AU2024291721A1

AU2024291721A1 - ANTI-FACTOR XII/XIIa ANTIBODIES AND USES THEREOF

Info

Publication number: AU2024291721A1
Application number: AU2024291721A
Authority: AU
Inventors: Dan CHALOTHORN; Kehdih LAI; Lyndon MITNAUL; Lori C. MORTON; George D. Yancopoulos
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2023-07-19
Filing date: 2024-07-19
Publication date: 2026-01-22
Also published as: IL325613A; WO2025019789A1

Abstract

The present invention provides monoclonal antibodies that bind to the Factor XII (FXII) protein, and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to FXII and to the activated form of FXII (FXIIa). In some embodiments, the antibodies of the invention are useful for inhibiting or neutralizing FXII activity, thus providing a means of treating or preventing a disease, disorder or condition associated with thrombosis in humans.

Description

ANTI-FACTOR Xll/Xlla ANTIBODIES AND USES THEREOF

RELATED APPLICATIONS

[001] The instant application claims priority to U.S. Provisional Application No. 63/514,392, filed July 19, 2023, the entire contents of which are expressly incorporated by reference herein.

SEQUENCE LISTING

[002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on July 18, 2024, is named “118003-11720. xml” and is 56,608 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

FIELD

[003] The present disclosure is related to antibodies and antigen-binding fragments of antibodies that specifically bind to coagulation factor XII, and therapeutic and diagnostic methods of using those antibodies.

BACKGROUND

[004] Coagulation factors, along with platelets, are blood components relevant in the process of haemostasis during vessel injury. It is well-established that these components can be drivers of thrombosis when imbalances occur in regulation (i.e., production and/or activity). Thrombotic diseases were believed to primarily arise from aberrant activation in the extrinsic pathway (via tissue factor), but more recently, preclinical studies with F12 deficient mice and molecules that target activated FXII (FXIIa) suggested that the intrinsic pathway (also known as the contact pathway) of coagulation is also involved (Renne et al 2005, J.

Exp. Med. 202: 271-81; Larsson et al 2014, Sci. Transl. Med. 6: 222ra17). FXIIa is central to the contact pathway and can drive coagulation via cleavage of FXI or can drive inflammation via cleavage of plasma prekallikrein. Thus, inhibition of FXII activity may lead to reduced thrombotic coagulation and inflammation associated with contact pathway activation (Schousboe 2007, Biochem. Pharmacol. 75: 1007-13; Danese et al 2016, Semin. Thromb. Hemostat. 42: 682-8; Weitz 2016, Thromb. Res. 141 Suppl. 2: S40-5). The anti-coagulants used presently in the art, e.g., heparin, are effective anti-thrombotics yet they also increase the risk for bleeding. Thus, it is desirable to develop anti-coagulants that prevent thrombosis without increasing the risk of bleeding. [005] Monoclonal antibodies to FXII are known in the art and have been described, for example, in US Patent/Publication Nos. 4963657, 5500349, 8119137, 8715672, 9856326, 9856325, 9518127, 20130095108, 20140072572, 20140072600, and in W02006066878, W02013014092, EP1830924B1, EP27345522A1, and EP2623110A1.

[006] Fully human antibodies that specifically bind to (activated) FXII protein with high affinity and prevent thrombosis without increasing the risk of bleeding could be important in the prevention and treatment of various FXII-associated diseases (e.g., thrombosis, embolism, edema).

BRIEF SUMMARY

[007] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind the activated form of coagulation Factor XII protein (FXIIa). In certain embodiments, the antibodies also bind the zymogen form of Factor XII (FXII). In certain embodiments, the antibodies are fully human antibodies that bind to FXII and also to FXIIa (“dual FXII/FXIIa binders”) with high affinity. The antibodies of the present disclosure are useful, inter alia, for inhibiting or neutralizing the activity of FXII protein. In certain embodiments, the antibodies are useful in preventing, treating or ameliorating at least one symptom or indication of a FXII-associated disease or disorder in a subject. In certain embodiments, the antibodies may be administered prophylactically or therapeutically to a subject having or at risk of having a FXII-associated disease or disorder. In certain preferred embodiments. In specific embodiments, the antibodies prevent thrombosis without increasing bleeding risk in a subject. Such antibodies can be used as anti-coagulation therapy without increasing risk of bleeding when administered to a subject in need thereof, along with less frequent dosing in a subject with a FXII-associated disease or disorder.

[008] The antibodies of the disclosure can be full-length (for example, an lgG1 or lgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab’)2 or scFv fragment), and may be modified to affect functionality, e.g., to increase persistence in the host or to eliminate residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925- 1933). In certain embodiments, the antibodies may be bispecific.

[009] In a first aspect, the present disclosure provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that bind specifically to Factor XI I (FXII) and/or activated Factor XII (FXIIa). In some embodiments, the antibodies are fully human monoclonal antibodies. In certain embodiments, the antibodies are “dual binders,” being able to bind FXII and FXIIa.

[010] Exemplary anti-FXI l/FXIIa antibodies of the present disclosure are listed in Tables 1 and 2 herein. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDRs) (HCDR1 , HCDR2 and HCDR3), and light chain complementarity determining regions (LCDRs) (LCDR1 , LCDR2 and LCDR3) of exemplary antibodies. Table 2 sets forth the nucleic acid sequence identifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 of the exemplary antibodies. [011] The present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1 , or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[012] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1 , or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[013] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCVR/LCVR amino acid sequence pair contained within any of the exemplary anti-FXI l/FXI la antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from one of SEQ ID NOs: 2/22 (e.g., REGN9533) or 22/30 (e.g., REGN9534).

[014] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence listed in Table 1 having no more than twelve amino acid substitutions, and/or said LCVR comprising an amino acid sequence listed in Table 1 having no more than ten amino acid substitutions. For example, the present disclosure provides antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, said amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions. In another example, the present disclosure provides antibodies or antigenbinding fragments thereof comprising a HCVR and a LCVR, said LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30, said amino acid sequence having one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions. In one embodiment, the present disclosure provides anti-FXI l/FXI la antibodies or antigen-binding fragments thereof comprising a HCVR and a LCVR, said HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, said amino acid sequence having at least one amino acid substitution, and/or said LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30, said amino acid sequence having at least one amino acid substitution.

[015] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[016] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[017] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[018] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [019] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [020] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. [021] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acid sequence pair contained within any of the exemplary anti-FXII antibodies listed in Table 1. In certain embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2/22 (e.g., REGN9533) or 22/30 (e.g., REGN9534).

[022] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, HCDR2 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, and HCDR3 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid. In certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, LCDR2 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid, and LCDR3 comprising an amino acid sequence differing from an amino acid sequence listed in Table 1 by 1 amino acid. For example, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4 by 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence differing from SEQ ID NO: 6 by 1 amino acid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 8 or an amino acid sequence differing from SEQ ID NO: 8 by 1 amino acid. In another exemplary embodiment, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO : 12 or an amino acid sequence differing from SEQ ID NO: 12 by 1 amino acid, LCDR2 comprising an amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 1 amino acid, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by 1 amino acid. [023] As another example example, the present disclosure provides antibodies, or antigenbinding fragments thereof, comprising a HCVR and a LCVR, said HCVR comprising HCDR1 comprising an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence differing from SEQ ID NO: 24 by 1 amino acid, HCDR2 comprising an amino acid sequence of SEQ ID NO: 26 or an amino acid sequence differing from SEQ ID NO: 26 by 1 amino acid, and HCDR3 comprising an amino acid sequence of SEQ ID NO: 28 or an amino acid sequence differing from SEQ ID NO: 28 by 1 amino acid. In another exemplary embodiment, the present disclosure provides antibodies, or antigen-binding fragments thereof, comprising a HCVR and a LCVR, said LCVR comprising LCDR1 comprising an amino acid sequence of SEQ ID NO: 32 or an amino acid sequence differing from SEQ ID NO: 32 by 1 amino acid, LCDR2 comprising an amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 1 amino acid, and LCDR3 comprising an amino acid sequence of SEQ ID NO: 34 or an amino acid sequence differing from SEQ ID NO: 34 by 1 amino acid.

