assay, a one-stage clotting assay, or the combination thereof. In some aspects, 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 otherwise achieved by FVIII in the same assay. In some aspects, the bispecific molecule is capable of generating thrombin from prothrombin, fibrin from fibrinogen, and/or fibrin clot in vitro or in vivo. In some aspects, the bispecific molecule concurrently binds to both FIXa and FX, as determined by BLI. In some aspects, the bispecific molecule is of the IgG isotype. In some aspects, the IgG isotype is of the IgG1 subclass. In some aspects, the IgG isotype is of the IgG4 subclass. In some aspects, the bispecific molecule is of a bispecific IgG format and is selected from the group consisting of the antibodies in TABLE 2. In some aspects, the bispecific molecule is of a bispecific heterodimeric format. In some aspects, the bispecific molecule comprises two different heavy chains and two different light chains. In some aspects, the bispecific molecule comprises two identical light chains and two different heavy chains. In some aspects, the bispecific molecule is capable of controlling or reducing the incidence of bleeding episodes in a subject having hemophilia. In some aspects, the bispecific molecule is capable of maintaining homeostasis or in a subject having hemophilia. In some aspects, the bispecific molecule is capable of providing routine prophylaxis in a subject having hemophilia. In some aspects, the subject has developed or is expected to develop neutralizing antibodies against Factor VIII.

The present disclosure also provides an immunoconjugate comprising a bispecific molecule disclosed herein linked to an agent, e.g., a therapeutic agent. Also provided is a composition comprising (i) a bispecific molecule disclosed herein or an immunoconjugate comprising the bispecific molecule, and (ii) a carrier. Also provide is a kit comprising (i) a bispecific molecule disclosed herein or an immunoconjugate comprising the bispecific molecule, and (ii) instructions for use. Also provided is a nucleic acid sequence encoding the bispecific molecule disclosed herein. Also provided are a vector comprising the nucleic acid, and a host cell comprising the vector. In some aspects, 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. Also provided is a method of producing a bispecific molecule disclosed herein, comprising culturing a host cell disclosed herein under conditions that allow the expression of the bispecific molecule. In some aspects, the method of producing a bispecific molecule disclosed herein further comprises using conditions that enhance heterodimerization.

The present disclosure also provides a method of promoting FX activation in a subject in need thereof, 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 which comprises or encodes a bispecific molecule disclosed herein.

The present disclosure also provides a method of reducing the frequency or degree of a bleeding episode in a subject in need thereof, 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 which comprises or encodes a bispecific molecule disclosed herein. In some aspects, the subject has developed or has a tendency to develop an inhibitor against Factor VIII (“FVIII”). In some aspects, the inhibitor against FVIII is a neutralizing antibody against FVIII. In some aspects, the bleeding episode is the result of hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, or any combinations thereof.

The present disclosure also provides a method of treating a blood coagulation disorder in a subject in need thereof, 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 which comprises or encodes a bispecific molecule disclosed herein. In some aspects, the blood coagulation disorder is hemophilia A or hemophilia B. In some aspects, the subject is a human subject. In some aspects, the subject is undergoing or has undergone FVIII replacement therapy. In some aspects, the bispecific molecule is administered in combination with a hemophilia therapy. In some aspects, the hemophilia therapy is a FVIII replacement therapy. In some aspects, the bispecific molecule, immunoconjugate, composition, nucleic acid, vector, or host cell is administered before, during or after administration of the hemophilia therapy. In some aspects, the bispecific molecule, immunoconjugate, composition, nucleic acid, vector, or host cell is administered intravenously or subcutaneously. In some aspects, administration of the bispecific molecule, immunoconjugate, composition, nucleic acid, vector, or host cell reduces the frequency of break-through bleeding episodes, spontaneous bleeding episodes, or acute bleeding. In some aspects, administration of the bispecific molecule, immunoconjugate, composition, nucleic acid, vector, or host cell reduces the annualized bleed rate by 5%, 10%, 20%, 30%, or 50%.

The present disclosure also provides an anti-FIXa antibody, or antigen binding portion thereof, which binds to the same epitope as BIIB-9-1336. Also provides is an anti-FIXa antibody, or antigen binding portion thereof, which binds to an epitope overlapping BIIB-9-1336 epitope.

The present disclosure also provides an anti-FIXa antibody, or antigen binding portion thereof, which binds to an epitope region comprising at least one amino acid located between chymotrypsinogen numbering positions (i) 91 and 101, (ii) 125 and 128, (iii) 165 and 179, or (iv) 232 and 241 in the sequence of the heavy chain of FIXa. Also provided is an anti-FIXa antibody, or antigen binding portion thereof, which binds to an epitope comprising at least one of chymotrypsinogen numbering 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 sequence of the heavy chain of FIXa. In some aspects, the epitope comprises chymotrypsinogen numbering amino acid residues N93, R165, N178, and R233 of the sequence of the heavy chain of FIXa. In other aspects, the epitope comprises chymotrypsinogen numbering 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 sequence of the heavy chain of FIXa. In some aspects, the epitope does not comprise at least one of chymotrypsinogen numbering amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the sequence of the heavy chain of FIXa. In some aspects, the epitope does not comprise chymotrypsinogen numbering amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the sequence of the heavy chain of FIXa. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to at least one amino acid residue in the light chain of FIXa (SEQ ID NO:756). In some aspects, the amino acid residue in the light chain of FIXa (SEQ ID NO:756) is K100. In some aspects, the epitope overlaps the binding site of FVIIIa to FIXa. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, cross-competes with FVIIIa for binding to FIXa. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, blocks binding of FVIIIa to FIXa.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein (i) the VH CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in TABLE 7 or the VH CDR1 with one or two mutations; and/or, (ii) the VH CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in TABLE 7 or the VH CDR2 with one or two mutations; and/or, (iii) the VH CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in TABLE 7 or the VH CDR3 with one or two mutations; and/or, (iv) the VL CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in TABLE 7 or the VL CDR1 with one or two mutations; and/or, (v) the VL CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in TABLE 7 or the VL CDR2 with one or two mutations; and/or, (vi) the VL CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in TABLE 7 or the VL CDR3 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDXIGGYAGYYGMDV (SEQ ID NO: 2196), wherein X₁is L or V. In some aspects, (i) the VH CDR1 comprises the amino acid sequence FTFX₁SX₂X₃MX₄(SEQ ID NO: 2194), wherein X₁is S, G or E, X₂is Y or F, X₃is S, E, G, or D, and X₄is N, V, A, or T; and/or (ii) the VH CDR2 comprises the amino acid sequence X₅ISX₆X₇X₈X₉X₁₀IYYADSVKG (SEQ ID NO: 2195), wherein X₅is S, A, Y, or G, X₆is S or A, X₇is S, A, or G, X₈is S, G, or D, X₉is S, T, or G, and X₁₀is Y or T.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VL CDR1, CDR2 and CDR3, wherein the VL CDR3 comprises the amino acid sequence QQYANFPYT (SEQ ID NO:2168). In some aspects, (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).

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

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence X₁RDVX₂GYAGX₃YGMDV (SEQ ID NO: 2198), wherein X₁is A or V, X₂is G or S, and X₃is Y or F. In some aspects, (i) the VH CDR1 comprises the amino acid sequence FTFGSYDMN (SEQ ID NO: 2048); and/or (ii) the VH CDR2 comprises the amino acid sequence SISX₁X₂X₃SYIX₄YAX₅SVKG (SEQ ID NO: 2197), wherein X₁is S or D, X₂is G or S, X₃is E or A, X₄is Y or A, and X₅is E or D.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VL CDR3 comprises the amino acid sequence X₁QYAX₂FPYT (SEQ ID NO: 2201), wherein X₁is Q or S, and X₂is N or R. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VL CDR1, CDR2 and CDR3, wherein (i) the VL CDR1 comprises the amino acid sequence X₁AX₂X₃X₄IX₅X₆YLN (SEQ ID NO: 2199), wherein X₁is Q, G, or E, X₂is S or N, X₃is Q or E, X₄is D or Y, X₅is A or S, X₆is N or D; and/or (ii) the VL CDR2 comprises the amino acid sequence DAX₇NLX₈X₉(SEQ ID NO: 2200), wherein X₇is S or A, X₈is E, H or Q, and X₉is T or Y.

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

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDGPX₁X₂X₃DYYMDV (SEQ ID NO: 2204), wherein X₁is R or Q, X₂is V, D, L or E, and X₃is S or V. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2 and CDR3, wherein (i) the VH CDR1 comprises the amino acid sequence YTFX₁X₂YX₃MH (SEQ ID NO: 2202), wherein X₁is T or H, X₂is S, G, or H, and X₃is Y or P; and/or (ii) the VH CDR2 comprises the amino acid sequence X₄INPSX₅GX₆TX₇YAQKFQG (SEQ ID NO: 2203), wherein X₄is I or S, X₅is G or R, X₆is S or R, and X₇is S or E.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VL CDR3 comprises the amino acid sequence QQRDNWPFT (SEQ ID NO:2116). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VL CDR1, CDR2 and CDR3, wherein (i) the VL CDR1 comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO:2116); and/or, (ii) the VL CDR2 comprises the amino acid sequence DASNRAT (SEQ ID NO:2116).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises (i) VH CDR1, CDR2, and CDR3, wherein VH CDR1 is selected from SEQ ID NOs: 2053 to 2057, VH CDR2 is selected from SEQ ID NOs: 2079 to 2083, and VH CDR3 is selected from SEQ ID NOs: 2105 to 2109, and/or (ii) VL CDR1, CDR2, and CDR3, wherein VL CDR1 is selected from SEQ ID NOs: 2131 to 2135, VL CDR2 is selected from SEQ ID NOs: 2157 to 2161, and VL CDR3 is selected from SEQ ID NOs: 2183 to 2187.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDKYQDYSX₁DI (SEQ ID NO: 2207), wherein X₁is F or V. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises VH CDR1, CDR2 and CDR3, wherein (i) the VH CDR1 comprises the amino acid sequence GSIX₁SX₂X₃YX₄WX₅(SEQ ID NO: 2205), wherein X₁is S or A, X₂is S, T, G, or V, X₃is S or A, X₄is Y or A, and X₅is G, V, N, or S; and/or (ii) the VH CDR2 comprises the amino acid sequence X₆IX₇X₈X₉GX₁₀TX₁₁YNPSLKS (SEQ ID NO: 2206), wherein X₆is S or Y, X₇is S, Y, R, T or Q, X₈is Y, G, P or A, X₉is S or Q, X₁₀is S or K, and X₁₁is Y or Q.

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

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises (i) VH CDR1, CDR2, and CDR3, wherein VH CDR1 is selected from SEQ ID NOs: 2058 to 2063, VH CDR2 is selected from SEQ ID NOs: 2084 to 2089, and VH CDR3 is selected from SEQ ID NOs: 2110 to 2115, and/or (ii) VL CDR1, CDR2, and CDR3, wherein VL CDR1 is selected from SEQ ID NOs: 2136 to 2141, VL CDR2 is selected from SEQ ID NOs: 2162 to 2167, and VL CDR3 is selected from SEQ ID NOs: 2188 to 2193.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises a VH and a VL, wherein (i) the VH comprises an amino acid sequence which 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 the 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 which 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 the 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.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, disclosed herein which specifically binds to FIXa, comprises a VH and a VL, wherein (a1) the VH and the VL comprise SEQ ID NOs: 1935 and 1937, respectively (BIIB-9-3595); (a2) the VH and the VL comprise SEQ ID NOs: 1939 and 1941, respectively (BIIB-9-3601); (a3) the VH and the VL comprise SEQ ID NOs: 1943 and 1945, respectively (BIIB-9-3604); (a4) the VH and the VL comprise SEQ ID NOs: 1947 and 1949, respectively (BIIB-9-3617); (a5) the VH and the VL comprise SEQ ID NOs: 1951 and 1953, respectively (BIIB-9-3618); (a6) the VH and the VL comprise SEQ ID NOs: 1955 and 1957, respectively (BIIB-9-3621); (a7) the VH and the VL comprise SEQ ID NOs: 1959 and 1961, respectively (BIIB-9-3647); (a8) the VH and the VL comprise SEQ ID NOs: 1963 and 1965, respectively (BIIB-9-3649); (a9) the VH and the VL comprise SEQ ID NOs: 1967 and 1969, respectively (BIIB-9-3650); (a10) the VH and the VL comprise SEQ ID NOs: 1971 and 1973, respectively (BIIB-9-3654); (a11) the VH and the VL comprise SEQ ID NOs: 1975 and 1977, respectively (BIIB-9-3753); (a12) the VH and the VL comprise SEQ ID NOs: 1979 and 1981, respectively (BIIB-9-3754); (a13) the VH and the VL comprise SEQ ID NOs: 1983 and 1985, respectively (BIIB-9-3756); (a14) the VH and the VL comprise SEQ ID NOs: 1987 and 1989, respectively (BIIB-9-3764); (a15) the VH and the VL comprise SEQ ID NOs: 1991 and 1993, respectively (BIIB-9-3766); (a16) the VH and the VL comprise SEQ ID NOs: 1995 and 1997, respectively (BIIB-9-3707); (a17) the VH and the VL comprise SEQ ID NOs: 1999 and 2001, respectively (BIIB-9-3709); (a18) the VH and the VL comprise SEQ ID NOs: 2003 and 2005, respectively (BIIB-9-3720); (a19) the VH and the VL comprise SEQ ID NOs: 2007 and 2009, respectively (BIIB-9-3727); (a20) the VH and the VL comprise SEQ ID NOs: 2011 and 2013, respectively (BIIB-9-3745); (a21) the VH and the VL comprise SEQ ID NOs: 2015 and 2017, respectively (BIIB-9-3780); (a22) the VH and the VL comprise SEQ ID NOs: 2019 and 2021, respectively (BIIB-9-3675); (a23) the VH and the VL comprise SEQ ID NOs: 2023 and 2025, respectively (BIIB-9-3681); (a24) the VH and the VL comprise SEQ ID NOs: 2027 and 2029, respectively (BIIB-9-3684); (a25) the VH and the VL comprise SEQ ID NOs: 2031 and 2033, respectively (BIIB-9-3698); or, (a26) the VH and the VL comprise SEQ ID NOs: 2035 and 2037, respectively (BIIB-9-3704).

EMBODIMENTS

Embodiment 1. An isolated antibody, or an antigen binding portion thereof, that specifically binds to activated factor IX (FIXa) (“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).

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

Embodiment 3. An isolated anti-FIXa antibody, or antigen binding portion thereof, which binds to FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz.

Embodiment 4. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to FIXa with a K_Dof 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 as determined by a Bio-Layer Interferometry (BLI) assay.

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

Embodiment 6. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, wherein the FIXz comprises non-activatable Factor IX (FIXn).

Embodiment 7. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3A, FIG. 3B, and FIG. 3C.

Embodiment 8. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3A, FIG. 3B, and FIG. 3C.

Embodiment 9. The anti-FIXa antibody, or antigen binding portion thereof, of 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.

Embodiment 10. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which preferentially binds to FIXa-SM compared to free FIXa or FIXz and/or binds to FIXa-SM with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to free FIXa or FIXz.

Embodiment 11. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3A.

Embodiment 12. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3A.

Embodiment 13. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 11 or 12, wherein the reference antibody is selected from BIIB-9-484, BIIB-9-440, BIIB-9-460, and any combination thereof.

Embodiment 14. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which preferentially binds to free FIXa compared to FIXa-SM or FIXz and/or binds to free FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXa-SM or FIXz.

Embodiment 15. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3B.

Embodiment 16. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3B.

Embodiment 17. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which preferentially binds to free FIXa or FIXa-SM compared to FIXz and/or binds to free FIXa or FIXa-SM with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz.

Embodiment 18. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3C. 19. Embodiment The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3C.

Embodiment 20. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 18 or 19, wherein the reference antibody is selected from BIIB-9-882, BIIB-9-433, and the combination thereof.

21. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR3 with one or two mutations.

Embodiment 22. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 1 to 21, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises ARDX₁X₂X₃X₄X₅X₆YYX₇MDV (SEQ ID NO:753), wherein X₁is V or G, X₂is G or V, X₃is G or R, X₄is Y or V, X₅is A or S, X₆is G or D, X₇is G or none.

Embodiment 23. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 22, wherein the 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).

Embodiment 24. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR1 with one or two mutations.

Embodiment 25. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 3A, FIG. 3B, and FIG. 3C or the VH CDR2 with one or two mutations.

Embodiment 26. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR1 with one or two mutations.

Embodiment 27. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR2 with one or two mutations.

Embodiment 28. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 3A, FIG. 3B, and FIG. 3C or the VL CDR3 with one or two mutations.

Embodiment 29. An isolated anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 comprise VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, CDR2, and CDR3 of FIG. 3A, FIG. 3B, and FIG. 3C, respectively.

Embodiment 30. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 29, wherein the anti-FIXa antibody or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 815, 860, and 905, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 950, 995, and 1040, respectively (BIIB-9-484).

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

Embodiment 32. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 29, wherein the antibody 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 comprising SEQ ID NO: 957, SEQ ID NO: 1002, and SEQ ID NO: 1047, respectively (BIIB-9-619).

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

Embodiment 34. The anti-FIXa antibody, or antigen binding portion thereof, of any preceding embodiments, comprising VH and VL, wherein the VH comprises an amino acid sequence which 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 the 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.

Embodiment 35. The anti-FIXa antibody, or antigen binding portion thereof of any preceding embodiments, comprising VH and VL, wherein the VL comprises an amino acid sequence which 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 the 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.

Embodiment 36. The anti-FIXa antibody, or antigen binding portion thereof of any preceding embodiments, which 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.

Embodiment 37. The anti-FIXa antibody, or antigen binding portion thereof of any preceding embodiments, which 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.

Embodiment 38. The anti-FIXa antibody, or antigen binding portion thereof of any preceding embodiments, comprising VH and VL, wherein (a1) VH and VL comprise SEQ ID NOs: 31 and 221, respectively (BIIB-9-484); (a2) VH and VL comprise SEQ ID NOs: 19 and 209, respectively (BIIB-9-440); (a3) VH and VL comprise SEQ ID NOs: 115 and 301, respectively (BIIB-9-882); (a4) VH and VL comprise SEQ ID NOs: 23 and 213, respectively (BIIB-9-460); (a5) VH and VL comprise SEQ ID NOs: 127 and 313, respectively (BIIB-9-433); (a6) VH and VL comprise SEQ ID NOs: 45 and 235, respectively (BIIB-9-619); (a7) VH and VL comprise SEQ ID NOs: 185 and 371, respectively (BIIB-9-578); (a8) VH and VL comprise SEQ ID NOs: 87 and 221, respectively (BIIB-9-1335); or, (a9) VH and VL comprise SEQ ID NOs: 89 and 221, respectively (BIIB-9-1336).

Embodiment 39. An isolated antibody, or an antigen binding portion thereof, which specifically binds to FIXz (“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 a binding affinity of the anti-FIXz antibody or antigen binding portion thereof to free FIXa or FIXa-SM.

Embodiment 40. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which cross-competes with a reference antibody selected from the group consisting of the antibodies in FIG. 3D.

Embodiment 41. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 3D.

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

Embodiment 43. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 3D or the VH CDR3 with one or two mutations.

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

Embodiment 45. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 3D or the VH CDR1 with one or two mutations.

Embodiment 46. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 3D or the VH CDR2 with one or two mutations.

Embodiment 47. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 3D or the VL CDR1 with one or two mutations.

Embodiment 48. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 3D or the VL CDR2 with one or two mutations.

Embodiment 49. The anti-FIXz antibody, or antigen binding portion thereof, of any preceding embodiments, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 3D or the VL CDR3 with one or two mutations.

Embodiment 50. The anti-FIX antibody, or antigen binding portion thereof, of any one of the preceding embodiments and embodiments 169 to 204, wherein the antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4 or a variant thereof.

Embodiment 51. The anti-FIX antibody, or antigen binding portion thereof, of embodiment 50, wherein the antibody is an IgG4 antibody.

Embodiment 52. The anti-FIX antibody, or antigen binding portion thereof, of embodiment 50, wherein the antibody comprises an effectorless IgG4 Fc.

Embodiment 53. The anti-FIX antibody, or antigen binding portion thereof, of any one of the preceding embodiments and embodiments 169 to 204, comprises a heavy chain constant region.

Embodiment 54. The anti-FIX antibody of any one of the preceding embodiments and embodiments 169 to 204, wherein the antibody is a human antibody, an engineered antibody, or a humanized antibody.

Embodiment 55. The anti-FIX antigen binding portion thereof of any one of the preceding embodiments and embodiments 169 to 204, wherein the antigen binding portion thereof comprises an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv (scFv).

Embodiment 56. A bispecific molecule comprising the anti-FIX antibody or antigen binding portion thereof of any one of the preceding embodiments and embodiments 169 to 204 linked to a molecule having a second binding specificity.

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

Embodiment 58. An expression vector comprising the nucleic acid molecule of embodiment 57.

Embodiment 59. A cell transformed with an expression vector of embodiment 58.

Embodiment 60. An immunoconjugate comprising the antibody or 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, linked to an agent.

Embodiment 61. A composition comprising the antibody, or antigen binding portion thereof of any one of embodiment 1 to 55 and embodiments 169 to 204, the bispecific molecule of embodiment 56, or the immunoconjugate of embodiment 60, and a carrier.

Embodiment 62. A kit comprising the antibody, or antigen binding portion thereof of 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.

Embodiment 63. A method of preparing an anti-FIX antibody, or antigen binding portion thereof, comprising expressing the antibody, or antigen binding portion thereof, in the cell of embodiment 59 and isolating the antibody, or antigen binding portion thereof, from the cell.

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

Embodiment 65. The method of embodiment 64, wherein the sample is blood or serum.

Embodiment 66. An isolated antibody, or an antigen binding portion thereof, that specifically binds to factor X zymogen (FXz) (“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).

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

Embodiment 68. An isolated anti-FXz antibody, or antigen binding portion thereof, which binds to FXz with a binding affinity higher than a binding affinity of the antibody or antigen binding portion thereof to FXa.

Embodiment 69. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 68, which binds to FXz with a KD 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 1 nM or less as measured by BLI.

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

Embodiment 71. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 70, wherein the FXz comprises non-activatable Factor X (FXn).

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

Embodiment 73. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 72, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 12A and FIG. 12B.

Embodiment 74. The anti-FXz antibody, or antigen binding portion thereof, of embodiment 73, which 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.

Embodiment 75. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 74, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 12A and FIG. 12B or the VH CDR3 with one or two mutations.

Embodiment 76. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 75, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises ARX₁X₂X₃RX₄X₅X₆X₇FDX₈(SEQ ID NO: 766), wherein X₁is G or L, X₂is R or G, X₃is F or Y, X₄is P or G, X₅is R or A, X₆is G or S, X₇is R or A, and X₈is Y or I.

Embodiment 77. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 76, which comprises CDR1, CDR2, and CDR3, wherein the 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).

Embodiment 78. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 77, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 12A and FIG. 12B or the VH CDR1 with one or two mutations.

Embodiment 79. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 78, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 12A and FIG. 12B or the VH CDR2 with one or two mutations.

Embodiment 80. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 79, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 12A and FIG. 12B or the VL CDR1 with one or two mutations.

Embodiment 81. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 80, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 12A and FIG. 12B or the VL CDR2 with one or two mutations.

Embodiment 82. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 81, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 12A and FIG. 12B or the VL CDR3 with one or two mutations.

Embodiment 83. An isolated antibody, or antigen binding portion thereof, which specifically binds to FXz, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 comprise VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3 of FIG. 12A and FIG. 12B, respectively.

Embodiment 84. The anti-FXz antibody, or antigen binding portion thereof, of embodiment 83, wherein the antibody comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1393, 1483, or 1573, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1663, 1753, or 1843, respectively (BIIB-12-915).

Embodiment 85. The anti-FXz antibody, or antigen binding portion thereof, of embodiment 83, wherein the antibody comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1395, 1485, or 1575, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1665, 1755, or 1845, respectively (BIIB-12-917).

Embodiment 86. The anti-FXz antibody, or antigen binding portion thereof, of embodiment 83, wherein the antibody comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1409, 1499, or 1589, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1679, 1769, or 1859, respectively (BIIB-12-932).

Embodiment 87. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 86, comprising VH and VL, wherein the VH comprises an amino acid sequence which 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 the 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.

Embodiment 88. The anti-FXz antibody, or antigen binding portion thereof of any one of embodiments 66 to 87, comprising VH and VL, wherein the VL comprises an amino acid sequence which 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 the 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.

Embodiment 89. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 88, which 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.

Embodiment 90. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 89, which comprises a VH and a VL, wherein the VL is derived from a germline sequence 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.

Embodiment 91. The anti-FXz antibody, or antigen binding portion thereof, of any one of embodiments 66 to 90, comprising VH and VL, wherein

(b1) VH and VL comprise SEQ ID NOs: 423 and 611, respectively (BIIB-12-915);

(b2) VH and VL comprise SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or,

(b3) VH and VL comprise SEQ ID NOs: 455 and 643, respectively (BIIB-12-932).

Embodiment 92. An isolated antibody, or an antigen binding portion thereof, that specifically binds to activated factor X (FXa) (“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 a binding affinity of the antibody or antigen binding portion thereof to FXz.

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

Embodiment 94. The anti-FXa antibody, or antigen binding portion thereof, of embodiment 92 or 93, which binds to the same epitope as a reference antibody selected from the group consisting of the antibodies in FIG. 12C.

Embodiment 95. The anti-FXa antibody, or antigen binding portion thereof, of embodiment 94, which binds to the same epitope as a reference antibody selected from the group consisting of BIIB-12-925.

Embodiment 96. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 95, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in FIG. 12C or the VH CDR3 with one or two mutations.

Embodiment 97. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 96, which comprises CDR1, CDR2, and CDR3, wherein the CDR3 comprises AKGPRYYWYSWYFDL (SEQ ID NO: 1919, BIIB-12-925).

Embodiment 98. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 97, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in FIG. 12C or the VH CDR1 with one or two mutations.

Embodiment 99. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 98, which comprises VH CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in FIG. 12C or the VH CDR2 with one or two mutations.

Embodiment 100. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 99, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in FIG. 12C or the VL CDR1 with one or two mutations.

Embodiment 101. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 100, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in FIG. 12C or the VL CDR2 with one or two mutations.

Embodiment 102. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 101, which comprises VL CDR1, CDR2, and CDR3, wherein the CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in FIG. 12C or the VL CDR3 with one or two mutations.

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

Embodiment 104. The anti-FXa antibody, or antigen binding portion thereof, of embodiment 103, wherein the antibody comprises VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1911, 1915, or 1919, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1923, 1927, or 1931, respectively (BIIB-12-925).

Embodiment 105. The anti-FXa antibody, or antigen binding portion thereof, of any one of embodiments 92 to 104, comprising VH and VL, wherein the VH and VL comprise SEQ ID NOs: 559 and 747, respectively (BIIB-12-925).

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

Embodiment 107. The anti-FX antibody, or antigen binding portion thereof, of embodiment 106, wherein the antibody is an IgG4 antibody.

Embodiment 108. The anti-FX antibody, or antigen binding portion thereof, of embodiment 106, wherein the antibody comprises an effectorless IgG4 Fc.

Embodiment 109. The anti-FX antibody, or antigen binding portion thereof, of any one of embodiments 66 to 108, which comprises a heavy chain constant region.

Embodiment 110. The anti-FX antibody of any one of embodiments 66 to 109, wherein the antibody is a human antibody, an engineered antibody, or a humanized antibody.

Embodiment 111. The anti-FX antigen binding portion thereof of any one of embodiments 66 to 110, wherein the antigen binding portion thereof comprises an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv (scFv).

Embodiment 112. A bispecific molecule comprising the anti-FX antibody of any one of embodiments 66 to 111 linked to a molecule having a second binding specificity.

Embodiment 113. A nucleic acid encoding the heavy and/or light chain variable region of the antibody, or antigen binding portion thereof, of any one of embodiments 66-111 or the bispecific molecule of embodiment 112.

Embodiment 114. An expression vector comprising the nucleic acid molecule of embodiment 113.

Embodiment 115. A cell transformed with an expression vector of embodiment 114.

Embodiment 116. An immunoconjugate comprising the antibody, or antigen binding portion thereof, of any one of embodiments 66-111 or the bispecific molecule of embodiment 112, linked to an agent.

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

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

Embodiment 119. A method of preparing an anti-FX antibody, or antigen binding portion thereof, comprising 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.

Embodiment 120. A method of measuring zymogen FX (FXz) in a subject in need thereof comprising contacting the anti-FX antibody, or antigen binding portion thereof, of any one 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 to FXz in the sample.

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

Embodiment 122. A bispecific molecule comprising the anti-FIX antibody, or antigen binding portion thereof, of any one of embodiments 1 to 55 and embodiments 169 to 204 and (ii) the anti-FX antibody, or antigen binding portion thereof, of any one of embodiments 66 to 111.

Embodiment 123. The bispecific molecule of embodiment 122, which cross-competes with a reference bispecific antibody, wherein the reference bispecific antibody comprises a VH and a VL of an anti-FIX antibody selected from the group consisting of the anti-FIX antibodies in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D and a VH and a VL of an anti-FX antibody selected from the group consisting of the anti-FX antibodies in FIG. 12A, FIG. 12B, and FIG. 12C.

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

Embodiment 125. The bispecific molecule of any one of embodiments 122 to 124, wherein (i) the anti-FIX antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1s, VH CDR2s, and VH CDR3s and VL CDR1s, VL CDR2s, and VL CDR3s of the anti-FIX (BIIB-9) antibodies in FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D; and (ii) the anti-FX antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 and the VL CDR1, CDR2, and CDR3 are selected from the group consisting of VH CDR1s, VH CDR2s, and VH CDR3s and VL CDR1s, VL CDR2s, and VL CDR3s of the anti-FX (BIIB-12) antibodies in FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D.

Embodiment 126. The bispecific molecule of any one of embodiments 122 to 124, wherein (a) the anti-FIX antibody, or antigen binding portion thereof, comprises:

(a1) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 815, 860, or 905, respectively, and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 950, 995, or 1040, respectively (BIIB-9-484);

(a2) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 822, 867, and 912, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 957, 1002, and 1047, respectively (BIIB-9-619);

(a3) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1347, 1351, and 1355, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1359, 1363, and 1367, respectively (BIIB-9-578);

(a4) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 843, 888, and 933, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 978, 1023, and 1068, respectively (BIIB-9-1335); or,

(a5) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 844, 889, and 934, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 979, 1024, and 1069, respectively (BIIB-9-1336); and,

(b) the anti-FX antibody, or antigen binding portion thereof, comprises:

(b1) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1393, 1483, and 1573, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1663, 1753, and 1843, respectively (BIIB-12-915);

(b2) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1395, 1485, and 1575, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1665, 1755, and 1845, respectively (BIIB-12-917);

(b3) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1911, 1915, and 1919, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1923, 1927, and 1931, respectively (BIIB-12-925);

(b4) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1409, 1499, and 1589, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1679, 1769, and 1859, respectively (BIIB-12-932); or,

(b5) VH CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1433, 1523, and 1613, respectively, and/or VL CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 1703, 1793, and 1883, respectively (BIIB-12-1306).