[024] The present disclosure also provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (/.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16 (e.g.,REGN9533), or 24-26-28-32- 14-34 (e.g., REGN9534).

[025] In a related embodiment, the present disclosure provides antibodies, or antigenbinding fragments thereof, comprising a set of six CDRs (/.e., HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary antibodies listed in Table 1. For example, the present disclosure includes antibodies, or antigen-binding fragments thereof, comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10 (e.g., REGN9533) or 10/30 (e.g., REGN9534). Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et a!., J. Mol. Biol. 273:927-948 (1997); and Martin et a!., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.

[026] In certain embodiments, the present disclosure includes an antibody or antigen-binding fragment thereof that binds specifically to FXII and/or FXIIa, wherein the antibody or antigenbinding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein the HCVR comprises: (i) the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22, (iii) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22; or (iv) the amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 22 having no more than 12 amino acid substitutions; and the LCVR comprises: (i) the amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30, (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30, (iii) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30; or (iv) the amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 30 having no more than 10 amino acid substitutions.

[027] The present disclosure provides anti-FXI l/FXI la antibodies, or antigen-binding fragments thereof, comprising a heavy chain (HC) and a light chain (LC), wherein the heavy chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 18, and the light chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 20. In another embodiment, the heavy chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 36, and the light chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 38. In some ebodiments, the heavy chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 40, and the light chain comprises a sequence having at least 90% sequence identity to SEQ ID NO: 20. [028] In certain embodiments, the present disclosure provides anti-FXI l/FXIIa antibodies, or antigen-binding fragments thereof, comprising an Fc domain comprising one or more mutations that alter Fc region function. In some embodiments, the Fc domain comprises a mutation in the CH2 or a CH3 region that increase FcyR binding activity. In some embodiments, the Fc domain comprises at least one mutation in one or more amino acids selected from the group consisting of amino acids at positions 248, 250, 252, 254, 256. 257, 307, 311 , 376, 380, 428, 433 and 434. In some embodiments, the Fc domain comprises at least one mutation in one or more amino acids at positions 252, 254 and 256. In some embodiments, the at least one mutation in the Fc domain comprises mutating position 252 to Y, mutating position 254 to T; and/or mutating position 256 to E. In some embodiments, the at least one mutation in the Fc domain increases the half-life of the antibody, or antigenbinding fragment thereof, in blood plasma compared to an antibody, or an antigen-binding fragment thereof, without the mutation. In some embodiments, the half-life is increased by at least 1.2-fold, preferably at least 1.5-fold, compared to an antibody, or an antigen-binding fragment thereof, without the mutation. [029] The present disclosure includes anti-FXI l/FXIIa antibodies having a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[030] In certain embodiments, the present disclosure provides antibodies and antigenbinding fragments thereof that exhibit pH-dependent binding to FXII. For example, the present disclosure includes antibodies and antigen-binding fragment thereof that bind FXII with higher affinity at neutral pH than at acidic pH (/.e., reduced binding at acidic pH).

[031] The present disclosure also provides for antibodies and antigen-binding fragments thereof that compete for specific binding to FXII or to FXIIa with an antibody or antigenbinding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[032] The present disclosure also provides antibodies and antigen-binding fragments thereof that cross-compete for binding to FXII/FXIIa with a reference antibody or antigen-binding fragment thereof comprising the CDRs of a HCVR and the CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[033] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof comprising three CDRs of a HCVR and three CDRs of a LCVR, wherein the HCVR and LCVR each has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[034] In some embodiments, the antibody or antigen binding fragment thereof may bind specifically to FXII and/or FXIIa in an agonist manner, i.e. , it may enhance or stimulate FXII/FXIIa binding and/or activity; in other embodiments, the antibody may bind specifically to FXII and/or FXIIa in an antagonist manner, i.e., it may block FXII binding and/or activity. [035] The present disclosure also provides isolated antibodies and antigen-binding fragments thereof that block FXIIa binding to FXI. In some embodiments, the antibody or antigen-binding fragment thereof that blocks FXIIa binding to FXI may bind to the same epitope on FXIIa as FXI or may bind to a different epitope on FXIIa as FXI.

[036] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are bispecific comprising a first binding specificity to a first epitope of FXII/FXIIa and a second binding specificity to a second epitope of FXII/FXIIa wherein the first and second epitopes are distinct and non-overlapping.

[037] In certain embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that has one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to activated Factor XII (FXIIa); (c) binds to FXII with a dissociation constant (KD) of less than 1.5 nM at 25°C, as measured in a surface plasmon resonance assay; (d) binds to FXII with a KD of less than 17nM at 37°C, as measured in a surface plasmon resonance assay; (e) binds to FXIIa with a KD of less than 5nM, preferably less than 0.9 nM, at 25°C as measured in a surface plasmon resonance assay; (f) binds to FXIIa with a KD of less than 6.5nM, preferably less than 2.5nM, at 37°C as measured in a surface plasmon resonance assay; (g) blocks thrombin generation by intrinsic pathway at a concentration of less than 250nM, as measured by functional plasma assays; and/or (h) blocks thrombin generation by intrinsic pathway without blocking thrombin generation by extrinsic pathway, as measured by functional plasma assays.

[038] In a second aspect, the present disclosure provides nucleic acid molecules encoding anti-FXI l/FXI la antibodies or portions thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[039] The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[040] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1 ; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[041] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1 ; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. [042] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1 ; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[043] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[044] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[045] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[046] The present disclosure also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (/.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequence set is as defined by any of the exemplary antibodies listed in Table 1.

[047] The present disclosure also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (/.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequence set is as defined by any of the exemplary antibodies listed in Table 1.

[048] The present disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1 , and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1 , or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti- FXII antibody listed in Table 1.

[049] In a related aspect, the present disclosure provides recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an antibody. For example, the present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 2. In certain embodiments, the present disclosure provides expression vectors comprising: (a) a nucleic acid molecule comprising a nucleic acid sequence encoding a HCVR of an antibody that binds FXI l/FXIIa, wherein the HCVR comprises an amino acid sequence selected from the group consisting of sequences listed in Table 1; and/or (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a LCVR of an antibody that binds FXII/FXIIa, wherein the LCVR comprises an amino acid sequence selected from the group consisting of sequences listed in Table 1. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced. In certain embodiments, the host cells comprise a mammalian cell or a prokaryotic cell. In certain embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell or an Escherichia coli (E. coli) cell. In certain embodiments, the present disclosure provides methods of producing an antibody or antigen-binding fragment thereof of the disclosure, the methods comprising introducing into a host cell an expression vector comprising a nucleic acid sequence encoding a HCVR and/or LCVR of an antibody or antigen-binding fragment thereof operably linked to a promoter; culturing the host cell under conditions favorable for expression of the nucleic acid sequence; and isolating the antibody or antigen-binding fragment thereof from the culture medium and/or host cell. The isolated antibody or antigen-binding fragment thereof may purified using any of the methods known in prior art.

[050] In a third aspect, the disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigenbinding fragment thereof which specifically binds FXII/FXIIa and a pharmaceutically acceptable carrier. In a related aspect, the disclosure features a composition which is a combination of an anti-FXI l/FXI la antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-FXII/FXI la antibody. Exemplary agents that may be advantageously combined with an anti-FXI l/FXI la antibody include, without limitation, other agents that bind and/or inhibit FXII activity (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents which do not directly bind FXII but nonetheless treat or ameliorate at least one symptom or indication of a FXII-associated disease or disorder. Additional combination therapies and co-formulations involving the anti-FXII antibodies of the present disclosure are disclosed elsewhere herein.