Embodiment 127. The bispecific molecule of any one of embodiments 122 to 124, wherein (a) the anti-FIX antibody, or antigen binding portion thereof, comprises:

(a1) a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively (BIIB-9-484);

(a2) a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively (BIIB-9-619);

(a3) a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively (BIIB-9-578);

(a4) a VH and a VL comprising SEQ ID NOs: 87 and 221, respectively (BIIB-9-1335); or

(a5) a VH and a VL comprising SEQ ID NOs: 89 and 221, respectively (BIIB-9-1336); and, (b) the anti-FX antibody, or antigen binding portion thereof, comprises:

(b1) a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915);

(b2) a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917);

(b3) a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925);

(b4) a VH and VL comprising SEQ ID NOs: 455 and 643, respectively (BIIB-12-932); or,

(b5) a VH and a VL comprising SEQ ID NOs: 503 and 691, respectively (BIIB-12-1306).

Embodiment 128. The bispecific molecule of any one of embodiments 122 to 127, (i) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively; (BIIB-9-484) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); (ii) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively; (BIIB-9-484) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); (iii) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively; (BIIB-9-484) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925); (iv) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 31 and 221, respectively; (BIIB-9-484) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 455 and 643, respectively (BIIB-12-932); (v) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively; (BIIB-9-578) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 423 and 611, respectively (BIIB-12-915); (vi) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 185 and 371, respectively; (BIIB-9-578) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); (vii) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively; (BIIB-9-619) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 427 and 615, respectively (BIIB-12-917); or, (viii) wherein the anti-FIX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 45 and 235, respectively; (BIIB-9-619) and the anti-FX antibody, or antigen binding portion thereof, comprises a VH and a VL comprising SEQ ID NOs: 559 and 747, respectively (BIIB-12-925).

Embodiment 129. The bispecific molecule of any one of embodiments 122 to 128, which functionally mimics activated factor VIII (FVIIIa) cofactor in at least one FVIIIa activity assay.

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 the combination thereof.

Embodiment 131. The bispecific molecule of embodiment 129 or 130, wherein 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 otherwise achieved by FVIII in the same assay.

Embodiment 132. The bispecific molecule of any one of embodiments 122 to 131, wherein the bispecific molecule is capable of generating thrombin from prothrombin, fibrin from fibrinogen, and/or fibrin clot in vitro or in vivo.

Embodiment 133. The bispecific molecule according to any one of embodiments 122 through 132, wherein the bispecific molecule concurrently binds to both FIXa and FX, as determined by BLI.

Embodiment 134. The bispecific molecule of any one of embodiments 122 to 133, wherein the bispecific molecule is of the IgG isotype.

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

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

Embodiment 137. The bispecific molecule of any one of embodiments 122 to 136, wherein the bispecific molecule is of a bispecific IgG format and is selected from the group consisting of the antibodies in TABLE 2.

Embodiment 138. The bispecific molecule of embodiment 137, wherein the bispecific molecule is of a bispecific heterodimeric format.

Embodiment 139. The bispecific molecule of any one of embodiments 122 to 138, wherein the bispecific molecule comprises two different heavy chains and two different light chains.

Embodiment 140. The bispecific molecule according to any one of embodiments 122 through 138, wherein the bispecific molecule comprises two identical light chains and two different heavy chains.

Embodiment 141. The bispecific molecule of 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.

Embodiment 142. The bispecific molecule of any one of embodiments 122 to 140, wherein the bispecific molecule is capable of maintaining homeostasis or in a subject having hemophilia.

Embodiment 143. The bispecific molecule of any one of embodiments 122 to 140, wherein the bispecific molecule is capable of providing routine prophylaxis in a subject having hemophilia.

Embodiment 144. The bispecific molecule of any one of embodiments 122 to 143, wherein the subject has developed or is expected to develop neutralizing antibodies against Factor VIII.

Embodiment 145. An immunoconjugate comprising the bispecific molecule of any one of embodiments 122 to 144, linked to an agent.

Embodiment 146. A composition comprising the bispecific molecule of any one of embodiments 122 to 144 or the immunoconjugate, of embodiment 145, and a carrier.

Embodiment 147. A kit comprising the bispecific molecule of any one of embodiments 122 to 144 or the immunoconjugate of embodiment 145 and instructions for use.

Embodiment 148. A nucleic acid sequence encoding the bispecific molecule of any one of embodiments 122 to 144.

Embodiment 149. A vector comprising the nucleic acid according to embodiment 148.

Embodiment 150. A host cell comprising the vector of embodiment 149.

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.

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

Embodiment 153. The method of producing the bispecific molecule of any one of embodiments 122 to 144, further comprising conditions that enhance heterodimerization.

Embodiment 154. A method of promoting FX activation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific molecule of any one of embodiments 122 through 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.

Embodiment 155. A method of reducing the frequency or degree of a bleeding episode in a subject in need thereof, comprising administering to the subject an effective amount of the bispecific molecule of any one of embodiments 122 through 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.

Embodiment 156. The method of embodiment 155, wherein the subject has developed or has a tendency to develop an inhibitor against Factor VIII (“FVIII”).

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

Embodiment 158. The method of any one of embodiments 155 to 157, wherein the bleeding episode is the result of hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, or any combinations thereof.

Embodiment 159. A method of treating a blood coagulation disorder in a subject in need thereof, comprising administering to the subject an effective amount of the bispecific molecule of any one of embodiments 122 through 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.

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

Embodiment 161. The method of any one of embodiments 154 to 160, wherein the subject is a human subject.

Embodiment 162. The method of any one of embodiments 154 to 160, wherein the subject is undergoing or has undergone FVIII replacement therapy.

Embodiment 163. The method of any one of embodiments 154 through 162, wherein the bispecific molecule is administered in combination with a hemophilia therapy.

Embodiment 164. The method of embodiment 163, wherein the hemophilia therapy is a FVIII replacement therapy.

Embodiment 165. The method of embodiment 163 or 164, wherein the bispecific molecule is administered before, during or after administration of the hemophilia therapy.

Embodiment 166. The method of any one of embodiments 154 through 165, wherein the bispecific molecule is administered intravenously or subcutaneously.

Embodiment 167. The method of any one of embodiments 154 through 166, wherein administration of the bispecific molecule reduces the frequency of break-through bleeding episodes, spontaneous bleeding episodes, or acute bleeding.

Embodiment 168. The method of embodiment 167, wherein administration of the bispecific molecule reduces the annualized bleed rate by 5%, 10%, 20%, 30%, or 50%.

Embodiment 169. An anti-FIXa antibody, or antigen binding portion thereof, which binds to the same epitope as BIIB-9-1336.

Embodiment 170. An anti-FIXa antibody, or antigen binding portion thereof, which binds to an epitope overlapping BIIB-9-1336 epitope.

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

Embodiment 172. An anti-FIXa antibody, or antigen binding portion thereof, which binds to an epitope comprising at least one of chymotrypsinogen numbering 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 sequence of the heavy chain of FIXa.

Embodiment 173. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 171 or 172, wherein the epitope comprises chymotrypsinogen numbering amino acid residues N93, R165, N178, and R233 of the sequence of the heavy chain of FIXa.

Embodiment 174. The anti-FIXa antibody, or antigen binding portion thereof, of embodiments 171 to 173 wherein the epitope comprises chymotrypsinogen numbering 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 sequence of the heavy chain of FIXa.

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

Embodiment 176. The anti-FIXa antibody, or antigen binding portion thereof, of embodiments 171 to 175, wherein the epitope does not comprise chymotrypsinogen numbering amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 of the sequence of the heavy chain of FIXa.

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

Embodiment 178. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 177, wherein the amino acid residue in the light chain of FIXa (SEQ ID NO:756) is K100.

Embodiment 179. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 169 to 178, wherein the epitope overlaps the binding site of FVIIIa to FIXa.

Embodiment 180. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 169 to 179, which cross-competes with FVIIIa for binding to FIXa.

Embodiment 181. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 169 to 180, wherein the antibody or antigen binding portion thereof blocks binding of FVIIIa to FIXa.

Embodiment 182. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 1 to 12, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein

(i) the VH CDR1 comprises a VH CDR1 selected from the group consisting of VH CDR1s in TABLE 7 or the VH CDR1 with one or two mutations; and/or,

(ii) the VH CDR2 comprises a VH CDR2 selected from the group consisting of VH CDR2s in TABLE 7 or the VH CDR2 with one or two mutations; and/or,

(iii) the VH CDR3 comprises a VH CDR3 selected from the group consisting of VH CDR3s in TABLE 7 or the VH CDR3 with one or two mutations; and/or,

(iv) the VL CDR1 comprises a VL CDR1 selected from the group consisting of VL CDR1s in TABLE 7 or the VL CDR1 with one or two mutations; and/or,

(v) the VL CDR2 comprises a VL CDR2 selected from the group consisting of VL CDR2s in TABLE 7 or the VL CDR2 with one or two mutations; and/or,

(vi) the VL CDR3 comprises a VL CDR3 selected from the group consisting of VL CDR3s in TABLE 7 or the VL CDR3 with one or two mutations.

Embodiment 183. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 182 which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDXIGGYAGYYGMDV (SEQ ID NO: 2196), wherein X₁is L or V.

Embodiment 184. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 183, wherein

(i) the VH CDR1 comprises the amino acid sequence FTFX₁SX₂X₃MX₄(SEQ ID NO: 2194), wherein X₁is S, G or E, X₂is Y or F, X₃is S, E, G, or D, and X₄is N, V, A, or T; and/or

(ii) the VH CDR2 comprises the amino acid sequence X₅ISX₆X₇X₈X₉X₁₀IYYADSVKG (SEQ ID NO: 2195), wherein X₅is S, A, Y, or G, X₆is S or A, X₇is S, A, or G, X₃is S, G, or D, X₉is S, T, or G, and X₁₀is Y or T.

Embodiment 185. The isolated anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 182 to 184, which comprises VL CDR1, CDR2 and CDR3, wherein VL CDR3 comprises the amino acid sequence QQYANFPYT (SEQ ID NO:2168).

Embodiment 186. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 185, which comprises VL CDR1, CDR2 and CDR3, wherein

(i) VL CDR1 comprises the amino acid sequence QASQDIANYLN (SEQ ID NO:2116); and/or,

(ii) VL CDR2 comprises the amino acid sequence DASNLET (SEQ ID NO:2142).

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

Embodiment 188. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 182, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence X₁RDVX₂GYAGX₃YGMDV (SEQ ID NO: 2198), wherein X₁is A or V, X₂is G or S, and X₃is Y or F.

Embodiment 189. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 188, wherein (i) the VH CDR1 comprises the amino acid sequence FTFGSYDMN (SEQ ID NO: 2048); and/or (ii) the VH CDR2 comprises the amino acid sequence SISX₁X₂X₃SYIX₄YAX₅SVKG (SEQ ID NO: 2197), wherein X₁is S or D, X₂is G or S, X₃is E or A, X₄is Y or A, and X₅is E or D.

Embodiment 190. The isolated anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 182, 188 or 189, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VL CDR3 comprises the amino acid sequence X₁QYAX₂FPYT (SEQ ID NO: 2201), wherein X₁is Q or S, and X₂is N or R.

Embodiment 191. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 190, which comprises VL CDR1, CDR2 and CDR3, wherein

(i) the VL CDR1 comprises the amino acid sequence X₁AX₂X₃X₄IX₅X₆YLN (SEQ ID NO: 2199), wherein X₁is Q, G, or E, X₂is S or N, X₃is Q or E, X₄is D or Y, X₅is A or S, X₆is N or D; and/or

(ii) the VL CDR2 comprises the amino acid sequence DAX₇NLX₈X₉(SEQ ID NO: 2200), wherein X₇is S or A, X₈is E, H or Q, and X₉is Tor Y.

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

Embodiment 193. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 182, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDGPX₁X₂X₃DYYMDV (SEQ ID NO: 2204), wherein X₁is R or Q, X₂is V, D, L or E, and X₃is S or V.

Embodiment 194. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 193, which comprises VH CDR1, CDR2 and CDR3, wherein

(i) the VH CDR1 comprises the amino acid sequence YTFX₁X₂YX₃MH (SEQ ID NO: 2202), wherein X₁is T or H, X₂is S, G, or H, and X₃is Y or P; and/or

(ii) the VH CDR2 comprises the amino acid sequence X₄INPSX₈GX₆TX₇YAQKFQG (SEQ ID NO: 2203), wherein X₄is I or S, X₅is G or R, X₆is S or R, and X₇is S or E.

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

Embodiment 196. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 195, which comprises VL CDR1, CDR2 and CDR3, wherein

(i) the VL CDR1 comprises the amino acid sequence RASQSVSSYLA (SEQ ID NO:2116); and/or,

(ii) the VL CDR2 comprises the amino acid sequence DASNRAT (SEQ ID NO:2116).

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

Embodiment 198. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 182, which specifically binds to FIXa, comprising VH CDR1, CDR2, and CDR3 and VL CDR1, CDR2, and CDR3, wherein the VH CDR3 comprises the amino acid sequence ARDKYQDYSX₁DI (SEQ ID NO: 2207), wherein X₁is F or V.

Embodiment 199. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 198, which comprises VH CDR1, CDR2 and CDR3, wherein

(i) the VH CDR1 comprises the amino acid sequence GSIX₁SX₂X₃YX₄WX₅(SEQ ID NO: 2205), wherein X₁is S or A, X₂is S, T, G, or V, X₃is S or A, X₄is Y or A, and X₅is G, V, N, or S; and/or

(ii) the VH CDR2 comprises the amino acid sequence X₆IX₇X₈X₉GX₁₀TX₁₁YNPSLKS (SEQ ID NO: 2206), wherein X₆is S or Y, X₇is S, Y, R, T or Q, X₈is Y, G, P or A, X₉is S or Q, X₁₀is S or K, and X₁₁is Y or Q.

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

Embodiment 201. The isolated anti-FIXa antibody, or antigen binding portion thereof, of embodiment 200, which comprises VL CDR1, CDR2 and CDR3, wherein

(i) the VL CDR1 comprises the amino acid sequence RASQGIDSWLA (SEQ ID NO:2136); and/or,

(ii) the VL CDR2 comprises the amino acid sequence AASSLQS (SEQ ID NO:2162).

Embodiment 202. The anti-FIXa antibody, or antigen binding portion thereof, of embodiment 182, wherein the anti-FIXa antibody or antigen binding portion thereof comprises VH CDR1, CDR2, and CDR3 comprising a VH CDR1 selected from SEQ ID NOs: 2058 to 2063, a VH CDR2 selected from SEQ ID NOs: 2084 to 2089, and a VH CDR3 selected from SEQ ID NOs: 2110 to 2115, and/or VL CDR1, CDR2, and CDR3 comprising a VL CDR1 selected from SEQ ID NOs: 2136 to 2141, a VL CDR2 selected from SEQ ID NOs: 2162 to 2167, and a VL CDR3 selected from SEQ ID NOs: 2188 to 2193.

Embodiment 203. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 182 to 202, comprising a VH and a VL, wherein

(i) the VH comprises an amino acid sequence which 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 the 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 which 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 the 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.