[051] In a fourth aspect, the disclosure provides therapeutic methods for treating a disease or disorder associated with FXII in a subject using an anti-FXII/FXI la antibody or antigenbinding portion of an antibody of the disclosure, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the disclosure to the subject in need thereof. The disorder treated is any disease or condition which is improved, ameliorated, inhibited or prevented by inhibition of FXII activity. In certain embodiments, the disclosure provides methods to prevent, or treat a FXII-associated disease or disorder comprising administering a therapeutically effective amount of an anti-FXI l/FXI la antibody or antigen-binding fragment thereof of the disclosure to a subject in need thereof. In some embodiments, the antibody or antigen-binding fragment thereof may be administered prophylactically or therapeutically to a subject having or at risk of having a FXII-associated disease or disorder. In certain embodiments, the antibody or antigen-binding fragment thereof the disclosure is administered in combination with a second therapeutic agent to the subject in need thereof. The second therapeutic agent may be selected from the group consisting of an anti-coagulant, a direct thrombin inhibitor, a thrombolytic drug, a fibrinolytic drug, an anti-platelet drug, an anti-inflammatory drug, an anti-hypertensive drug, a second anti-FXII antibody, a lipid-lowering drug, mechanical clot retrieval, catheter-guided thrombolysis, compression stockings, surgery and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent may be an agent that helps to counteract or reduce any possible side effect(s) associated with an antibody or antigenbinding fragment thereof of the disclosure, if such side effect(s) should occur. The antibody or fragment thereof may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof may be administered at a dose of about 0.1 mg/kg of body weight to about 100 mg/kg of body weight of the subject. In certain embodiments, an antibody of the present disclosure may be administered at one or more doses comprising between 10mg to 600mg.

[052] The present disclosure also includes use of an anti-FXI l/FXI la antibody or antigen- binding fragment thereof of the disclosure in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from the blockade of FXII/FXIIa binding and/or activity.

[053] Other embodiments will become apparent from a review of the ensuing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[054] FIG. 1A shows the time course of mAb concentrations and hFXI l/FXIIa concentrations measured for each individual mouse dosed with a single dose of 10 mg/kg of anti- hFXII/FXI la parental antibody (REGN9533). FIG. 1B shows the time course of mAb concentrations and hFXI l/FXI la concentrations measured for each individual mouse dosed with a single dose of 10 mg/kg of anti-hFXII/FXIIa YTE-variant antibody (REGN17653). FIG. 1C shows the time course of mAb concentrations and hFXI l/FXI la concentrations measured for each individual mouse dosed with a single dose of 10 mg/kg of anti-Fel D1 control antibody (REGN1945).

DETAILED DESCRIPTION

[055] Before the present methods are described, it is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[056] 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

Definitions

[057] The term “FXH”, also called “Factor XII” refers to coagulation Factor XII, also known as Hageman Factor. The 596-amino acid FXH protein is the zymogen form of Factor Xlla, a beta-globulin plasma serine protease composed of an amino-terminal heavy chain and a carboxy-terminal light chain. Its heavy chain contains two fibronectin-type domains (type I and II), two epidermal growth factor-like domains, a Kringle domain, and a proline-rich region, and its light chain contains the protease domain. Exposure of blood to negatively charged substances or artificial surfaces triggers thrombin generation and fibrin formation via a series oif reactions known as contact activation. Factor XI I activates Factor XI and prekallikrein in the coagulation cascade. Factor XII itself is activated to Factor XI la by plasma kallikrein, platelet or bacterial polyphosphates, extracellular DNA or RNA, heparins released from mast cells, amyloid peptides, misfolded protein aggregates and negatively charged surfaces, such as glass. This is the starting point of the intrinsic pathway. The amino acid sequence of full-length FXII protein is exemplified by the amino acid sequence provided in UniProtKB/Swiss-Prot as accession number P00748.3. The amino acid sequence of full-length FXII protein is also shown as SEQ ID NO: 65 herein. The term “FXII” includes recombinant FXII protein or a fragment thereof. The term also encompasses FXII protein or a fragment thereof coupled to, for example, histidine tag, mouse or human Fc, or a signal sequence such as ROR1.

[058] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (/.e., "full antibody molecules"), as well as multimers thereof (e.g. IgM) or antigen-binding fragments thereof. Each heavy chain is comprised of a heavy chain variable region (“HCVR” or “VH”) and a heavy chain constant region (comprised of domains CH1 , CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and a light chain constant region (CL). The VH and L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each H and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the disclosure, the FRs of the antibody (or antigen binding fragment thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[059] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428). [060] CDR residues not contacting antigen can be identified based on previous studies (for example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs lying outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative substitutions.

[061] The fully human anti-FXII monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies, and antigenbinding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1 , CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (/.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[062] The present disclosure also includes fully human anti-FXII monoclonal antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-FXII antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

[063] The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.

The human mAbs of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences {e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse), have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal, or in cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[064] The term “recombinant”, as used herein, refers to antibodies or antigen-binding fragments thereof of the disclosure created, expressed, isolated or obtained by technologies or methods known in the art as recombinant DNA technology which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[065] The term "specifically binds," or “binds specifically to”, or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x1 O'⁸ M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, which bind specifically to FXII. Moreover, multi- specific antibodies that bind to one domain in FXII and one or more additional antigens or a bi-specific that binds to two different regions of FXII are nonetheless considered antibodies that “specifically bind”, as used herein.

[066] The term “high affinity” antibody refers to those mAbs having a binding affinity to FXII, expressed as KD, of at least 10'⁸ M; preferably 10'⁹ M; more preferably 10'¹°M, even more preferably 10'¹¹ M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA.

[067] By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from FXII, with a rate constant of 1 x 10'³ s^-1 or less, preferably 1 x 10'⁴s^-1 or less, as determined by surface plasmon resonance, e.g., BIACORE™.

[068] The term “half-life” of an antibody as used herein refers to the time it takes for half of a given amount of antibodies to be cieared from the bloodstream or eliminated from the body of a subject, in some embodiments, anti-hFXII/FXIIa mAbs of the disclosure have a half-life of at least 0.5 day, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 15 days or 20 days.

[069] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigen-binding fragment" of an antibody, or "antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to bind to FXII protein.

[070] In specific embodiments, antibody or antibody fragments of the disclosure may be conjugated to a moiety such a ligand or a therapeutic moiety (“immunoconjugate”), a second anti-FXII antibody, or any other therapeutic moiety useful for treating a FXII-associated disease or disorder.

[071] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities e.g., an isolated antibody that specifically binds FXII and/or FXIIa, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than FXII or FXIIa.

[072] A “blocking antibody” or a "neutralizing antibody", as used herein (or an "antibody that neutralizes FXII activity" or “antagonist antibody”), is intended to refer to an antibody whose binding to FXII/FXIIa results in inhibition of at least one biological activity of FXII. For example, an antibody of the disclosure may prevent or block coagulation by the intrinsic pathway.

[073] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). [074] The term "KD ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[075] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.

[076] The term “cross-competes”, as used herein, means an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations, i.e., a first antibody that binds and blocks binding of second antibody and vice-versa. In certain embodiments, the first antibody and second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different, but overlapping epitopes such that binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Cross-competition between antibodies may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Cross-competition between two antibodies may be expressed as the binding of the second antibody that is less than the background signal due to self-self binding (wherein first and second antibodies is the same antibody). Cross-competition between 2 antibodies may be expressed, for example, as % binding of the second antibody that is less than the baseline self-self background binding (wherein first and second antibodies is the same antibody).

[077] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[078] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 144345, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[079] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GOG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA {e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.

[080] By the phrase “therapeutically effective amount” is meant an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[081] As used herein, the term “subject” refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and/or treatment of a FXII- associated disease or disorder such as thrombosis or embolism. The term includes human subjects who have or are at risk of having such a disease or disorder.

[082] As used herein, the terms “treat”, “treating”, or “treatment” refer to the reduction or amelioration of the severity of at least one symptom or indication of a FXII-associated disease or disorder due to the administration of a therapeutic agent such as an antibody of the present disclosure to a subject in need thereof. The terms include inhibition of progression of disease or of worsening of a symptom/indication. The terms also include positive prognosis of disease, i.e. , the subject may be free of disease or may have reduced disease upon administration of a therapeutic agent such as an antibody of the present disclosure. The therapeutic agent may be administered at a therapeutic dose to the subject. [083] The terms “prevent”, “preventing” or “prevention” refer to inhibition of manifestation of a FXII-associated disease or disorder or any symptoms or indications of such a disease or disorder upon administration of an antibody of the present disclosure.

Antigen-Binding Fragments of Antibodies

[084] Unless specifically indicated otherwise, the term "antibody," as used herein, shall be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains i.e., "full antibody molecules") as well as antigenbinding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen- binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. The terms "antigenbinding fragment" of an antibody, or "antibody fragment”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to FXII protein. An antibody fragment may include a Fab fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or an isolated CDR. In certain embodiments, the term “antigen-binding fragment” refers to a polypeptide fragment of a multi-specific antigenbinding molecule. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[085] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding fragment," as used herein.

[086] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR, which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain H - VH, VH - VL or VL - VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. [087] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH -CH1 ; (ii) VH -C_H2; (iii) V_H -C_H3; (iv) V_H -C_H1-C_H2; (v) V_H -C_H1-CH2-C_H3; (vi) V_H -C_H2-C_H3; (vii) V_H -C_L; (viii) V_L -C_H1 ; (ix) V_L -C_H2; (X) V_L -C_H3; (xi) V_L -C_H1-C_H2; (xii) V_L -CH1-C_H2-C_H3; (xiii) V_L -C_H2-C_H3; and (xiv) VL -CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non- covalent association with one another and/or with one or more monomeric H or VL domain (e.g., by disulfide bond(s)).

[088] As with full antibody molecules, antigen-binding fragments may be mono-specific or multi-specific (e.g., bi-specific). A multi-specific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

Preparation of Human Antibodies

[089] Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present disclosure to make human antibodies that specifically bind to FXII and/or FXIIa.

[090] An immunogen comprising any one of the following can be used to generate antibodies to FXII and/or FXIIa protein. In certain embodiments, the antibodies of the disclosure are obtained from mice immunized with a full length, native FXII or FXIIa protein (See, for example, UniProtKB/Swiss-Prot accession number P00748.3) or with DNA encoding the protein or fragment thereof. Alternatively, the protein or a fragment thereof may be produced using standard biochemical techniques and modified and used as immunogen.

[091] In some embodiments, the immunogen may be a recombinant FXII protein or fragment thereof expressed in E. coli or in any other eukaryotic or mammalian cells such as Chinese hamster ovary (CHO) cells.

[092] Using VELOCIMMUNE® technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies to FXII are initially isolated having a human variable region and a mouse constant region. The VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

[093] Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[094] Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate the fully human antibody of the disclosure, for example wild-type or modified IgG 1 or lgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.

Bioequivalents

[095] The anti-FXII antibodies and antibody fragments of the present disclosure encompass proteins having amino acid sequences that vary from those of the described antibodies, but that retain the ability to bind FXII protein. Such variant antibodies and antibody fragments comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Likewise, the antibody-encoding DNA sequences of the present disclosure encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody fragment that is essentially bioequivalent to an antibody or antibody fragment of the disclosure.

[096] Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antibodies will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.

[097] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[098] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.

[099] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[0100] Bioequivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antibody. [0101] Bioequivalent variants of the antibodies of the disclosure may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.

Anti-FXII Antibodies Comprising Fc Variants

[0102] According to certain embodiments of the present disclosure, anti-FXII antibodies are provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, the present disclosure includes anti-FXII antibodies comprising a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment {e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 e.g., E or Q); 250 and 428 {e.g., L or F); 252 {e.g., L/Y/F/W or T), 254 {e.g., S or T), and 256 {e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 {e.g., H/L/R/S/P/Q or K) and/or 434 {e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification at position 250 and/or 428; or a modification at position 307 or 308 {e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L {e.g., M428L) and 434S {e.g., N434S) modification; a 428L, 259I {e.g., V259I), and 308F {e.g., V308F) modification; a 433K {e.g., H433K) and a 434 {e.g., 434Y) modification; a 252, 254, and 256 {e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification {e.g., T250Q and M428L); and a 307 and/or 308 modification {e.g., 308F or 308P). In yet another embodiment, the modification comprises a 265A {e.g., D265A) and/or a 297A {e.g., N297A) modification. In one embodiment, the mutation comprises a 252Y, 254T, and 256E modification (“YTE” variant). In one embodiment, mutations in the Fc domain {e.g., 252Y, 254T, and 256E) extend the half-life of the antibody in blood plasma, when compared to an unmodified antibody. In one embodiment, mutations in the Fc domain {e.g., 252Y, 254T, and 256E) decrease clearance rates of the antibody in blood plasma, when compared to an unmodified antibody.

[0103] For example, the present disclosure includes anti-FXII antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 2571 and 3111 (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.

[0104] The present disclosure also includes anti-FXII antibodies comprising a chimeric heavy chain constant (CH) region, wherein the chimeric CH region comprises segments derived from the CH regions of more than one immunoglobulin isotype. For example, the antibodies of the disclosure may comprise a chimeric CH region comprising part or all of a CH2 domain derived from a human lgG1, human lgG2 or human lgG4 molecule, combined with part or all of a CH3 domain derived from a human lgG1, human lgG2 or human lgG4 molecule. According to certain embodiments, the antibodies of the disclosure comprise a chimeric CH region having a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues from positions 216 to 227 according to Ell numbering) derived from a human lgG1, a human lgG2 or a human lgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues from positions 228 to 236 according to Ell numbering) derived from a human lgG1 , a human lgG2 or a human lgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG 1 or a human lgG4 upper hinge and amino acid residues derived from a human lgG2 lower hinge. An antibody comprising a chimeric CH region as described herein may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., U.S. Patent Application Publication 2014/0243504, the disclosure of which is hereby incorporated by reference in its entirety).

Biological Characteristics of the Antibodies

[0105] In general, the antibodies of the present disclosure function by binding to FXII protein and preventing its cleavage to FXIIa and FXIIb. In certain embodiments, the antibodies bind to the activated form of Factor XII (FXIIa) (“dual FXII/FXIIa binders”). For example, the present disclosure includes antibodies and antigen-binding fragments of antibodies that bind FXII protein (e.g., at 25°C or at 37°C) with a KD of less than 20nM as measured by surface plasmon resonance, e.g., using the assay format as defined in the Examples herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind FXII with a KD of less than about 20nM, less than about 17nM, less than about 10nM, less than about 5nM, less than about 1nM, less than 0.5nM, or less than 0.3nM as measured by surface plasmon resonance, e.g., using the assay format as defined in the Examples herein, or a substantially similar assay.

[0106] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind human FXII protein with a dissociative half-life (t¹X) of greater than about 1 minute as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in the Examples herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind FXII protein with a t¹X of greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, greater than about 120 minutes, or greater than about 125 minutes, as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in Example 3 herein, or a substantially similar assay.

[0107] The present disclosure includes antibodies and antigen-binding fragments of antibodies that bind FXIIa protein (e.g., at 25°C or at 37°C) with a KD of less than 9nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind FXIIa with a KD of less than about 9nM, less than about 5nM, less than about 2nM, less than about 1nM, less than about 500pM, or less than about 350pM, as measured by surface plasmon resonance, e.g., using the assay format as defined in the Examples herein, or a substantially similar assay.

[0108] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind human FXIIa protein with a dissociative half-life (t¹X) of greater than about 1 minute as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in the Examples herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind FXIIa protein with a t¹X of greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, or greater than about 70 minutes, as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in the Examples herein, or a substantially similar assay. [0109] The present disclosure includes antibodies and antigen-binding fragments of antibodies that bind FXIIab protein (e.g., at 25°C or at 37°C) with a KD of less than 5nM as measured by surface plasmon resonance, e.g., using the assay format as defined in Example 3 herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind FXIIab with a KD of less than about 5nM, less than about 3mM, less than about 1nM, less than about 500pM, or less than about 300pM, as measured by surface plasmon resonance, e.g., using the assay format as defined in the Examples herein, or a substantially similar assay.

[0110] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind human FXIIab protein with a dissociative half-life (t¹X) of greater than about 1 minute as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in the Examples herein, or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure bind FXIIa protein with a t¹X of greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, or greater than about 50 minutes, as measured by surface plasmon resonance at 25°C or at 37°C, e.g., using an assay format as defined in the Examples herein, or a substantially similar assay.

[0111] The present disclosure also includes antibodies and antigen-binding fragments thereof that inhibit thrombin generation by intrinsic pathway at a concentration of less than 250nM, less than 200nM or less than 150nM, as measured, e.g., using an assay format as described in the Examples herein, or a substantially similar assay. In certain embodiments, the present disclosure includes antibodies and antigen-binding fragments thereof that inhibit thrombin generation by intrinsic pathway without blocking thrombin generation by extrinsic pathway, as measured, e.g., using an assay format as described in the Examples herein, or a substantially similar assay.