Embodiment 204. The anti-FIXa antibody, or antigen binding portion thereof, of any one of embodiments 182 to 203, comprising a VH and a VL, wherein

(a1) the VH and the VL comprise SEQ ID NOs: 1935 and 1937, respectively (BIIB-9-3595);

(a2) the VH and the VL comprise SEQ ID NOs: 1939 and 1941, respectively (BIIB-9-3601);

(a3) the VH and the VL comprise SEQ ID NOs: 1943 and 1945, respectively (BIIB-9-3604);

(a4) the VH and the VL comprise SEQ ID NOs: 1947 and 1949, respectively (BIIB-9-3617);

(a5) the VH and the VL comprise SEQ ID NOs: 1951 and 1953, respectively (BIIB-9-3618);

(a6) the VH and the VL comprise SEQ ID NOs: 1955 and 1957, respectively (BIIB-9-3621);

(a7) the VH and the VL comprise SEQ ID NOs: 1959 and 1961, respectively (BIIB-9-3647);

(a8) the VH and the VL comprise SEQ ID NOs: 1963 and 1965, respectively (BIIB-9-3649);

(a9) the VH and the VL comprise SEQ ID NOs: 1967 and 1969, respectively (BIIB-9-3650);

(a10) the VH and the VL comprise SEQ ID NOs: 1971 and 1973, respectively (BIIB-9-3654);

(a11) the VH and the VL comprise SEQ ID NOs: 1975 and 1977, respectively (BIIB-9-3753);

(a12) the VH and the VL comprise SEQ ID NOs: 1979 and 1981, respectively (BIIB-9-3754);

(a13) the VH and the VL comprise SEQ ID NOs: 1983 and 1985, respectively (BIIB-9-3756);

(a14) the VH and the VL comprise SEQ ID NOs: 1987 and 1989, respectively (BIIB-9-3764);

(a15) the VH and the VL comprise SEQ ID NOs: 1991 and 1993, respectively (BIIB-9-3766);

(a16) the VH and the VL comprise SEQ ID NOs: 1995 and 1997, respectively (BIIB-9-3707);

(a17) the VH and the VL comprise SEQ ID NOs: 1999 and 2001, respectively (BIIB-9-3709);

(a18) the VH and the VL comprise SEQ ID NOs: 2003 and 2005, respectively (BIIB-9-3720);

(a19) the VH and the VL comprise SEQ ID NOs: 2007 and 2009, respectively (BIIB-9-3727);

(a20) the VH and the VL comprise SEQ ID NOs: 2011 and 2013, respectively (BIIB-9-3745);

(a21) the VH and the VL comprise SEQ ID NOs: 2015 and 2017, respectively (BIIB-9-3780);

(a22) the VH and the VL comprise SEQ ID NOs: 2019 and 2021, respectively (BIIB-9-3675);

(a23) the VH and the VL comprise SEQ ID NOs: 2023 and 2025, respectively (BIIB-9-3681);

(a24) the VH and the VL comprise SEQ ID NOs: 2027 and 2029, respectively (BIIB-9-3684);

(a25) the VH and the VL comprise SEQ ID NOs: 2031 and 2033, respectively (BIIB-9-3698); or,

(a26) the VH and the VL comprise SEQ ID NOs: 2035 and 2037, respectively (BIIB-9-3704).

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A is a schematic representation of the domain organization of FIX zymogen and activated FIX with and without a substrate mimic bound in the active site (FIXa+EGR-CMK) (free FIXa and FIXa-SM, respectively). FIG. 1B is a schematic representation of the domain organization of FX zymogen and activated FX with and without a substrate mimic bound in the active site (FXa+EGR-CMK). In this representation, HC is the heavy chain of FIX, FIXa, FX, or FXa. LC is the light chain of FIX, FIXa, FX, or FXa.

FIG. 2 shows a schematic representation of the antibody generation, antibody characterization, and functional characterization for certain embodiments described herein.

FIGS. 3A-3D depict 95 anti-FIX antibodies disclosed herein, discovered by the antibody generation process described in FIG. 2. The germline, CDR length, CDR amino acid sequences, and SEQ ID NO are provided for both the VH and VL the antibodies. The full sequences of the VH and VL are provided in TABLE 4. The SEQ ID number for each CDR sequence is represented in TABLE 4. FIGS. 3A-3C present antibodies that preferentially bind to activated clotting factor IX (FIXa) (e.g., free FIXa and/or FIXa covalently modified by EGR- or LTR-CMK (FIXa-SM) compared to FIX zymogen (e.g., non-activatable FIX). In particular, FIG. 3A lists antibodies preferentially bind to FIXa-SM compared to free FIXa or FIXa zymogen (e.g., non-activatable FIX) (Class I). FIG. 3B lists antibodies preferentially bind to free FIXa compared to FIXa-SM or FIX zymogen (e.g., non-activatable FIX) (Class II). FIG. 3C lists antibodies that bind to either FIXa-SM or free FIXa, but do not appreciably bind to FIX zymogen (e.g., non-activatable FIX) (Class III). FIG. 3D lists antibodies that preferentially bind to FIX zymogen (e.g., non-activatable FIX) compared to free FIXa or FIXa-SM) (Class IV).

FIGS. 4A and 4B shows binding by Bio-Layer Interferometry (BLI) measurements of sensor-associated IgG to the indicated antigen, FIX zymogen (e.g., non-activatable FIX) (Haematologic Technologies, Inc., Essex Junction, Vt., USA) or FIXa (Haematologic Technologies, Inc., Essex Junction, Vt., USA). The maximum BLI response (nm) for each antibody is plotted on the y-axis.

FIG. 5 is a table presenting apparent monovalent affinity values (K_D) to free FIXa for each of the listed antibodies as determined by 1:1 fitting algorithms provided in the ForteBio Data Analysis 9.0 software.

FIGS. 6A-6E depict the measurement of binding by BLI of sensor associated IgG to the indicated antigen (free FIXa or FIX zymogen (e.g., non-activatable FIX)). FIG. 6A shows the measurement of binding for antibody BIIB-9-484 (VH: SEQ ID NO:31; VL: SEQ ID NO:221). FIG. 6B shows the measurement of binding of antibody BIIB-9-440 (VH: SEQ ID NO:19; VL: SEQ ID NO:209). FIG. 6C shows the measurement of binding of antibody BIIB-9-882 (VH: SEQ ID NO:115; VL: SEQ ID NO:301). FIG. 6D shows the measurement of binding of antibody BIIB-9-460 (VH: SEQ ID NO:23; VL: SEQ ID NO:213). FIG. 6E shows the measurement of binding for antibody BIIB-9-433 (VH: SEQ ID NO: 127; VL: SEQ ID NO: 313). The plots show the BLI response (nm) for association and dissociation as a function of time. FIG. 6F depicts a table of apparent monovalent affinity (K_D) to FIX zymogen (e.g., non-activatable FIX) and free FIXa, respectively, for each of the listed antibodies (i.e., BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, and BIIB-9-433) described in FIGS. 6A-6E, as determined by 1:1 fitting algorithms provided in the ForteBio Data Analysis 9.0 software.

FIG. 7 shows the measurement of binding by BLI of sensor-associated IgG to the indicated antigen, free FIXa (Haematologic Technologies, Inc., Essex Junction, Vt., USA) or FIXa-SM (e.g., FIXa+EGR-CMK (Haematologic Technologies, Inc., Essex Junction, Vt., USA)). The maximum BLI response (nm) for each antibody is plotted on the y-axis.

FIGS. 8A-8E exhibit the measurement of binding by BLI of sensor associated IgG to the indicated antigens (FIXa-SM, e.g., FIXa+EGF-CMK, and free FIXa). FIG. 8A shows the measurement of binding for antibody BIIB-9-484 (VH: SEQ ID NO:31; VL: SEQ ID NO:221). FIG. 8B shows the measurement of binding of antibody BIIB-9-440 (VH: SEQ ID NO:19; VL: SEQ ID NO:209). FIG. 8C shows the measurement of binding of antibody BIIB-9-882 (VH: SEQ ID NO:115; VL: SEQ ID NO:301). FIG. 8D shows the measurement of binding of antibody BIIB-9-460 (VH: SEQ ID NO:23; VL: SEQ ID NO:213). FIG. 8E shows the measurement of binding for antibody BIIB-9-433 (VH: SEQ ID NO: 127; VL: SEQ ID NO: 313). The provided plots show the BLI response (nm) for association and dissociation as a function of time. FIG. 8F depicts a table of apparent monovalent affinity (K_D) to (i) free FIXa or (ii) FIXa-SM (e.g., FIXa+EGR-CMK) for each of the listed antibodies (i.e., BIIB-9-484, BIIB-9-440, BIIB-9-882, BIIB-9-460, and BIIB-9-433) as determined by 1:1 fitting algorithms provided in the ForteBio Data Analysis 9.0 software.

FIGS. 9A-9D display the measurement of binding by BLI of listed sensor-associated IgG to the indicated antigen FIXn (non-activatable FIX), free FIXa, or FXa-SM (e.g., FIXa+EGR-CMK) (all obtained from Haematologic Technologies, Inc., Essex Junction, Vt., USA) as a function of time. The maximum BLI response (nm) is provided on each sensorgram. FIG. 9A shows the measurement of binding for representative antibodies in Class I (FIG. 3A), e.g., antibody BIIB-9-484 and antibody BIIB-9-460 (VH: SEQ ID NO: 23; VL: SEQ ID NO:213). FIG. 9B shows the measurement of binding for representative antibodies in Class II (FIG. 3B), e.g., antibody BIIB-9-416 (VH: SEQ ID NO:93; VL: SEQ ID NO:279) and antibody BIIB-9-885 (VH: SEQ ID NO:97; VL: SEQ ID NO:283). FIG. 9C shows the measurement of binding for a representative antibody in Class III (FIG. 3C), e.g., antibody BIIB-9-1287 (VH: SEQ ID NO:181; VL: SEQ ID NO:367). FIG. 9D shows the measurement of binding for a representative antibody in Class IV (FIG. 3D), e.g., antibody BIIB-9-397 (VH: SEQ ID NO:183; VL: SEQ ID NO:369).

FIG. 10 shows a table listing 95 antibodies disclosed herein, in addition to their assigned class based on antigen binding profiles as determined by BLI binding assays. Antibodies were assigned to classes if they exhibited a 0.1 or greater difference in BLI response to one antigen over another using the assay parameters defined in the examples. Class I antibodies corresponds to the antibodies in FIG. 3A. Class II antibodies correspond to the antibodies in FIG. 3B. Class III antibodies correspond to the antibodies in FIG. 3C. Class IV antibodies correspond to the antibodies in FIG. 3D.

FIG. 11 shows a table listing the maximum wavelength (nm) of each indicated antibody as determined by screening for propensity to self-interact by AC-SINS. A threshold value of 540 nm is set based on internal controls, with antibodies exceeding the threshold shaded in black (indicating a potential for self-interaction).

FIGS. 12A-12C are tables listing 94 antibodies disclosed herein, discovered by the antibody generation process. The germline, CDR length, CDR amino acid sequences, and SEQ ID NOs are provided for both the VH and VL of each of the 94 antibodies. The sequences of the complete VH and VL for each antibody are presented in TABLE 4. FIG. 12A and FIG. 12B present antibodies that preferentially bind to FX zymogen (e.g., non-activatable FX) compared to activated clotting factor FX (FXa) (e.g., FXa covalently modified by EGR- or LTR-CMK (FXa-SM) (Class V). FIG. 12C presents antibodies that preferentially bind to activated clotting factor FX (FXa) (e.g., FXa covalently modified by EGR- or LTR-CMK (FXa-SM) compared to FX zymogen (e.g., non-activatable FX) (Class VI).

FIGS. 13A and 13B depict the measurement of binding by BLI of sensor-associated IgG to the indicated antigen, FX zymogen or FXa-SM (e.g., FXa+EGR-CMK). The maximum BLI response (nm) for each antibody is plotted on the y-axis.

FIG. 14 shows a table of apparent monovalent affinity (K_D) to FX zymogen in M for each of the listed antibodies as determined by 1:1 fitting algorithms provided in the ForteBio Data Analysis 9.0 software.

FIG. 15 shows a table listing the maximum wavelength (nm) of each indicated antibody as determined by screening for propensity to self-interact by AC-SINS. A threshold value of 540 nm was set based on internal controls. Antibodies exceeding the threshold are shaded in black, which indicates a potential for self-interaction.

FIGS. 16A-16D show 202 bispecific antibodies identified as having the ability to replace FVIIIa-like function in a chromogenic Factor Xa generation assay. The 202 bispecific antibodies are separated into four groups, and four corresponding sub-figures. FIG. 16A shows the rates of FXa chromogenic substrate cleavage for the first group of bispecific antibodies (1-51 of 202). FIG. 16B shows the rates of FXa chromogenic substrate cleavage for the second group of bispecific antibodies (52-102 of 202). FIG. 16C shows the rates of FXa chromogenic substrate cleavage for the third group of bispecific antibodies (103-152 of 202). FIG. 16D shows the rates of FXa chromogenic substrate cleavage for the fourth group of bispecific antibodies (153-202 of 202). In each case, the mean baseline rate in the absence of bispecific antibody is indicated by the dashed line.

FIG. 17 depicts the rates of FXa chromogenic substrate cleavage for a subset of bispecific antibodies in the IgG4 format. The mean baseline rate in the absence of bispecific antibody is indicated by the dashed line.

FIG. 18 illustrates the kinetics of FXa chromogenic substrate cleavage in the presence of three representative bispecific antibodies. Compared to the baseline control (in which no antibody was added to the reaction mixture), BIIB-9-484/BIIIB-12-915, BIIB-9-619/BIIB-12-925, and BIIB-9-578/BIIB-12-917 were able to increase the rate of FXa chromogenic substrate cleavage, as indicated by the increase in OD over time.

FIG. 19 illustrates the ability of BIIB-9-484/BIIB-12-917, BIIB-9-484/BIIB-12-915, and BIIB-9-484/BIIB-12-1306 to replace the function of FVIIIa in a one-stage clotting assay in FVIII deficient plasma (as indicated by the decrease in clotting time). FVIII-deficient plasma with no bispecific antibody is shown for comparison.

FIG. 20 shows that the ability of a bispecific antibody, BIIB-9-484/BIIB-12-917, to replace FVIIIa-function in a one-stage clotting assay in FVIII deficient plasma is dependent on the bispecific format. Homodimeric BIIB-9-484, homodimeric BIIB-12-917, and a mixture of the two homodimers were unable to replace FVIIIa-like function. FVIII deficient plasma with no antibody is shown for comparison.

FIG. 21 exhibits the BLI-binding assay to assess co-binding of each target antigen to the indicated bispecific antibody using dip and read streptavidin biosensors. The sensorgram plots the BLI response (nm) as a function of time, with each stage of the experiment separated by a black vertical line. An increase in response at a particular stage is indicative of protein loading.

FIG. 22A lists the CDR sequences of BIIB-9-484 and two affinity matured daughters, BIIB-9-1335 and BIIB-9-1336. A CLUSTAL format multiple sequence alignment by MAFFT (v7.205) of the VH segments of BIIB-9-484, BIIB-9-1335, BIIB-9-1336 is provided. Degree of amino acid conservation is indicating above the alignment (“*”=identical; “:”=strongly conserved; “.”=poorly conserved), as well as the bars below the alignment. The VH and VL CDRs are underlined. The sequence before VH-CDR1 is framework region (FR) 1; the sequence after VH-CDR1 and before VH-CDR2 is FR2; the sequence after VH-CDR2 and before VH-CDR3 is FR3; and the sequence after VH-CDR3 is FR4. The sequence before VL-CDR1 is framework region (FR) 1; the sequence after VL-CDR1 and before VL-CDR2 is FR2; the sequence after VL-CDR2 and before VL-CDR3 is FR3; and the sequence after VL-CDR3 is FR4. FIGS. 22B-22D show the BLI-binding profile of free FIXa to BIIB-9-484, BIIB-9-1335, and BIIB-9-1336, respectively.

FIG. 23 shows that increasing the affinity of the anti-FIXa arm in the context of a bispecific antibody with FVIIIa-like activity results in higher activity in a one-stage clotting assay. An example of this is illustrated in the plot above, in which bispecific antibodies with high affinity anti-FIXa arms (BIIB-9-1335/BIIB-12-917 and BIIB-9-1336/BIIB-12-917) show a further decrease in clotting time compared to the bispecific antibody with a lower affinity anti-FIXa arm (BIIB-9-484/BIIB-12-917).

FIG. 24A shows the binding affinity of emicizumab (ACE910) sequence identical bispecific antibody (“emicizumab biosimilar”) to FIX zymogen, activated FIX, FX zymogen, and activated FX (i.e., 1 μM, 1 μM, 1 μM, and 1 μM, respectively). FIG. 24B shows the binding affinity of a bispecific molecule (BS-027125) to FIX zymogen, activated FIX, FX zymogen, and activated FX (i.e., 8 nM, 2 nM, 20 nM, and not detected, respectively).

FIG. 25 shows the clotting time (sec) of FVIII (inverted triangle), BS-025 FIXa homodimer (big diamond), BS-027 FX homodimer (small diamond), BS-027025 homodimer mixture (triangle), and BS-027025 bispecific (square) over various concentrations (IU/mL or μM).

FIG. 26 shows the FVIII activity (FVIII equivalent %) by BS-027125 (Square), BS-027125 FIXa homodimer (triangle), and BS-027125 FX homodimer (circle). The FVIII activity was measured by one-stage clotting assay.

FIG. 27 shows the nM FXa generation by rFVIII (big circle), BS-027125 (square), BS-027125 FIXa homodimer (triangle), BS-027125 FX homodimer (inverted triangle), and BS-027125 (no PL) (small circle) measured by chromogenic factor Xa (FXa) generation assay.

FIG. 28A shows the results of thrombin generation assay of BS-027125. The left panel shows the lag time (min), and the right panel shows the peak.

FIG. 28B shows the amount of thrombin generated by rFVIII (left panel) and BS-027125 (right panel).

FIG. 29 shows nM FXa generated in the presence of rFVIII, emicizumab biosimilar, or BS-027125 with either PC/PS (80%/20%) or PC/PEPS (40%/40%/20%) phospholipid vesicles (upper panels) and the fold change in activity contributed by PC/PEPS over PC/PS phospholipid vesicles (lower panels).

FIG. 30 shows the results of thrombin generation assay on PC/PEPS phospholipids in the presence of rFVIII, emicizumab biosimilar, and BS-027125. The upper panels show the lag time (min); the middle panels show the peak thrombin (nM); and the lower panels show the endogenous thrombin potential (ETP) (nM*min).

FIGS. 31A, 31B, 31C and 31D show the epitope binning of 47 different anti-FIXa antibodies against each other as determined by biolayer interferometry. FIG. 31A and FIG. 31B show the Octet profiles for non-competitive and competitive binders, respectively. FIG. 31C summarizes the 47×47 interactions tested and whether the antibodies pairs result in competitive or non-competitive binding. Dark gray squares indicate antibody pairs that cross-block, indicating that they fall into the same bin. Medium gray squares indicate antibody pairs that do not cross-block, indicating that they fall into different bins. White squares indicate unidirectional conflicts and light gray squares indicate antibodies for which data could not be analyzed. FIG. 31D shows node analysis of the binning network determined in FIG. 31C. The closer together two antibodies are on this map, the more similar their binning profiles are.

FIG. 32A shows the binding profile of BIIB-9-484 to FIXa in the presence and absence of calcium using biolayer interferometry. The dashed line indicates the end of the association phase and the start of the dissociation phase.

FIG. 32B shows the binding profile of BIIB-9-1336 to FIXa in the presence and absence of calcium using biolayer interferometry. The dashed line indicates the end of the association phase and the start of the dissociation phase.

FIG. 33A shows the rate of substrate cleavage by FIXa (250 nM) alone or in the presence of increasing concentrations of two different anti-FIXa antibodies (BIIB-9-1336 and BIIB-9-579) or an anti-FX antibody control (BIIB-12-917). BIIB-9-1336 was tested as a homodimeric, bivalent antibody (“BIIB-9-1336’), as a one armed antibody (“BIIB-9-1336 one-armed”) and in a bispecific configuration with BIIB-12-917 (“BIIB-9-1336/BIIB-12-917”). The rate of substrate cleavage is expressed in mOD/minute and increasing amounts of antibody are expressed in terms of the concentration (nM) of FIXa-Ab complex.

FIG. 33B shows the fold increase in FIXa amidolytic activity that is seen for 500 nM FIXa in the presence of a panel of BIIB-9-484, BIIB-9-1336, BIIB-9-619, and BIIB-9-578 antibodies.

FIG. 33C shows the rate of substrate cleavage by FIXa across varying concentrations of substrate in the presence or absence of saturating amounts of BIIB-9-1336.

FIG. 33D shows the K_Mand V_maxfor FIXa in the presence and absence of BIIB-9-1336.

FIG. 34A and FIG. 34B show the rate of ATIII inhibition of FIXa in the presence or absence of anti-FIXa antibodies. FIG. 34A shows the appearance of a band at 75 kDa corresponding to ATIII-FIXa complex. The rate of complex formation is indicative of ATIII inhibition of FIXa and is increased in the presence of BIIB-9-1335 and BIIB-9-1336. FIG. 34B shows the band intensity of ATIII-FIXa complex formation quantified and graphed at different time points.

FIG. 35 is cartoon representation of the crystal structure of the Fab region of BIIB-9-1336 in complex with the EGF2 and serine protease domain of FIXa shown in two orientations.

FIG. 36 shows the serine protease domain of FIXa in surface representation with the epitopes of BIIB-9-1336 (1336 epitope) and FVIIIa (FVIIIa epitope) mapped onto the surface. The BIIB-9-1336 epitope on FIXa and the FVIIIa epitope on FIXa are colored in black. Residues shared between the two epitopes are outlined in white.

FIG. 37 lists the specific amino acids residues in the FIXa heavy chain that constitute the BIIB-9-1336 and FVIIIa epitopes and correspond to the residues highlighted in FIG. 36. Residues shared between the two epitopes are shown underlined and in bold. Residues shown in parentheses are not shown in FIG. 36 as they do not agree across published reports. The numbering of the amino acid residues are based on the chymotrypsinogen numbering. BIIB-9-1336 also contacts one residue in the light chain of FIXa and is indicated by the asterisk. Light chain contacts for FVIIIa are not listed.

FIG. 38A shows the binding of BIIB-12-917 to a panel of FX variants including wild type Factor X zymogen, wild type activated FX, activated FX that retains the activation peptide, zymogen FX lacking the activation peptide, and a chimeric FIX construct in which the FIX activation peptide has been replaced by the FX activation peptide, by biolayer interferometry. The dashed line indicates the end of the association phase and the start of the dissociation phase.

FIG. 38B shows cartoon representations of each of the above-mentioned FX variants. The + and − signs indicate whether BIIB-12-917 binds. These data show that the epitope of BIIB-12-917 is in the activation peptide region of FX.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides antibodies that preferentially bind to specific forms of clotting factors. In particular, the disclosure provides antibodies and antigen binding portion thereof that specifically binds to FIX (e.g., antibodies and antigen binding portion thereof that preferentially bind to activated factor IX (FIXa) in the presence of FIXa and factor IX zymogen (FIXz)). The 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.

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

The present disclosure also provides, for example, compositions comprising the binding molecules, e.g., 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, methods of making, methods of treatment and diagnosis, immunoconjugates, and kits.

The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be defined by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions or process steps, as such can vary.

I. Definitions

In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

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”), as well as 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 “multiple.”

Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass 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).

Unless defined otherwise, 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 is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

Wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.

Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invention. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation. Nucleotides are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

About: The term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is f 10%.

Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

Administered in combination: As used herein, the term “administered in combination,” “combined administration,” or “combination therapy” means that two or more agents, e.g., a binding molecule disclosed herein, and a second agent, are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

Affinity: The term “affinity” refers to the degree to which a binding molecule, e.g., an antibody, binds to an antigen so as to shift the equilibrium of antigen and binding molecule toward the presence of a complex formed by their binding. Thus, where an antigen and binding molecule are combined in relatively equal concentration, a binding molecule of high affinity will bind to the available antigen so as to shift the equilibrium toward high concentration of the resulting complex. Binding molecules, e.g., antibodies, or antigen-binding fragments, variants or derivatives thereof of the present disclosure can also be described or specified in terms of their binding affinity to an antigen. The affinity of binding molecule, e.g., an antibody, for an antigen can be determined experimentally using any suitable method. (See, 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 methods described herein).

The measured affinity of a particular binding molecule-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., K_D, K_a, K_d) are preferably made with standardized solutions of binding molecule and antigen, and a standardized buffer.

The “high affinity” for a binding molecule, e.g., an antibody, refers to an equilibrium association constant (K_aff) of at least about 1×10⁷liters/mole, or at least about 1×10⁸liters/mole, or at least about 1×10⁹liters/mole, or at least about 1×10¹⁰liters/mole, or at least about 1×10¹¹liters/mole. or at least about 1×10¹²liters/mole, or at least about 1×10¹³liters/mole, or at least about 1×10¹⁴liters/mole or greater. “High affinity” binding can vary for antibody isotypes.

K_D, the equilibrium dissociation constant, is a term that is also used to describe antibody affinity and is the inverse of Kn. K_Dis obtained from the ratio of k_dto k_a(i.e., k_d/k_a) and is expressed as a molar concentration (M). K_Dvalues for antibodies can be determined using methods well established in the art. Available methods for determining the K_Dof an antibody include a Bio-Layer Interferometry (BLI) assay, surface plasmon resonance, a biosensor system such as a BIACORE® system or flow cytometry and Scatchard analysis. If K_Dis used, the term “high affinity” for an antibody refers to an equilibrium dissociation constant (K_D) of less than about 1×10⁻⁷M, or less than about 1×10⁻⁸M, or less than about 1×10⁻⁹M, or less than about 1×10⁻¹⁰M, or less than about 1×10⁻¹¹M, or less than about 1×10⁻¹²M, or less than about 1×10¹³M, less than about 1×10⁻¹⁴M, or lower.

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. An amino acid can be substituted in a parent or reference sequence (e.g., a wild type polypeptide sequence), for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, a reference to a “substitution at position X” refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position n, and Y and Z are alternative substituting amino acid residues that can replace A

In the context of the present disclosure, substitutions (even when they are referred to as amino acid substitution) are conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.

Affinity matured: The term “affinity matured” refers to a binding molecule, e.g., an antibody, that has undergone affinity maturation, a process by which binding molecules, e.g., antibodies, with increased affinity for a target antigen are produced. Thus, an affinity matured antibody is an antibody with one or more alterations in one or more CDRs thereof which result an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.

Any one or more methods of preparing and/or using affinity maturation libraries available in the art may be employed in order to generate affinity matured antibodies in accordance with various embodiments of the invention disclosed herein. Exemplary affinity maturation methods include random mutagenesis, bacterial mutator strains passaging, site-directed mutagenesis, mutational hotspots targeting, parsimonious mutagenesis, antibody shuffling, light chain shuffling, heavy chain shuffling, CDR1 and/or CDR1 mutagenesis, and methods of producing and using affinity maturation libraries amenable to implementing methods and uses in accordance with various embodiments of the invention disclosed herein, include, for example, those disclosed in: 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 herein 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; Jackson et al. (1995) J. Immunol. 154(7):3310-9; and Hawkins et al. (1992) J. Mol. Biol. 226:889-896. Mutation at selective mutagenesis positions, contact or hypermutation positions, with an activity enhancing amino acid residue is described in U.S. Pat. No. 6,914,128.

Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). 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, genetically-engineered animal, or a clone.

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

A typical antibody comprises at least two heavy chains (“HC”) and two light chains (“L”) interconnected by disulfide bonds.

Each “heavy chain” is comprised of a “heavy chain variable region” (abbreviated herein as “VH”) and a “heavy chain constant region” (abbreviated herein as “CH”). The heavy chain constant region in an unmodified antibody is comprised of three constants domains, CH1, CH2, and CH3.

Each “light chain” is comprised of a “light chain variable region” (abbreviated herein as “VL”) and a “light chain constant region”. The light chain constant region in an unmodified antibody is comprised of one constant domain, “CL”. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FW”).

Each VH and VL is composed of three CDRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The present disclosure presents VH and VL sequences as well as the subsequences corresponding to CDR1, CDR2, and CDR3. Accordingly, a person skilled in the art would understand that the sequences of FW1, FW2, FW3 and FW4 are equally disclosed. For a particular VH, FW1 is the subsequence between the N-terminus of the VH and the N-terminus of VH-CDR1, FW2 is the subsequence between the C-terminus of VH-CDR1 and the N-terminus of VH-CDR2, FW3 is the subsequence between the C-terminus of VH-CDR2 and the N-terminus of VH-CDR3, and FW4 is the subsequence between the C-terminus of VH-CDR3 and the C-terminus of the VH. Similarly, for a particular VL, FW1 is the subsequence between the N-terminus of the VL and the N-terminus of VL-CDR1, FW2 is the subsequence between the C-terminus of VL-CDR1 and the N-terminus of VL-CDR2, FW3 is the subsequence between the C-terminus of VL-CDR2 and the N-terminus of VL-CDR3, and FW4 is the subsequence between the C-terminus of VL-CDR3 and the C-terminus of the VL.

The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin 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, an scFv is covalently linked (for example, via peptidic bonds or via a chemical linker) to the N-terminus of either the heavy chain and/or the light chain of a typical antibody, or intercalated in the heavy chain and/or the light chain of a typical antibody.

As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain variable fragment (scFv), disulfide stabilized scFvs, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies and/or antigen binding portions thereof, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.

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

There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs. The Kabat numbering system is generally used when referring to a residue in the variable domain (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 Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

The phrases “amino acid position numbering as in Kabat,” “Kabat position,” and grammatical variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FW residue 82. See TABLE 1.

TABLE 1

Loop
Kabat
AbM
Chothia

L1
L24-L34
L24-L34
L24-L34

L2
L50-L56
L50-L56
L50-L56

L3
L89-L97
L89-L97
L89-L97

H1
H31-H35B
H26-H35B
H26-H32 . . . 34

(Kabat Numbering)

H1
H31-H35
H26-H35
H26-H32

(Chothia Numbering)

H2
H50-H65
H50-H58
H52-H56

H3
H95-H102
H95-H102
H95-H102

The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

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

For all heavy chain constant region amino acid positions discussed in the present invention, numbering is according to the EU index first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63(1):78-85, describing the amino acid sequence of myeloma protein EU, which is the first human lgG1 sequenced. The EU index of Edelman et al. is also set forth in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. Thus, the phrases “EU index as set forth in Kabat” or “EU index of Kabat” and “position . . . according to the EU index as set forth in Kabat,” and grammatical variants thereof refer to the residue numbering system based on the human igG1 EU antibody of Edelman et al. as set forth in Kabat 1991.

The numbering system used for the variable domains (both heavy chain and light chain) and light chain constant region amino acid sequence is that set forth in Kabat 1991.

As used herein the Fc region includes the polypeptides 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-terminal to these domains. For IgA and IgM Fc can include the J chain. For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2).

Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Kabat. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.

Polymorphisms have been observed at a number of different positions within antibody constant regions (e.g., Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 as numbered by the EU index as set forth in Kabat), and thus slight differences between the presented sequence and sequences in the prior art can exist. Polymorphic forms of human immunoglobulins have been well-characterized. At present, 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, pp. 43-78 (1990); Lefranc et al. (1979) Hum. Genet.: 50, 199-211. It is specifically contemplated that the antibodies of the present invention may be incorporate any allotype, isoallotype, or haplotype of any immunoglobulin gene, and are not limited to the allotype, isoallotype or haplotype of the sequences provided herein.

Antibody binding site: The term “antibody binding site” refers to a region in the antigen (e.g., FIXa or FXz) comprising a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds. Thus, the antibody binding site can contain additional areas in the antigen which are beyond the epitope and which can determine properties such as binding affinity and/or stability, or affect properties such as antigen enzymatic activity or dimerization. Accordingly, even if two antibodies bind to the same epitope within an antigen, if the antibody molecules establish distinct intermolecular contacts with amino acids outside of the epitope, such antibodies are considered to bind to distinct antibody binding sites.

Antigen binding portion: As used herein the term “antigen binding portion” can be interchangeably used with “antigen binding fragment” and refers to a portion of an intact antibody capable of specific binding to the same epitope as the intact antibody. In particular, it refers to a portion or portions of an intact antibody comprising one or more CDRs of an 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.