[0112] In one embodiment, the present disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof that binds specifically to FXII protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) binds to activated Factor XII (FXIIa); (c) binds to FXII with a dissociation constant (KD) of less than 1.5nM at 25°C, as measured in a surface plasmon resonance assay; (d) binds to FXII with a KD of less than 17nM at 37°C, as measured in a surface plasmon resonance assay; (e) binds to FXIIa with a KD of less than 5nM, preferably less than 0.9nM, at 25°C as measured in a surface plasmon resonance assay; (f) binds to FXIIa with a KD of less than 6.5nM, preferably less than 2.5nM, at 37°C as measured in a surface plasmon resonance assay; (g) blocks thrombin generation by intrinsic pathway at a concentration of less than 250nM, as measured by functional plasma assays; and (h) blocks thrombin generation by intrinsic pathway without blocking thrombin generation by extrinsic pathway, as measured by functional plasma assays.

[0113] The antibodies of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combinations thereof. Other biological characteristics of the antibodies of the present disclosure will be evident to a person of ordinary skill in the art from a review of the present disclosure including the working Examples herein.

Epitope Mapping and Related Technologies

[0114] The present disclosure includes anti-FXI l/FXIIa antibodies which interact with one or more amino acids found within one or more regions of the FXI l/FXIIa protein molecule including, the heavy chain and the light chain. The epitope to which the antibodies bind may consist of a single contiguous sequence of 3 or more (e.g. , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the FXII protein molecule (e.g. a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within either or both of the aforementioned domains of the protein molecule (e.g. a conformational epitope).

[0115] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, routine cross-blocking assays, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium- labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A. [0116] The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0117] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see US 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.

[0118] In certain embodiments, the present disclosure includes anti-FXII antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the heavy and/or light chain of FXIIa. The epitope(s) may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within heavy chain and/or light chain of FXIIa. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within FXIIa.

[0119] The present disclosure includes anti-FXII antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in T able 1. Likewise, the present disclosure also includes anti-FXII antibodies that compete for binding to FXII protein or a fragment thereof with any of the specific exemplary antibodies listed in Table 1. For example, the present disclosure includes anti-FXII antibodies that crosscompete for binding to FXII protein with one or more antibodies listed in Table 1.

[0120] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference anti-FXII antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference anti-FXII antibody of the disclosure, the reference antibody is allowed to bind to a FXII protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the FXII protein molecule is assessed. If the test antibody is able to bind to FXII following saturation binding with the reference anti-FXII antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-FXII antibody. On the other hand, if the test antibody is not able to bind to the FXII protein following saturation binding with the reference anti-FXII antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-FXII antibody of the disclosure.

[0121] To determine if an antibody competes for binding with a reference anti-FXII antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a FXII protein under saturating conditions followed by assessment of binding of the test antibody to the FXII molecule. In a second orientation, the test antibody is allowed to bind to a FXII molecule under saturating conditions followed by assessment of binding of the reference antibody to the FXII molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the FXII molecule, then it is concluded that the test antibody and the reference antibody compete for binding to FXII. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0122] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0123] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. Immunoconjugates

[0124] The disclosure encompasses a human anti-FXII monoclonal antibody conjugated to a therapeutic moiety (“immunoconjugate”), to treat a FXII-associated disease or disorder (e.g., thrombosis). As used herein, the term “immunoconjugate” refers to an antibody which is chemically or biologically linked to a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a peptide or protein or a therapeutic agent. The antibody may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any location along the molecule so long as it is able to bind its target. Examples of immunoconjugates include antibody drug conjugates and antibodytoxin fusion proteins. In one embodiment, the agent may be a second different antibody to FXII protein. The type of therapeutic moiety that may be conjugated to the anti-FXII antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable agents for forming immunoconjugates are known in the art; see for example, WO 05/103081.

Multi-specific Antibodies

[0125] The antibodies of the present disclosure may be mono-specific, bi-specific, or multispecific. Multi-specific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et a/., 2004, Trends Biotechnol. 22:238-244.

[0126] Any of the multi-specific antigen-binding molecules of the disclosure, or variants thereof, may be constructed using standard molecular biological techniques (e.g., recombinant DNA and protein expression technology), as will be known to a person of ordinary skill in the art.

[0127] In some embodiments, FXII-specific antibodies are generated in a bi-specific format (a "bi-specific") in which variable regions binding to distinct domains of FXII protein are linked together to confer dual-domain specificity within a single binding molecule. Appropriately designed bi-specifics may enhance overall FXII-protein inhibitory efficacy through increasing both specificity and binding avidity. Variable regions with specificity for individual domains, (e.g., segments of the N-terminal domain), or that can bind to different regions within one domain, are paired on a structural scaffold that allows each region to bind simultaneously to the separate epitopes, or to different regions within one domain. In one example for a bi-specific, heavy chain variable regions ( H) from a binder with specificity for one domain are recombined with light chain variable regions (Vi_) from a series of binders with specificity for a second domain to identify non-cognate VL partners that can be paired with an original VH without disrupting the original specificity for that VH. In this way, a single VL segment {e.g., L1) can be combined with two different H domains e.g., VH1 and VH2) to generate a bi-specific comprised of two binding "arms" (VH1- VL1 and VH2- VL1). Use of a single VL segment reduces the complexity of the system and thereby simplifies and increases efficiency in cloning, expression, and purification processes used to generate the bi-specific (See, for example, USSN13/022759 and US2010/0331527).

[0128] Alternatively, antibodies that bind more than one domains and a second target, such as, but not limited to, for example, a second different anti-FXII antibody, may be prepared in a bi-specific format using techniques described herein, or other techniques known to those skilled in the art. Antibody variable regions binding to distinct regions may be linked together with variable regions that bind to relevant sites on, for example, the extracellular domain of FXI I , to confer dual-antigen specificity within a single binding molecule. Appropriately designed bi-specifics of this nature serve a dual function. Variable regions with specificity for the extracellular domain are combined with a variable region with specificity for outside the extracellular domain and are paired on a structural scaffold that allows each variable region to bind to the separate antigens.

[0129] An exemplary bi-specific antibody format that can be used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of lgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of lgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of lgG4 antibodies. Variations on the bi-specific antibody format described above are contemplated within the scope of the present disclosure.

[0130] Other exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG- scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, lgG1/lgG2, dual acting Fab (DAF)-lgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide/nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub'. Dec. 4, 2012]).

Therapeutic Administration and Formulations

[0131] The disclosure provides therapeutic compositions comprising the anti-FXII antibodies or antigen-binding fragments thereof of the present disclosure. Therapeutic compositions in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0132] The dose of antibody may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like. When an antibody of the present disclosure is used for treating a disease or disorder in an adult patient, or for preventing such a disease, it is advantageous to administer the antibody of the present disclosure normally at a single dose of about 0.1 to about 100 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the antibody or antigen-binding fragment thereof of the disclosure can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0133] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, Langer (1990) Science 249:1527-1533).

[0134] The use of nanoparticles to deliver the antibodies of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M., et al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389, 24 pages, doi: 10.1155/2009/439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995, each incorporated herein in its entirety.

[0135] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose.

[0136] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known.

[0137] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0138] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 300 mg and in about 10 to about 300 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

[0139] The antibodies of the present disclosure are useful for the treatment, and/or prevention of a disease or disorder or condition associated with coagulation (including thrombosis and embolism) or edema (e.g., hereditary angioedema) and/or for ameliorating at least one symptom associated with such disease, disorder or condition. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure may be administered at a therapeutic dose to a patient with a disease or disorder or condition associated with coagulation or with edema. In certain embodiments, an antibody or antigenbinding fragment thereof of the disclosure is administered to a subject in need thereof to prevent thrombosis without increasing risk of bleeding.

[0140] In certain embodiments, the antibodies of the present disclosure are useful for treating or preventing at least one symptom or indication of a FXII-associated disease or disorder selected from the group consisting of venous thrombosis, arterial thrombosis, device thrombosis, thromboembolism, hereditary angioedema, stroke, thrombophilia, cardiac ischemia, atherosclerotic plaque rupture, use of mechanical valve prostheses, use of bloodcontacting medical devices, use of blood-contacting extracorporeal circuits, venous thromboembolism, pulmonary embolism, deep vein thrombosis, portal vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, renal vein thrombosis, cerebral venous sinus thrombosis, jugular vein thrombosis, cavernous sinus thrombosis, hepatic artery thrombosis, limb ischemia and myocardial infarction. [0141] It is also contemplated herein to use one or more antibodies of the present disclosure prophylactically to subjects at risk for thrombosis such as subjects using extracorporeal membrane oxygenation (heart-lung machine). An antibody of the disclosure may be used to prevent thrombotic occlusions of the oxygenator and tubing in the extracorporeal circuit. [0142] In a further embodiment of the disclosure, the present antibodies are used for the preparation of a pharmaceutical composition or medicament for treating patients suffering from a disease, disorder or condition disclosed herein. In another embodiment of the disclosure, the present antibodies are used as adjunct therapy with any other agent or any other therapy known to those skilled in the art useful for treating or ameliorating a disease, disorder or condition disclosed herein.

Combination Therapies

[0143] Combination therapies may include an antibody of the disclosure and any additional therapeutic agent that may be advantageously combined with an antibody of the disclosure, or with a biologically active fragment of an antibody of the disclosure. The antibodies of the present disclosure may be combined synergistically with one or more drugs or therapy used to treat a disease or disorder associated with thrombosis, specifically to prevent thrombosis or to treat a subject at risk of thrombosis due to an underlying disease, disorder or condition (described elsewhere herein). In some embodiments, the antibodies of the disclosure may be combined with a second therapeutic agent to ameliorate one or more symptoms of said disease or condition.

[0144] Depending upon the disease, disorder or condition, the antibodies of the present disclosure may be used in combination with one or more additional therapeutic agents including, but not limited to, an anti-coagulant (e.g., warfarin, heparin, phenindione, fondaparinux, idraparinux), a thrombin inhibitor (e.g., argatroban, lepirudin, bivalirudin, or dabigatran), a thrombolytic drug, an anti-platelet drug (e.g., aspirin), an antihypertensive (e.g., an angiotensin-converting enzyme inhibitor, a beta blocker, a calcium channel blocker), an immunosuppressive agent (e.g., vincristine, cyclosporine A, or methotrexate), a fibrinolytic agent, a cholesterol-lowering agent (e.g., a statin or a PCSK9 inhibitor such as alirocumab), an anti-inflammatory drug (e.g., a corticosteroid, or a non-steroidal antiinflammatory drug), a second anti-FXII antibody, mechanical clot retrieval, catheter-guided thrombolysis and surgery.

[0145] As used herein, the term “in combination with” means that additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti-FXII antibody of the present disclosure. The term “in combination with” also includes sequential or concomitant administration of an anti-FXII antibody and a second therapeutic agent.

[0146] The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-FXII antibody of the present disclosure. For example, a first component may be deemed to be administered "prior to" a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, or less than 30 minutes before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-FXII antibody of the present disclosure. For example, a first component may be deemed to be administered "after" a second component if the first component is administered 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after or more after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti-FXII antibody of the present disclosure. "Concurrent" administration, for purposes of the present disclosure, includes, e.g., administration of an anti-FXII antibody and an additional therapeutically active component to a subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-FXII antibody and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route (e.g., the anti-FXII antibody may be administered intravenously, and the additional therapeutically active component may be administered orally). In any event, administering the components in a single dosage from, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered "concurrent administration," for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-FXII antibody "prior to", "concurrent with," or "after" (as those terms are defined herein above) administration of an additional therapeutically active component is considered administration of an anti-FXII antibody "in combination with" an additional therapeutically active component.

[0147] The present disclosure includes pharmaceutical compositions in which an anti-FXII antibody of the present disclosure is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein. Diagnostic Uses of the Antibodies

[0148] The antibodies of the present disclosure may be used to detect and/or measure FXII in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present disclosure in assays to detect a FXII-associated-disease or disorder. Exemplary diagnostic assays for FXII may comprise, e.g., contacting a sample, obtained from a patient, with an anti-FXII antibody of the disclosure, wherein the anti-FXII antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate FXII from patient samples. Alternatively, an unlabeled anti-FXII antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵l; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, p-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure FXII in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0149] Samples that can be used in FXII diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of either FXII protein, or fragments thereof, under normal or pathological conditions. Generally, levels of FXII protein in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with FXII) will be measured to initially establish a baseline, or standard, level of FXII. This baseline level of FXII can then be compared against the levels of FXII measured in samples obtained from individuals suspected of having a FXII-associated condition, or symptoms associated with such condition.

[0150] The antibodies specific for FXII protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing an N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

EXAMPLES

[0151] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used {e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, room temperature is about 25°C, and pressure is at or near atmospheric.

Example 1 : Generation of Human Antibodies to Factor Xll/activated Factor XII (FXII/FXIIa) proteins

[0152] Human antibodies to FXII/FXIIa proteins were generated in a VELOCIMMUNE® mouse comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions. The mice were immunized with plasma purified human FXII and FXIIa proteins (Enzyme Research Laboratories).

[0153] Anti-FXII antibodies were isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Patent 7582298, herein specifically incorporated by reference in its entirety. Using this method, several fully human anti-FXII antibodies (/.e., antibodies possessing human variable domains and human constant domains) were obtained; exemplary antibodies generated in this manner were designated as REGN9533 and REGN9534.

[0154] The biological properties of the exemplary antibodies generated in accordance with the methods of this Example are described in detail in the Examples set forth below.

Example 2: Amino Acid and Nucleotide Sequences

[0155] Table 1 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-FXII antibodies of the disclosure.

Table 1: Amino Acid Sequence Identifiers

[0156] The corresponding nucleic acid sequence identifiers are set forth in Table 2. Table 2: Nucleic Acid Sequence Identifiers

[0157] Antibodies referred to herein typically have fully human variable regions, but may have human or mouse constant regions. As will be appreciated by a person of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG 1 Fc can be converted to an antibody with a human I gG4, etc.), but in any event, the variable domains (including the CDRs) - which are indicated by the numerical identifiers shown in Table 2 - will remain the same, and the binding properties to antigen are expected to be identical or substantially similar regardless of the nature of the Fc domain. In certain embodiments, selected antibodies with a mouse IgG 1 Fc are converted to antibodies with human lgG4 Fc. In one embodiment, the lgG4 Fc domain comprises 2 or more amino acid changes as disclosed in LIS20100331527. In one embodiment, the human lgG4 Fc comprises a serine to proline mutation in the hinge region (S108P) to promote dimer stabilization.

Control Constructs used in the Following Examples

[0158] The following control constructs (anti-FXII antibodies) were included in the experiments disclosed herein, for comparative purposes: "Comparator 1," a monoclonal antibody against human FXII/FXIIa having VH/VL sequences of antibody “3F7” according to EP20110175105 (CSL Behring GmbH); and "Comparator 2," a human monoclonal antibody against human FXII having VH/VL sequences of antibody “15H8” according to WO 2014/089493 (Vanderbilt Univ./Aronora).

Example 3: Biacore binding kinetics of anti- FXII monoclonal antibodies binding to different FXII reagents measured at 25°C

[0159] Equilibrium dissociation constants (KDs) for different FXII reagents binding to purified anti-FXII monoclonal antibodies were determined using a real-time surface plasmon resonance based Biacore 8K biosensor. All binding studies were performed in 10mM HEPES, 300mM NaCI, and 0.05% v/v Surfactant Tween-20, pH 7.4 (HBS-P) running buffer at 25°C. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with the monoclonal mouse anti-human Fc antibody (REGN2567) to capture anti-FXII monoclonal antibodies and an irrelevant isotype control. Binding studies were performed on human FXII, FXIIa, and FXIIab. Different concentrations of hFXII, hFXIIa and hFXIIab (30nM - 1.11 nM; 3- fold serial dilution) were first prepared in HBS-P running buffer and were injected over antihuman Fc captured anti-FXII monoclonal antibody surface for 3 minutes at a flow rate of 30pL/minute, while the dissociation of monoclonal antibody bound FXII reagent was monitored for 8 minutes in HBS-P running buffer. The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgrams to a 1 :1 binding model with mass transport limitation using Biacore Insight Evaluation Software. Binding dissociation equilibrium constant (KD) and dissociative half-life (t¹X) were calculated from the kinetic rates as: fed hi (2)

KD (M) = fax , and t¹X (min) = 60^-fe Binding kinetics parameters for hFXIIa, hFXIIab, or hFXII, binding to different anti-FXII monoclonal antibodies of the invention at 25°C are shown in Tables 3 through 5.