Antigen binding molecule: The terms “antigen binding molecule” and “binding molecule” are used interchangeable in the present disclosure and encompass antibodies as defined herein, as well as other molecular entities comprising at least one of the CDRs of the antibodies disclosed herein which are capable of binding to the same epitopes. For example, the term includes antibody mimics based on the scaffold of the fibronectin type III domain (monobodies), other scaffolding systems (e.g., tenascin) in which one or more CDRs are grafted, aptamers, etc. In some aspects, the antigen binding molecule can be bispecific, i.e., a “bispecific binding molecule” or a “bispecific molecule”.

Approximately: As used herein, the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” 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) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Associated with: As used herein with respect to a disease, the term “associated with” means that the symptom, measurement, characteristic, or status in question is linked to the diagnosis, development, presence, or progression of that disease. As association may, but need not, be causatively linked to the disease.

When used with respect to two or more moieties, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An “association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated.

Binding affinity: “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_D). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.

The terms “higher binding affinity” or “greater affinity” as applied to any of the antibodies of the invention refer to increased binding affinity (as measured, for example, by the K_D) with respect to a reference antibody. In some embodiments, the reference antibody is an corresponding antibody that has not been affinity matured. In some embodiments, the reference antibody is another antibody with the same specificity (e.g., for an anti-FIXa disclosed herein, a reference antibody could be another anti-FIX or anti-FIXa antibody known in the art). In some embodiments, increased binding affinity can be 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), form of the factor (e.g., FIXa or FXz), or antigen binding site (e.g., epitope). In some embodiments, increased binding affinity can be 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), form of the factor (e.g., FIXa or FXz), or antigen binding site (e.g., epitope).

Binding: The term “binding” refers to a physical interaction between two molecules, for example, an antibody and an antigen.

(i) Binding specificity: The term “specificity” refers to the ability of an binding molecule, e.g., an antibody, to bind preferentially to one antigenic site (e.g., an epitope) versus a different antigenic site and does not necessarily imply high affinity. The terms “binding specificity” and “specificity” are used interchangeably and can refer both to (i) a specific portion of a binding molecule and (ii) the ability of the binding molecule to specifically bind (see definition of “specific binding” below) to a particular epitope. For example, in some embodiments, a bispecific antibody disclosed herein comprises two binding specificities, a first binding specificity for example to FIXa and a second binding specificity for example to FXz (in this context, a “binding specificity” such as a specific region of a bispecific antibody binding to the a particular antigenic determinant would be equivalent to a “binding domain”).

(ii) Specific binding: An binding molecule, e.g., an antibody, “specifically binds” when there is an immunological reaction of specific interaction between an antigen and the binding molecule. The term “specifically binds” means that the antibody has been generated to bind to the antigen through its variable region. The term “non-specific binding” means that the antibody has not been generated to specifically bind to the antigen but does somehow bind the antigen through non-specific means. As one example, an antibody will non-specifically bind to an Fc receptor through the Fc portion of the antibody molecule. As another example, certain antibodies may inadvertently cross-react with antigens to which they were not generated.

(iii) Preferential binding: A binding molecule, e.g., an antibody, “preferentially binds” to an antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that preferentially binds to an FIXa epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other FIXa epitopes or non-FIXa epitopes. For example, an anti-FIX antibody preferentially binds to activated FIX over FIX zymogen if more than 50%, 60%, 70%, 80%, 90%, or 95% of the anti-FIX antibody binds to FIXa in the presence of both FIXa and FIXz. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that preferentially binds to a first target may or may not preferentially bind to a second target. As such, “preferential binding” does not necessarily require (although it can include) exclusive binding. Thus, in some aspects, “preferential binding” can be “exclusive binding.” To exemplify these concepts, if 50% of an anti-FIX specifically binds to FIX zymogen and 50% specifically binds to FIXa, such binding would be “non-selective” or “non-preferential.” If less than 50% of the anti-FIX binds to FIX zymogen and more than 50% binds to FXa, the anti-FIX would “preferentially bind” to FIXa. If the anti-FIX does not bind to FIX zymogen and only binds to FIXa, the anti-FIX would “exclusively bind” to FIXa.

Biological sample: The term “biological sample” as used herein refers to any sample obtained from an subject, cell line, tissue culture, or other source potentially comprising a molecule comprising an antigen specifically recognized by the binding molecules disclosed herein. In some aspects, 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.

Bispecific antibody: A “bispecific antibody” is a particular type of “bispecific molecule” or “bispecific binding molecule.” The term “bispecific antibody” means an antibody that is able to bind to at least two antigenic determinants (e.g., epitopes) through two different antigen binding sites. In certain embodiments, the bispecific antibody is capable of concurrently binding two antigenic determinants (e.g., epitopes). In some embodiments, a bispecific antibody binds one antigen (or epitope) on one of its binding arms (one pair of heavy chain/light chain), and binds a different antigen (or epitope) on its second binding arm (a different pair of heavy chain/light chain). In some embodiments, a bispecific antibody can have two distinct antigen binding arms (in both specificity and CDR sequences), and is monovalent for each antigen to which it binds. Bispecific antibodies include, e.g., those generated by quadroma technology (Milstein & 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 approaches which introduces mutations in the Fc region (Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-6448).

A wide variety of recombinant bispecific antibody formats have been developed in the recent past, e.g. by fusion of, e.g. an IgG antibody format and single chain domains (see Kontermann R E, mAbs 4:2, (2012) 1-16). Bispecific antibodies wherein the variable domains VL and VH or the constant domains CL and CH1 are replaced by each other are described in WO2009080251 and WO2009080252.

An approach to circumvent the problem of mispaired byproducts, which is known as ‘knobs-into-holes’, aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids were replaced by amino acids with short side chains to create a ‘hole’. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a ‘knob’. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (‘knob-hole’) versus homodimer formation (‘hole-hole’ or ‘knob-knob’) was observed (Ridgway J B, Presta L G, Carter P; and WO1996027011). The percentage of heterodimer could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A. M, et al, Nature Biotech 16 (1998) 677-681; Anwell S, Ridgway J B, Wells J A, Carter P., J Mol Biol 270 (1997) 26-35). New approaches for the knobs-into-holes technology are described in e.g. in EP 1870459A1. Xie, Z., et al, J Immunol Methods 286 (2005) 95-101 refers to a format of bispecific antibody using scFvs in combination with knobs-into-holes technology for the FC part.

The modular architecture of antibodies has been exploited to create more than 60 different bispecific antibody formats. See Spiess et al. (2015) Molecular Immunology 67:95-106, which is herein incorporated by reference in its entirety. Accordingly, in some aspects, the bispecific antibody format is selected from crossMab, DAF (Dual Action Fab) (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Knobs-in-holes common LC, Knobs-in-holes assembly, Charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y (bispecific antibody with a leucize zipper inducing 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, Zybody, DVI-IgG (four-in-one), Nanobody, Nanobody-HSA, BiTE (bispecific T cell engager), Diabody, DART (dual-affinity-retargeting), TandAb (tandem antibody), scDiabody, scDiabody-CH3, Triple Body, Miniantibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)2, F(ab′)2-ScFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HC Ab, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Locck, ImmTAC, HSAbody, scDiabody-HSA, Tandem scFv-Toxin, IgG-IgG, Cov-X-Body, and scFv1-PEG-scFV2.

In some aspects, the bispecific antibody is an asymmetric (e.g., heterdimeric) antibody, comprising a chain A and a chain B, wherein

- (i) Chain A comprises a T336W mutation, and chain B comprises T366W, L368A, and Y407V mutations (Knows-in holes format);
- (ii) Chain A comprises a F405L mutation, and chain B comprises a K409R mutation (duobody format);
- (iii) Chain A comprises T350V, L351Y, F405A, and Y407V mutations, and chain B comprises T350V, T366L, K392L and T394W mutations (azymetric format);
- (iv) Chain A comprises K409D and K392D mutations, and chain B comprises D399K and E356K mutations (Charge pair format);
- (v) Chain A comprises D221E, P228E, and L368E mutations, and chain B comprises D221R, P228R and K409R mutations (Charge pair format);
- (vi) Chain A comprises S364H and F405A mutations, and chain B comprises Y349T and T394F mutations (HA-TF format); or,
- (vii) Chain A comprises an IgG/A chimera, and chain B also comprises an IgG/A chimera (SEEDbody format).

In some aspects, the bispecific antibody is a monospecific antibody engineered for bispecificity by appending either the amino or carboxy termini of either the light or heavy chains with additional antigen-binding units. Alternatives for 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 aspects, a bispecific molecule of the present invention comprises a bispecific antibody fragment. Numerous bispecific fragment forms lacking some or all of the bispecific antibody constant domains are known in the art. In some aspects, the bispecific molecule of the invention is a bispecific fusion protein, e.g., an ImmTAC (scFv linked to an affinity matured receptor). In other aspects, the bispecific molecule is a bispecific antibody conjugate.

Bispecific molecule: See the definition of “Antigen binding molecule”/“Binding molecule” above.

Chimeric antibody: The term “chimeric antibody” and grammatical variants thereof refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, and/or affinity, and/or capability while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

Complementarity determining region: The term “complementarity determining region” or “CDR” refers to variable regions of either H (heavy) or L (light) chains contains the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as “hypervariable regions.” See definition of “Antibody” above.

Conservative amino acid substitution: A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including 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, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

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

Other amino acid substitutions can be readily identified by workers of ordinary skill. For example, for the amino acid alanine, a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement 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 can be expected to induce changes in the properties of isolated polypeptides are those in which (i) a polar residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties.

Conserved: As used herein, the term “conserved” refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completely conserved” or “identical” if they are 100% identical to one another. 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 one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. 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 one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an polynucleotide or polypeptide or may apply to a portion, region or feature thereof.

Cross-compete: The terms “compete” or “cross-compete”, as used herein with regard to a binding molecule, e.g., an antibody, means that a first binding molecule, e.g., a first antibody or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second binding molecule, e.g., a second antibody or an antigen-binding portion thereof, such that the result of binding of the first binding molecule with its cognate epitope is detectably decreased 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. The alternative, where the binding of the second binding molecule to its epitope is also detectably decreased in the presence of the first binding molecule, can, but need not be the case. That is, a first binding molecule can inhibit the binding of a second binding molecule to its epitope without that second molecule inhibiting the binding of the first binding molecule to its respective epitope. However, where each binding molecule detectably inhibits the binding of the other binding molecule with its cognate epitope, whether to the same, greater, or lesser extent, the binding molecules are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing binding molecules are encompassed by the present invention.

Binding molecules, e.g., antibodies, are said to “bind to the same epitope” or “comprising the same binding site” or have “essentially the same binding” characteristics, if the binding molecules cross-compete so that only one antibody can bind to the epitope at a given point of time, i.e., one binding molecule prevents the binding or modulating effect of the other.

Competition herein means a greater relative inhibition 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% as determined by competition ELISA analysis or by ForteBio analysis, e.g., as described in the Examples section. It may be desirable to set a higher threshold of relative inhibition as criteria of what is a suitable level of competition in a particular context. Thus, for example, it is possible to set criteria for the competitive binding, wherein at least about 40% relative inhibition is detected, 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 even about 100%, before an antibody is considered sufficiently competitive.

Effective Amount: As used herein, the term “effective amount” of an agent, e.g., a therapeutic agent such as an antibody, is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering a therapeutic agent that treats bleeding, an effective amount of an agent is, for example, an amount sufficient to reduce or decrease in bleeding occurrences, as compared to the response obtained without administration of the agent. The term “effective amount” can be used interchangeably with “effective dose,” “therapeutically effective amount,” or “therapeutically effective dose.”

Effector function: The “effector function” of an antibody is the ability to bind complement proteins which can assist in lysing the target antigen, for example, a cellular pathogen, in a process termed complement-dependent cytotoxicity (CDC). Another effector activity of the Fc region is to bind to Fc receptors (e.g., FcγRs) on the surface of immune cells, or so-called effector cells, which have the ability to trigger other immune effects. The effector function of an antibody can be avoided, e.g., by using antibody fragments lacking the Fc region (e.g., such as a Fab, F(ab′)2, or single chain Fv (scFv)), by removing sugars that are linked to particular residues in the Fc region (aglycosylated antibodies), or by employing Fc regions from an IgG4 antibody (an “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 cellular cytotoxicity than IgG1.

Engineered antibody: As used herein, embodiments of the invention are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type, or native molecule. In this respect, an “engineered antibody” is, for example, an antibody in which substitutions/mutations have been made to improve affinity, plasma half-life, etc., the format of the antibody has been modified (e.g., by generating an scFv or a bispecific antibody), or the antibody has been subjected to affinity maturation.

Epitope: The term “epitope” as used herein refers to an antigenic protein determinant (e.g., an amino acid subsequence of FIXa or FXz) capable of binding to a binding molecule, e.g., an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. The part of an antibody or binding molecule that recognizes the epitope is called a paratope. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen.

Expression vector: An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An “expression system” usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product. The antibodies (e.g., bispecific antibodies) according to the invention are preferably produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity.

Germline sequence: As used herein, the term “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin may be used. The term “germline” refers to the sequences of the V, D, and J minigenes, prior to the exposure of an antibody to an antigen. Rearranged “V-regions” describe the genetic element which results from the rearrangement event between V, D, and J (for heavy chains) or V and J minigenes (for light chains). An “antibody V-region” refers to the polypeptide region encoded by the V, D, and J element. An antibody V-region is encoded by rearranged V, D, and J minigenes. The term “V(D)J Recombination” refers to any process wherein a V, D, or J minigene is recombined to another V, D, or J minigene. A V-region may be part of a full length antibody, an Fab, a scFv, or any other derivative of an antibody (see definition of antibody below). A “germline V-region” refers to the sequence of rearranged V, D, and J minigenes prior to significant mutagenic events. A germline V-region may have random insertions or deletions at the junctions of the V-D, D-J, or V-J minigenes. A non-germline V-region (or a “mature” V-region) will differ from the germline sequences of the minigenes by usually more than 5 residues (not including the junctional deletions or insertions).

Homology: As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Generally, the term “homology” implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present invention, the term homology encompasses both to identity and similarity.

In some embodiments, polymeric molecules are considered to be “homologous” to one another 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 are similar (conservative substitutions). The term “homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

Human antibody: The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art (e.g., recombinant expression in cultures cells, or expression in transgenic animals). Thus, the term human antibody also encompasses an antibody having an amino acid sequence corresponding to an antibody originally produced by a human (or an engineered variant or derivative thereof) but expressed in a non-human system (e.g., produced by chemical synthesis; recombinantly expressed in microbial, mammal, or insect cells; or expressed in an animal subject). Accordingly, an antibody obtained from a human subject or from human cells (e.g., hybridoma or cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal, e.g., mice, is considered a human antibody. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

Humanized antibody: The term “humanized antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and capability (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 a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, and/or affinity, and/or capability.

The humanized antibody can be further modified by the substitution of additional residues either in the FW regions and/or within the replaced non-human residues to refine and optimize antibody specificity, and/or affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FW regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. Nos. 5,225,539 or 5,639,641.

Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polypeptide molecules or polynucleotide molecules (e.g. DNA molecules and/or RNA molecules). The term “identical” without any additional qualifiers, e.g., protein A is identical to protein B, implies the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., “70% identical,” is equivalent to describing them as having, e.g., “70% sequence identity.”

Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence 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 corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (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, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

In certain aspects, the percentage identity (% ID) or 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 nucleobases) scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven 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., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

Immunoconjugate: The term “immunoconjugate” as used herein refers to a compound comprising a binding molecule (e.g., an anti-FIXa, anti-FXz, or biospecific anti-FIXa/anti-FXz) and one or more moieties, e.g., therapeutic or diagnostic moieties, chemically conjugated to the binding molecule. In general an immunoconjugate is defined by a generic formula: A-(L-M)n wherein A is a binding molecule (e.g., an antibody), L is an optional linker, and M is a heterologous moiety which can be for example a therapeutic agent, a detectable label, etc., and n is an integer. Immunoconjugates can also be defined by the generic formula in reverse order. In some aspects, 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 conjugates molecules. The term “immunoconjugate” as used in the present disclosure also includes genetic fusions.