At 25°C, anti-FXII monoclonal antibodies bound to hFXIIa with KD values ranging from 322pM to 2.09nM, as shown in Table 3. At 25°C, anti-FXII monoclonal antibodies bound to hFXIIab with KD values ranging from 264pM to 2.59nM, and REGN4026 did not bind hFXIIab as shown in Table 4. At 25°C, anti-FXII monoclonal antibodies bound to hFXII with KD values ranging from 233pM to 27.5nM, REGN3059 did not bind hFXII as shown in Table 5.

Table 3: Binding kinetics parameters of hFXIIa binding to FXII monoclonal antibodies at 25°C.

Table 4: Binding kinetics parameters of hFXIIab binding to FXII monoclonal antibodies at 25°C.

NB = No binding (~2 fold above REGN1945 iso-type control bound RU)

Table 5: Binding kinetics parameters of hFXII binding to FXII monoclonal antibodies at 25°C.

NB = No binding (~2 fold above REGN1945 iso-type control bound Rll)

Example 4: Octet cross-competition between different anti-FXII monoclonal antibodies using pre-complex method

[0160] Binding competition between anti-FXII monoclonal antibodies was determined using a real time, label-free bio-layer interferometry assay on an OctetHTX biosensor (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 0.01 M HEPES pH7.4, 0.15M NaCI, 0.05% v/v Surfactant Tween-20, 0.1mg/mL BSA (HBS-P buffer) with the plate shaking at the speed of lOOOrpm. To assess whether 2 antibodies were able to compete with one another for binding to their respective epitopes on a human FXIIa (ERL) around 1.38-2.32 nm of anti-human FXII monoclonal antibody was first captured onto anti-hFc antibody coated Octet biosensor tips (Pall ForteBio Corp., # 18-5060) by submerging the tips for 5 minutes into wells containing a 50 pg/mL solution of anti-human FXII monoclonal antibody (subsequently referred to as mAb-1). The antibody captured biosensor tips were then saturated with an irrelevant lgG4 isotype control monoclonal antibody (subsequently referred to as blocking mAb) by dipping into wells containing 200 pg/mL solution of blocking mAb for 10 minutes. The biosensor tips were then subsequently dipped into wells containing a co-complexed solution of 25 nM hFXIIa and 1 pM of a second anti-human FXII monoclonal antibody (subsequently referred to as mAb-2), that had been pre-incubated for 2 hours. The biosensor tips were washed in HBS-P buffer in between every step of the experiment. The real-time binding response was monitored during the course of the experiment and the binding response at the end of every step was recorded. The response of human FXII precomplexed mAb-2 binding to captured mAb-1 was corrected for background binding, compared and competitive/non-competitive behavior of different anti-FXII monoclonal antibodies was determined.

[0161] Table 6 explicitly defines the relationships of antibodies competing in both directions, independent of the order of binding.

Table 6: Cross-competition of anti-hFXI I antibodies for binding to human FXII.

Example 5: In vitro functional characterization of REGN9533 and REGN9534 in clotting assays (aPTT, PT), thrombin generation assay (TGA), clot lysis assay, and plasma kallikrein activity assay with human, cyno and F12^hu/hu mouse plasma

Determination of aPTT

[0162] The aPTT was determined on Diagnostica Stago STart4 Hemostasis Analyzer in the following manner: A total of 50 ul of pooled normal human plasma was added to a cuvette at 37°C. After 1 min, 5 ul of a 2x serially diluted test article (antibody or small molecule inhibitor) in PBS was added to the cuvette and allowed to incubate for 5 min. Then 50 ul of APPT-XL Ellagic Acid (Thermo Scientific) was added and allowed to incubate for 300 seconds before 50 ul of 20 mM calcium chloride (Thermo Scientific) was added to start the reaction. The measured clot time for the test article concentration was normalized to baseline (no drug) plasma clot time and plotted against log molar concentration of the test article. The results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5 software (GraphPad) to obtain doubling time concentration (C2xt).

Determination of PT

[0163] The PT was determined on Diagnostica Stago STart4 Hemostasis Analyzer in the following manner. A total of 50 ul of pooled normal human plasma was added to a cuvette at 37°C. After 1 min, 5 ul of a 2x serially diluted test article (antibody or small molecule inhibitor) in PBS was added to the cuvette and allowed to incubate for 5 min. Then 100 ul of Tissue Factor (TriniCLOT PT Excel, Diagnostica Stago) was added to start the reaction. The measured clot time for the test article concentration was normalized to baseline (no drug) plasma clot time and plotted against log molar concentration of the test article. The results were analyzed using nonlinear regression (4-parameter logistics) with Prism 5 software (GraphPad) to obtain doubling time concentration (C2xt).

Determination of TGA with Ellagic Acid

[0164] The thrombin generation profile was determined in a Diagnostica Stago Calibrated Automated Thrombogram in the following manner: A total of 55 ul of pooled normal human plasma was added to a well of a microplate at 37°C. Then 5 ul of a 2x serially diluted test article (antibody or small molecule inhibitor) in PBS was added to the well of the microplate and allowed to incubate for 30 min. 15 ul of APPT-XL Ellagic Acid (Thermo Scientific) was diluted in MP reagent then added to the well and allowed to incubate for 30 min. Then 15 ul of Fluo Flu Cal substrate (Diagnostica Stago) was added immediately before a continuous 90 min reading of the microplate. The measured real-time thrombin concentration values were plotted against time to yield a thrombogram profile for each concentration of the test article used.

Determination of TGA with Tissue Factor

[0165] The thrombin generation profile was determined in a Diagnostica Stago Calibrated Automated Thrombogram in the following manner: A total of 55 ul of pooled normal human plasma was added to a well of a microplate at 37°C. Then 5 ul of a 2x serially diluted test article (antibody or small molecule inhibitor) in PBS was added to the well of the microplate and allowed to incubate for 30 min. 15 ul of Tissue Factor PPP Reagent (Diagnostica Stago) was added to the well and allowed to incubate for 30 min. Then 15 ul of Fluo Flu Cal substrate (Diagnostica Stago) was added immediately before a continuous 90 min reading of the microplate. The measured real-time thrombin concentration values were plotted against time to yield a thrombogram profile for each concentration of the test article used.

Determination of Clot lysis

[0166] The clot lysis was determined on a microplate reader capable of reading at OD405nm in kinetic mode for 3 hours at 37°C. A total of 50 ul of pooled normal human plasma was added to each experiment well on the 96-well microplate at 37°C. And 5 ul of a 2x serially diluted test article (antibody or small molecule inhibitor) in PBS was added to the experiment well and incubate at 37°C for 30 mins. Then add 50 uL of activation mixture to each well (The final cone, of Ellagic Acid in assay 62.5 nM or Tissue Factor in assay 0.125 pM, and with tPA 50 ng/mL in total 100 uL/well assay) and immediately read at every 30 s for 180 min at OD405 nm kinetic mode in a microplate reader (Molecular Devices i3 system). Clot Lysis Time (CLT) was defined by measuring the time between 50% clot formation curve and 50% clot lysis curve.

Determination of Plasma Kallikrein activity

[0167] Plasma kallikrein activity was determined on a microplate reader capable of reading at OD405nm in kinetic mode for 1 hour at 37°C. Plasma samples were diluted (500 times for human, 250 times for Cyno or 500 times for mouse) in 20 mM of HBS buffer. To each well, 100 ul of diluted plasma was added along with 20 ul of mAb and incubated at 37°C for 30 mins before a 10 min activation with Dextran Sulfate (DXS; 5 ul of 100 ug/ml DXS). 25 ul of 1 mM Kallikrein substrate (final cone. 0.167 mM in assay) was added to each well and then immediately read on a microplate reader to measure Vmax (maximum change in OD at 405 nm per min). Correct all Vmax readouts by subtracting the Vmax from the blank (HBS assay buffer) and then plotted against log molar concentration of the test article. The results were analyzed using nonlinear regression (4-parameter logistics) with Prism 8 software (GraphPad) to obtain IC50 (The half maximal inhibitory concentration).