Isolated: As used herein, the term “isolated” refers to a substance or entity (e.g., polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature) that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances (e.g., nucleotide sequence or protein sequence) can have varying levels of purity in reference to the substances from which they have been associated.

Isolated substances and/or entities can be separated from 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 of the other components with which they were initially associated.

In some embodiments, isolated agents are more 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 more than about 99% pure.

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 was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing 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 of the compound of the present disclosure, or salt thereof.

A polynucleotide (e.g., an antibody), vector, polypeptide, cell, or any composition disclosed herein which is “isolated” is a polynucleotide (e.g., an antibody), vector, polypeptide, cell, or composition which is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a polynucleotide, vector, polypeptide, or composition which is isolated is substantially pure.

Mimic 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 according to different methods known in the art. One such method is a chromogenic assay as described in the Examples section of this specification. In one aspect, a binding molecule disclosed herein (e.g., a bispecific antibody) is said to “mimic FVIIIa activity” is there is an observed rate of FXa substrate cleavage at least three standard deviations above the mean basal rate in the absence of the added binding molecule (e.g., a bispecific antibody). Another exemplary method is an activated Partial Thromboplastin Time (aPTT) assay. The term ‘Activated Partial Thromboplastin Time (APTT)’ derives from the original form of the test (devised in 1953) in which only the phospholipid concentration of the test was controlled (as opposed to the phospholipid and the surface activator concentrations) and the name ‘partial thromboplastin’ was applied at the time to phospholipid preparations which accelerated clotting but did not correct the prolonged clotting times of haemophilic plasma. Essentially the term ‘partial’ means phospholipid is present but no Tissue Factor. The aPIT is also known as: Kaolin Cephalin Clotting Time (KCCT) or Partial Thromboplastin Time with Kaolin (PTTK). Other useful methods include a one stage (OS) clotting assay which is modified from the traditional aPIT assay described above. The one stage clotting assay uses FVIII-deficient plasma and a diluted test sample, and can be quantitative for FVIII activity. See Example 4. In contrast, the aPTT assay uses sample plasma with the aPIT reagent and calcium and reports the clotting time.

Monoclonal Antibody: A “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain variable fragments (scFv), fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals (e.g., expression of a human antibody in a transgenic mouse).

Mutation: In the content of the present disclosure, the terms “mutation” and “amino acid substitution” as defined above (sometimes referred simply as a “substitution”) are considered interchangeable. In some aspects, the term mutation refers to the deletion, insertion, or substitution of any nucleotide, by chemical, enzymatic, or any other means, in a nucleic acid encoding an antibody germline gene such that the amino acid sequence of the resulting polypeptide is altered at one or more amino acid residues. In some aspects, a mutation in a nucleic acid sequence disclosed herein results in an amino acid substitution. In other aspects, the mutation of a codon in a nucleic acid sequence disclosed herein wherein the resulting codon is a synonymous codon does not result in an amino acid substitution. Accordingly, in some aspects, the nucleic acid sequences 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 the stability, half life, or other desirable property of an mRNA or a DNA encoding a binding molecule disclosed herein, wherein such mRNA or DNA is administered to a subject in need thereof.

Patient: As used herein, “patient” refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.

Pharmaceutical composition: The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of the active ingredient (e.g., a binding molecule disclosed herein, such as an antibody) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In general, approval by a regulatory agency of the Federal or state governments (or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia) for use in animals, and more particularly in humans implies that those compounds, materials, compositions, and/or dosage forms are pharmaceutically acceptable. Compounds, materials, compositions, and/or dosage forms that are generally acceptable as safe for therapeutically purposes are “therapeutically acceptable.” Compounds, materials, compositions, and/or dosage forms that are generally acceptable as safe for diagnostic purposes are “diagnostically acceptable.”

Pharmaceutically acceptable excipients: The phrase “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients can include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, 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.

Excipients that are generally accepted as safe for therapeutic purposes are “therapeutically acceptable excipients.” Excipients that are generally accepted as safe for diagnostic purposes are “diagnostically acceptable excipients.”

Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral 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, benzene sulfonic 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, 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 salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17^thed., 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 incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate: The term “pharmaceutically acceptable solvate,” as used herein, means a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), 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 referred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.

Polynucleotide: The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one unnatural nucleobase. In some aspects, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some aspects, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and U in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

The skilled artisan will appreciate that the T bases in the codon maps disclosed herein are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs. For example, a codon-nucleotide sequence disclosed herein in DNA form, e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA. In this respect, both codon-optimized DNA sequences (comprising T) and their corresponding RNA sequences (comprising U) are considered codon-optimized nucleotide sequence of the present invention. A skilled artisan would also understand that equivalent codon-maps can be generated by replaced one or more bases with non-natural bases. Thus, e.g., a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn would correspond to a ΨΨC codon (RNA map in which U has been replaced with pseudouridine).

Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the Ni and C6-NH₂, respectively, of adenosine and between the C2-oxy, N3 and C4-NH₂, of cytidine and the C2-NH₂, N′-H and C6-oxy, respectively, of guanosine. 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 modification results in a nucleoside base which will no longer effectively form a standard base pair with cytosine. However, modification of 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 nucleotide which will not effectively base pair with guanosine but will form a base pair with isoguanosine (U.S. Pat. No. 5,681,702 to Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the method described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2′-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467, and references cited therein; and isoguanine nucleotides can be prepared using the method described by Switzer et al., 1993, supra, and Mantsch et al. (1993) Biochem. 14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 to Collins et al. Other nonnatural base pairs can be synthesized by the method described in Piccirilli et al. (1990) Nature 343:33-37, for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotide units which form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al., supra.

Polypeptide: The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, omithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly 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 an artificial chemical analogue of a corresponding naturally occurring amino acid. In some embodiments, a “peptide” can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Preventing: As used herein, the term “preventing” refers to partially or completely delaying onset of an disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

Prophylactic: As used herein, “prophylactic” refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a bleeding episode, e.g., hemophilia.

Prophylaxis: As used herein, a “prophylaxis” refers to a measure taken to maintain health and prevent or delay the onset of a bleeding episode, or to prevent or delay symptoms associated with a disease or condition.

Recombinant: A “recombinant” polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. The polypeptides disclosed herein can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized.

Similarity: As used herein, the term “similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.

Subject: By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; 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; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. In other embodiments, a subject is a human patient. In a particular embodiment, a subject is a human patient or cells thereof whether in vivo, in vitro or ex vivo, amenable to the methods described herein.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differences between doses, the term means plus/minus 2%.

Substantially simultaneous: As used herein and as it relates to plurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) can be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Therapeutic Agent: The terms “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 elicits a desired biological and/or pharmacological effect. For example, in some embodiments, a bispecific antibody disclosed herein can be a therapeutic agent. In some embodiments, an agent is another molecule (e.g., a clotting factor, cofactor, etc.) which is co-administered as part of a combination therapy with at least one of the antibodies disclosed herein.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

Therapeutically effective outcome: As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

Treat. treatment, therapy: As used herein, the terms “treat” or “treatment” or “therapy” or grammatical variants thereof refer to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a bleeding disease, disorder, or condition, e.g., hemophilia. For example, “treating” a bleeding disorder can refer to prevent bleeding, decrease the frequency and/or severity of bleeding episodes, etc. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

Vector: A “vector” is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. In some aspects, the administration and/or expression of a nucleic acid (DNA or RNA, such as an mRNA) encoding a binding molecule disclosed herein can take place in vitro (e.g., during recombinant protein production), whereas in other cases it can take place in vivo (e.g., administration of an mRNA to a subject), or ex vivo (e.g., DNA or RNA introduced into an autologous or heterologous cells for administration to a subject in need thereof). Also included are vectors that provide more than one of the functions as described.

II. Anti-FIX and Anti-FX Binding Molecules

The present disclosure provides antibodies that bind to factor IX and to factor X, as well as antigen binding portions thereof. These antibodies are capable of preferential binding to specific functional forms of these clotting factors. For example, in some embodiments, the disclosed antibodies against FIX preferentially bind to activated FIX (FIXa), e.g., free FIXa or FIXa covalently linked to a substrate mimic in the active site (FXa+EGR-CMK). In other embodiments, the disclosed antibodies preferentially bind to FIXa-SM over free FIXa or FIX zymogen. In yet other embodiments, the disclosed antibodies preferentially bind to free FIXa over FIXa-SM or FIX zymogen. In contrast, in some embodiments, the disclosed antibodies against FX preferentially bind to FX zymogen (FXz) over activated FX (FXa). This preferential binding is critical to generate bispecific molecules comprising an anti-FIXa moiety and an anti-FXz moiety that can specifically and simultaneously bind to FIXa and FXz. Factor VIII is a cofactor for FIXa which, in the presence of Ca²⁺ and phospholipids forms a complex with FX that converts FX to the activated FXa. Therefore, the formation of an antibody-mediated complex between FIXa and FXz mimics the effect of FVIIIa.

In still other embodiments, some disclosed antibodies preferentially bind to FIX zymogen over free FIXa or FIXa-SM (“anti-FIXz antibody”). Therefore, the anti-FIXz antibody can be used to generate a bispecific molecule comprising the anti-FIXz antibody and an anti-FX antibody (e.g., an anti-FXz antibody or an anti-FXa antibody).

In certain embodiments, some disclosed anti-FX antibodies preferentially bind to FXa over FXz (“anti-FXa antibody”). The anti-FXa antibody can be used to generate a bispecific molecule comprising the anti-FXa antibody and an anti-FIX antibody (e.g., an anti-FIXa antibody or an anti-FIXz antibody).

The formation of antibody-mediated complex between FIX and FX can therefore be used to bypass FVIII replacement therapy, in particular, in subjects having developed antibodies against FVIII or at risk of developing antibodies against FVIII.

The present disclosure also provides a bispecific binding molecule that binds 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 anti-FIXa antibody or anti-FIXz antibody and any one of anti-FXa antibody or 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 different binding affinity (e.g., K_D). In other embodiments, the bispecific binding molecule binds with a K_Dless than 1 μM to each of FIXz, FIXa, and FXz (e.g., 8 nM, 2 nM, or 20 nM, respectively).

(a) Anti-FIXa Binding Molecules

The present disclosure provides anti-FIX binding molecules, e.g., anti-FIX antibodies or molecules comprising antigen binding portions thereof, that preferentially bind activated FIX (FIXa) over FIX zymogen.

Factor IX (FIX) is synthesized by liver hepatocytes as a pre-prozymogen that requires extensive posttranslational modification. The pre-prozymogen contains a pre-peptide (hydrophobic signal peptide) at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum. Once inside the ER, this signal peptide is cleaved by a signal peptidase. A pro-peptide functions as a recognition element for a vitamin K-dependent carboxylase (γ-glutamyl carboxylase) which modifies 12 glutamic acid residues to gamma-carboxyglutamyl (Gla) residues. These residues are required for the association with the anionic phospholipid surface through Ca2+-dependent binding.

The amino acid sequence of pre-pro-FIX zymogen is provided below (the signal sequence is underlined (1-28); the propeptide sequence (29-46) is bolded):

(SEQ ID NO: 764)

MQRVNMIMAESPGLITICLLGYLLSAEC
TVFLDHE

NANKILNRPKRYNSGKLEEFVQGNLERECMEEKCS

FEEAREVFENTERTTEFWKQYVDGDQCESNPCLNG

GSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGR

CEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVP

FPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETI

LDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLN

GKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAG

EHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHD

IALLELDEPLVLNSYVTPICIADKEYTNIFLKFGS

GYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRS

TKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVE

GTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIK

EKTKLT

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

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

It is known, however, that activity of free FIXa is similar to the activity of FIX zymogen. The complex formation with cofactor FVIIIa represents a critical second phase of activation, after which the intrinsic Xase complex reaches an approximately 200,000-fold enhanced activity that is strictly specific toward the physiological substrate FX and restricted to the surface of activated platelets (van Dieijen et al., J Biol Chem. 1981 Apr. 10; 256(7):3433-42). This macromolecular activation is assisted by low-molecular-weight agonists, including Ca2+(Mathur et al., Biol. Chem., 272 (1997), pp. 23418-23426). Several lines of evidence indicate that Ca2+ binding is accompanied by a conformational rearrangement (Bajaj et al., Proc. Natl. Acad. Sci. USA, 89 (1992), pp. 152-156, Enfield and Thompson, Blood, 64 (1984), pp. 821-831). This superactive FIXa in a tenase complex can be mimicked by covalently attaching a substrate mimic (e.g., Glu-Gly-Arg-chloromethyl ketone (EGR-CMK)) to the active site of FIXa (FXa+EGR-CMK, also called as FIXa-SM). Therefore, FIXa-SM can be used as an important tool to distinguish antibodies or antigen binding portion thereof that preferentially bind to superactive FIXa (in tenase complex) compared to free FIXa.

Tripeptide chloromethylketones are generally accepted in the field as substrate mimics, with the tripeptide sequence representing the native substrate sequence that is cleaved by a particular enzyme and the CMK part allows this tripeptide to be irreversibly locked into the active site since it reacts with the active site serine. As a consequence, if an enzyme is bound to a substrate-mimic, this should represent the substrate bound form, i.e. the true 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.

The term “FIX zymogen” can be used interchangeably herein with “FIXz,” “FIX precursor,” “unactivated FIX,” “non-activated FIX,” or “non-activated FIX precursor.” In one embodiment, FIX zymogen (FIXz) includes non-activated FIX precursor in which the activation peptide (e.g., 35 activation peptide that are represented as amino acids 146 to 180 of SEQ ID NO: 764 (mature numbering) is not cleaved from the precursor. FIX zymogen can include any naturally-occurring or engineered variants. A non-limiting example of FIX zymogen is shown in SEQ ID NO: 764. In another embodiment, FIX zymogen is non-activatable FIX (FIXn), which is engineered to be non-active in the presence of Factor XIa, activated plasma thromboplastin antecedent. An example of non-activatable FIX can be FIX carrying an arginine to alanine mutation at position 180 (mature numbering) preventing its activation and maintaining Factor IX in the zymogen form (FIXz). FIX zymogen can optionally contain a signal peptide and/or propeptide.

The term “activated FIX” can be used interchangeably herein with “FIXa”. In one embodiment, activated FIX is wild-type, naturally occurring FIXa (also referred to herein as “wild-type FIXa”). In another embodiment, FIXa comprises non-naturally occurring FIXa, e.g., FIXa conformational variant. For example, FIXa can be a FIXa-SM, which is designed to have the same conformation as wild-type, naturally occurring FIXa bound to its substrate, FX. In a particular embodiment, FIXa-SM is activated FIX with a substrate mimic covalently bound to the active site, which is intended to mimic the most active conformation of activated FIX.

FIX zymogen and FIXa can include FIX variants. In one embodiment, FIX variants have been cloned, as described in U.S. Pat. Nos. 4,770,999 and 7,700,734, and cDNA coding for human Factor IX has been isolated, characterized, and cloned into expression vectors (see, for example, 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 particular variant of FIX, the R338L FIX (Padua) variant, characterized by Simioni et al, 2009, comprises a gain-of-function mutation, which correlates with a nearly 8-fold increase in the activity of the Padua variant relative to native FIX. FIX variants can also include any FIX polypeptide having one or more conservative amino acid substitutions, which do not affect the FIX activity of the FIX polypeptide.

The present disclosure therefore 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 antigen binding portion thereof preferentially binds to FIXa in the presence of FIXa and FIX zymogen (“anti-FIXa antibody or antigen binding portion thereof”).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz. In one embodiment the binding affinity is expressed as K_D.

The present disclosure also provides an isolated anti-FIXa antibody, or antigen binding portion thereof, which binds to FIXa with a binding affinity higher than a binding affinity of the anti-FIXa antibody or antigen binding portion thereof to FIXz. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to FIXa with a K_Dof 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 Bio-Layer Interferometry (BLI) assay. In other embodiments, the anti-FIXa antibody, or antigen binding portion thereof, binds to FIXa with a K_Dof 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. In yet other embodiments, the anti-FIXa antibody, or antigen binding portion thereof, binds to FIXa with a K_Dof 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.

In some aspects, 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 aspects, 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 aspects, 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.

In further aspects, the anti-FIXa antibody or antigen binding portion thereof can further be classified into three classes:

Class I: anti-FIXa antibodies or antigen binding portion thereof that preferentially binds to FIXa-SM over free FIXa or FIXz (FIG. 3A antibodies);

Class II: anti-FIXa antibodies or antigen binding portion thereof that preferentially binds to free FIXa over FIXa-SM or FIXz (FIG. 3B antibodies); and

Class III: anti-FIXa antibodies or antigen binding portion thereof that binds to free FIXa and FIXa-SM with near equivalency, but do not appreciably associate with FIXz (FIG. 3C antibodies).

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.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR3 comprises

- (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3A, or
- (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3A except for 1, 2, or 3 amino acid substitutions.

In other aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR3 comprises

- (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3B, or
- (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3B except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR3 comprises

- (i) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3C, or
- (ii) a VH CDR3 sequence identical to a VH CDR3 sequence selected from the group consisting of VH CDR3 sequences in FIG. 3C except for 1, 2, or 3 amino acid substitutions.

In some aspects, the amino acid substitutions are conservative amino acid substitutions. In other aspects, the amino acid substitutions are back mutation.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR3 sequence comprises the amino acid sequence ARDX₁X₂X₃X₄X₅X₆YYX₇MDV (SEQ ID NO:753), wherein X₁is V or G, X₂is G or V, X₃is G or R, X₄is Y or V, X₅is A or S, X₆is G or D, X₇is G or none.

A person of skill in the art would understand that when a position is described as “none” or “absent” in a consensus sequence, such absence does not indicate a breakage in the polypeptide chain. These terms merely reflects the occurrence of insertions and deletions in the amino acid chains as 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 lacking the insertion will have an “absent” (“none”) amino acid at that position.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and a VH CDR3, wherein the VH CDR3 sequence comprises an amino acid sequence selected from ARDVGGYAGYYGMDV (SEQ ID NO: 905; VH CDR 3 for 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 aspects, the VH CDR3 sequences disclosed herein can comprise 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1 sequence comprises

- (i) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3A, or
- (ii) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3A except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1 sequence comprises

- (i) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3B, or
- (ii) a VH CDR1 sequence identical to a sequence selected from the group consisting of the VH CDR1 sequences disclosed in FIG. 3B except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR1 sequence comprises

- (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 except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR2 sequence comprises

- (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 except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR2 sequence comprises

- (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 except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH CDR1, a VH CDR2, and VH CDR3, wherein the VH CDR2 sequence comprises

- (i) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3C, or
- (ii) a VH CDR2 sequence identical to a sequence selected from the group consisting of the VH CDR2 sequences disclosed in FIG. 3C except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 sequence comprises

- (i) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3A, or
- (ii) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3A except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 sequence comprises

- (i) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B, or
- (ii) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3B except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 sequence comprises

- (i) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3C, or
- (ii) a VL CDR1 sequence identical to a sequence selected from the group consisting of the VL CDR1 sequences disclosed in FIG. 3C except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR2 sequence comprises

- (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in FIG. 3A, or
- (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in FIG. 3A except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR2 sequence comprises

- (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in FIG. 3B, or
- (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2 sequences in FIG. 3B except for 1, 2, or 3 amino acid substitutions.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR2 sequence comprises

- (i) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2s sequences in FIG. 3C, or
- (ii) a VL CDR2 sequence identical to a sequence selected from the group consisting of the VL CDR2s sequences in FIG. 3C except for 1, 2, or 3 amino acid substitutions.

In some aspects, 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 comprises

- (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3A, or
- (ii) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3A except for 1, 2, or 3 amino acid substitutions.

In some aspects, 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 comprises

- (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3B, or
- (ii) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3B except for 1, 2, or 3 amino acid substitutions.

In some aspects, 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 comprises

- (i) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3C, or
- (ii) a VL CDR3 sequence identical to a sequence selected from the group consisting of the VL CDR3 sequences disclosed in FIG. 3C except for 1, 2, or 3 amino acid substitutions.

The present disclosure also provides an isolated antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 comprise the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of an anti-FIXa antibody selected from the group consisting of the antibodies in FIG. 3A: 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, BIIB-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.

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

In other embodiments, the disclosure provides an isolated antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 comprise the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of an anti-FIXa antibody selected from the group consisting of the antibodies in FIG. 3C: 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 some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NOs: 800-844, SEQ ID NOs: 845-889, and SEQ ID NOs: 890-934, respectively (VH CDRs for Class I antibodies), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 935-979, SEQ ID NOs: 980-1024, and SEQ ID NO: 1025-1069, respectively (VL CDRs for Class I antibodies).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NOs: 1070-1073, SEQ ID NOs: 1074-1077, and SEQ ID NOs: 1078-1081, respectively (VH CDRs for Class II antibodies), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1082-1085, SEQ ID NOs: 1086-1089, and SEQ ID NO: 1090-1093, respectively (VL CDRs for Class II antibodies).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NOs: 1094-1135, SEQ ID NOs: 1136-1177, and SEQ ID NOs: 1178-1219, respectively (VH CDRs for Class III antibodies), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NOs: 1220-1261, SEQ ID NOs: 1262-1303, and SEQ ID NO: 1304-1345, respectively (VL CDRs for Class III antibodies).

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

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

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

In other aspects, the anti-FIXa antibody, or antigen binding portion thereof, cross-competes with antibodies BIIB-9-484, BIIB-9-1335, and BIIB-9-1336 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 and VL CDR1, VL CDR2, and VL CDR3, wherein the VH CDR3 comprises ARDVGGYAGYYGMDV (SEQ ID NO: 905, BIIB-9-484 VH CDR3), VH CDR2 comprises SISSX₁X₂SYIYYAX₃SVKG (SEQ ID NO: 754), wherein X₁is S, G, or any conservative substitution, X₂is S, E, or any conservative substitution, and X₃is D, E, or any conservative substitution, VH CDR1 comprises FTFX₄SYX₅MX₆(SEQ ID NO: 755), wherein X₄is S, G, or any conservative substitution, X₅is D, S, or any conservative substitution, and X₆is H, N, or any conservative substitution. The anti-FIXa antibody, or antigen binding portion thereof, can comprises 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 CDRs)

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises

(a1) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 809, SEQ ID NO: 854, and SEQ ID NO: 899, respectively (VH CDRs for BIIB-9-440 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 944, SEQ ID NO: 989, and SEQ ID NO: 1034, respectively (VL CDRs for BIIB-9-440 antibody);

(a2) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 1102, SEQ ID NO: 1144, and SEQ ID NO: 1186, respectively (VH CDRs for BIIB-9-882 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 1228, SEQ ID NO: 1270, and SEQ ID NO: 1312, respectively (VL CDRs for BIIB-9-882 antibody);

(a3) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 811, SEQ ID NO: 856, and SEQ ID NO: 901, respectively (VH CDRs for BIIB-9-460 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 946, SEQ ID NO: 991, and SEQ ID NO: 1036, respectively (VL CDRs for BIIB-9-460 antibody); or,

(a4) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 1108, SEQ ID NO: 1150, and SEQ ID NO: 1192, respectively (VH CDRs for BIIB-9-433 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 1234, SEQ ID NO: 1276, and SEQ ID NO: 1318, respectively (VL CDRs for BIIB-9-433 antibody);

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

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises

(i) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 843, SEQ ID NO: 888, and SEQ ID NO: 933, respectively (VH CDRs for BIIB-9-1335 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively, respectively (VL CDRs for BIIB-9-1335 antibody); or,

(ii) VH CDR1, VH CDR2, and VH CDR3 sequences comprising SEQ ID NO: 844, SEQ ID NO: 889, and SEQ ID NO: 934, respectively (VH CDRs for BIIB-9-1336 antibody), and/or VL CDR1, VL CDR2, and VL CDR3 sequences comprising SEQ ID NO: 950, SEQ ID NO: 995, and SEQ ID NO: 1040, respectively (VL CDRs for BIIB-9-1336 antibody).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH, wherein the VH comprises an amino acid sequence which 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 to 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 (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 I antibodies; SEQ ID NOs: 91, 93, 95, and 97 for Class II antibodies; and 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 for Class III antibodies).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VL, wherein the VL comprises an amino acid sequence which 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 an the 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 (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 I antibodies; SEQ ID NOs: 277, 279, 281, and 283 for Class II 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 for Class III antibodies).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH and a VL, wherein

(i) the VH comprises an amino acid sequence which 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 to 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 (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 I antibodies; SEQ ID NOs: 91, 93, 95, and 97 for Class II antibodies; and 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 for Class III antibodies); and,

(ii) the VL comprises an amino acid sequence which 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 to 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 (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 I antibodies; SEQ ID NOs: 277, 279, 281, and 283 for Class II 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 for Class III antibodies).

In certain aspects, an 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 V, D, or J germline sequences and/or the VL sequence of the anti-FIXa antibody can be derived from any one of kappa or lambda germline sequence.

In other aspects, provided herein are isolated FIXa antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region that is the 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 particular 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.

Antibodies described herein include those comprising a heavy chain variable region that is the product of or derived from one of the above-listed human germline VH genes and also comprising a light chain variable region that is the product of or derived from one of the above-listed human germline VK genes, as shown in the Figures.

As used herein, a human antibody comprises heavy and light chain variable regions that are “the product of or “derived from” a particular germline sequence if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is “the product of or “derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody. A human antibody that is “the product of” or “derived from” a particular human germline immunoglobulin sequence can contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation. However, a selected human antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as being human when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a human antibody can be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, the human antibody can display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, comprises a VH and a VL, wherein

(a1) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 31 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221 (VH and VL of BIIB-9-484, respectively);

(a2) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID 209 (VH and VL of BIIB-9-440, respectively);

(a3) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 301 (VH and VL of BIIB-9-882, respectively);

(a4) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 23 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 213 (VH and VL of BIIB-9-460, respectively);

(a5) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 127 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 313 (VH and VL of BIIB-9-433, respectively);

(a6) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 45 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 235 (VH and VL of BIIB-9-619, respectively);

(a7) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 87 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221 (VH and VL of BIIB-9-1335, respectively); or

(a8) VH comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 89 and VL comprises an amino acid sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 221 (VH and VL of BIIB-9-1336, respectively).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to the same epitope as BIIB-9-1336. In other aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope overlapping BIIB-9-1336's epitope. In some embodiments, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope region comprising at least one amino acid located between chymotrypsinogen numbering positions 91 and 101, 125 and 128, 165 and 179, or 232 and 241 in the sequence of the heavy chain of FIXa (corresponding to positions 76 to 88, 112 to 115, 153 to 167, and 222 to 231, respectively in SEQ ID NO: 758).

As used herein, the phrase “chymotrypsinogen numbering amino acid residue” and grammatical variants thereof refers to the description of certain amino acids in FIX by homology to the serine protease chymotrypsinogen. In the present disclosure the chymotrypsinogen numbering within the serine protease domain was used according to Hopfner et al. (EMBO J. 1997; 16:6626-35). For the present disclosure the chymotrypsinogen numbering is only used when explicitly indicated herein. The correspondence between the disclosed chymotripsinogen numbering amino acid residues and the amino acid position is SEQ ID NO: 758 is provided in the table below:

Correspondence between FIXa chymotrypsinogen

numbering and SEQ ID NO: 758 positions

FIXa chymotrypsinogen numbering
SEQ ID NO: 758

amino acid residue
amino acid residue

H91
H76

H92
H77

N93
N78

H101
H88

D125
D112

K126
K113

E127
E114

Y128
Y115

R165
R153

Y177
Y165

N178
N166

N179
N167

S232
S222

R233
R223

Y234
Y224

V235
V225

N236
N226

W237
W227

E240
E230

K241
K231

N100
N87

K132
K121

Y137
Y126

R170
R158

T172
T160

F174
F162

T175
T163

H185
H174

E202
E192

G205
G195

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprising at least one of chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acid residues selected from the group consisting chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprises chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope consisting of chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprising chymotrypsinogen numbering amino acid residues N93, R165, N178, and R233 in the sequence of the heavy chain of FIXa (corresponding to positions N78, R153, N166 and R223, respectively in SEQ ID NO: 758).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope which does not comprise at least one of chymotrypsinogen numbering amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 in the sequence of the heavy chain of FIXa (corresponding to positions N87, K121, Y126, R158, T160, F162, T163, H174, E192 and G195, respectively in SEQ ID NO: 758). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope which does not comprise chymotrypsinogen numbering amino acid residues N100, K132, Y137, R170, T172, F174, T175, H185, E202, and G205 in the sequence of the heavy chain of FIXa (corresponding to positions N87, K121, Y126, R158, T160, F162, T163, H174, E192 and G195, respectively in SEQ ID NO: 758).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprising at least one amino acid residue in the light chain of FIXa (SEQ ID NO:756). In some aspects, the epitope in the light chain of FIXa (SEQ ID NO:756) that the anti-FIXa antibody, or antigen binding portion thereof binds to is K100.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope comprising chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758) and amino acid residue K100 of the sequence of the light chain of FIXa (SEQ ID NO:756). In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope consisting of chymotrypsinogen numbering amino acid residues H91, H92, N93, H101, D125, K126, E127, Y128, R165, Y177, N178, N179, S232, R233, Y234, V235, N236, W237, E240, and K241 in the sequence of the heavy chain of FIXa (corresponding to positions H76, H77, N78, H88, D112, K113, E114, Y115, R153, Y165, N166, N167, S222, R223, Y224, V225, N226, W227, E230, and K231, respectively in SEQ ID NO: 758) and amino acid residue K100 of the sequence of the light chain of FIXa (SEQ ID NO:756).

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, binds to an epitope that overlaps the binding site of FVIIIa to FIXa. In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, cross-competes with FVIIIa for binding to FIXa. In some aspects, the anti-FXa antibody, or antigen binding portion thereof, blocks binding of FVIIIa to FIXa.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 sequences, wherein the VH CDR1 sequence comprises a VH CDR1 sequence selected from the group consisting of VH CDR1 sequences disclosed in TABLE 7 or a VH CDR1 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 sequences, wherein the VH CDR2 sequence comprises a VH CDR2 sequence selected from the group consisting of VH CDR2 sequences disclosed in TABLE 7 or a VH CDR2 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VH CDR1, CDR2, and CDR3 sequences, wherein the VH CDR3 sequence comprises a VH CDR3 sequence selected from the group consisting of VH CDR3 sequence disclosed in TABLE 7 or a VH CDR3 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VL CDR1, CDR2, and CDR3 sequences, wherein the VL CDR1 sequence comprises a VL CDR1 sequence selected from the group consisting of VL CDR1 sequences disclosed in TABLE 7 or a VL CDR1 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VL CDR1, CDR2, and CDR3 sequences, wherein the VL CDR2 sequence comprises a VL CDR2 sequence selected from the group consisting of VL CDR2 sequences disclosed in TABLE 7 or a VL CDR2 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the anti-FIXa antibody, or antigen binding portion thereof, which specifically binds to FIXa, comprises VL CDR1, CDR2, and CDR3 sequences, wherein the VL CDR3 sequence comprises a VL CDR3 sequence selected from the group consisting of VL CDR3 sequences disclosed in TABLE 7 or a VL CDR3 sequence disclosed in TABLE 7 with one or two mutations.

In some aspects, the isolated anti-FIXa antibody, or antigen binding portion thereof, comprises VH CDR1, CDR2, and CDR3 sequences, and VL CDR1, CDR2, and CDR3 sequences, wherein the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences comprise VH CDR1, CDR2, and CDR3 sequences and VL CDR1, CDR2, and CDR3 sequences disclosed in TABLE 7, respectively.