Results summary and conclusions:

[0168] Dose response curves were generated to determine the effect of anti-FXI l/FXI la on aPTT and PT in pooled normal “healthy” human plasma. The comparator, COMP3059, which is an anti-FXI la antibody prolonged aPTT to 3-fold from baseline without affecting PT up to a dose of 600 nM (Tables 7a and 7d). The Regeneron anti-FXI l/FXI la mAbs (REGN9533 and REGN9534) prolonged the aPTT to ~7-fold and ~6-fold, respectively, from baseline up to a dose of 600 nM without increasing PT (Tables 7b-7c, 7e-7f). The efficiency of the drug to inhibit coagulation activity is indexed by an arbitrary “doubling time” concentration, which is the concentration of drug required to prolong the clotting time by 2- fold over the baseline value. The Regeneron FXI l/FXI la mAbs REGN9533 and REGN9534 reached a doubling time with -125 and -200 nM, respectively, similar to COMP3059 that also required 200 nM. Both Regeneron anti-FXI l/FXI la mAbs were not found to double the PT clotting time at the maximal 600 nM tested; same was true for the COMP3059. In plasma from pooled normal “healthy” Cyno monkeys, the anti-FXI l/FXIIa mAbs were less efficacious at doses up to 600 nM since REGN9533 prolonged aPTT to -3.2-fold while REGN9534 prolonged aPTT to -1.9-fold whereas COMP3059 reached -1.5-fold above baseline (Tables 8a-8f). The doubling time for aPTT was 250 nM for REGN9533 and >600nM for REGN9534 and COMP3059. The PT was unchanged and did not reach a doubling time for all mAbs at doses up to 600 nM (Tables 8d-8f). In plasma from pooled normal “healthy” pi2^hu/hu mice, the anti-FXI l/FXIIa mAbs had reduced efficacy at doses up to 600 nM since REGN9533 and REGN9534 prolonged aPTT to ~2.X-fold while COMP3059 reached -1.9-fold above baseline (Tables 9a-9f). The doubling time for aPTT was -250 nM for REGN9533 and 300nM for REGN9534 but -2000 nM for COMP3059 (Tables 9a-9c). The PT was unchanged and did not reach a doubling time for all mAbs at doses up to 600 nM.

[0169] The effects of the anti-FXI l/FXI la mAbs were evaluated for their ability to inhibit thrombin generation (i.e., prolonged time to detection of thrombin = lag time, reduction in thrombin peak and reduction in total amount of thrombin generated = endogenous thrombin potential) when plasma was triggered by ellagic acid or Tissue Factor. In pooled normal “healthy” human plasma, both Regeneron FXII/FXIIa mAbs required at least 125 nM of drug to begin to affect thrombin generation triggered by ellagic acid activation but at doses of >250 nM the Regeneron anti-FXII/FXI la mAb completely inhibited thrombin production (Tables 10a-10f), indicating that the Regeneron mAbs suppress the intrinsic pathway of coagulation that in turn prevents downstream thrombin generation. COMP3059 showed a dose dependent effect on thrombin generation with a near complete inhibition at 500 nM. Thrombin generation triggered by Tissue Factor was minimally (<30% of baseline thrombin generation) affected by the Regeneron anti-FXII/FXI la mAbs or the COMP3059. The concentrations of test article required to extend the lag time by two-fold, reduce the thrombin peak and total amount of thrombin generation by half when coagulation is activated by ellagic acid or Tissue Factor are summarized in Tables 10a-f. In pooled normal “healthy” Cyno plasma, both Regeneron FXII/FXIIa mAbs and COMP3059 were less effective in inhibiting thrombin generation triggered by ellagic acid, even at a maximal dose that was 10 times greater than that used in human plasma. REGN9533 and REGN9534 were capable of reducing thrombin generation to -60% of baseline thrombin production while COMP3059 reduced thrombin generation by 50% (Tables 11a-11f). All anti-FXII/FXI la mAb had no noticeable effect on thrombin generation activated by Tissue Factor. The concentrations of anti-FXI l/FXI la required to extend the lag time by two-fold, reduce the thrombin peak and total amount of thrombin generation by half when coagulation is activated by ellagic acid or Tissue Factor in Cyno plasma are summarized in Tables 11a-f. In pooled normal “healthy” F12^hu/hu mouse plasma, both Regeneron FXII/FXIIa mAbs and COMP3059 were somewhat effective in inhibiting thrombin generation triggered by ellagic acid at doses up to 500 nM. REGN9533 and REGN9534 were capable of reducing thrombin generation to -25% of baseline thrombin production, respectively, while COMP3059 reduced thrombin generation by -30% (Table 12). All anti-FXI l/FXI la mAb had no noticeable effect on thrombin generation activated by Tissue Factor. The concentrations of anti-FXI l/FXI la required to extend the lag time by two-fold, reduce the thrombin peak and total amount of thrombin generation by half when coagulation is activated by ellagic acid or Tissue Factor in pi2^hu/hu mouse plasma are summarized in Tables 12a-12f. [0170] The anti-FXI l/FXI la mAbs were evaluated for their ability to affect fibrinolysis in a turbidity assay that evaluates clotting time and clot lysis time. In the presence of X pM of Tissue Factor (TF), all anti-FXI l/FXI la mAbs and the control isotype (REGN1945) generated similar clotting profiles as noted by a similar clot formation time of 4 min (Table 13), indicating that the anti-FXI l/FXI la mAbs do not affect extrinsic pathway mediated clotting. In the presence of tissue plasminogen activator (tPA 50ng/mL), following clotting induced by TF, the REGN9533, REGN9534 and REGN1945 showed a similar clot lysis time (i.e. , decline in optical signal) of 120-125 min whereas COMP3059 showed a slightly longer clot lysis time of 144 min (Table 13).

[0171] The mAbs were also evaluated for their ability to inhibit FXI la-mediated plasma kallikrein activity triggered by dextran sulfate (DXS). In pooled normal “healthy” human plasma activated with Dextran Sulfate (DXS), REGN9533 and REGN9534 inhibited plasma kallikrein activity with an IC50 of -0.35 nM (Table 14). REGN1945 did not have an effect while COMP3059 had an IC50 of 0.49 nM. The rate of inhibition at the mAb concentrations used are presented in Table 14. In pooled normal “healthy” Cyno plasma activated with DXS, REGN9533 and REGN9534 was less potent than in human plasma at inhibiting plasma kallikrein activity with an IC50 of -1.5 and 2.5 nM, respectively (Table 15). REGN1945 did not have an effect while COMP3059 had an IC50 of 9.2 nM. The rate of inhibition at the mAb concentrations used are presented in Table 15. In pooled normal F12^hu/hu,Serping1~^/~ mouse plasma activated with DXS, REGN9533 and REGN9534 was less potent than in human plasma at inhibiting plasma kallikrein activity with an IC50 of -0.7 and 1.6 nM, respectively (Table 16). REGN1945 did not have an effect while COMP3059 had an IC50 of 13.7 nM. The rate of inhibition at the mAb concentrations used are presented in Table 16.

[0172] The Regeneron anti-FXI l/FXI la mAbs REGN9533 and REGN9534 are potent at inhibiting intrinsic coagulation pathway activity (i.e., aPTT and TGA-EA) more potently in human than in Cyno monkey or pi2^hu/hu mouse plasma. There is no effect on extrinsic coagulation pathway activity (PT and TGA-TF). These mAbs did not affect clot lysis time in the presence of tPA. In addition, REGN9533 and REGN9534 were found to be effective at inhibiting the activation of plasma kallikrein (i.e., plasma kallikrein activity) induced by dextran sulfate.

Table 7a: aPTT tables for human plasma with COMP3Q59

Table 7b: aPTT tables for human plasma with REGN9533

Table 7c: aPTT tables for human plasma with REGN9534

Table 7e: PT tables for human plasma with REGN9533

Table 7f: PT tables for human plasma with REGN9534

Table 8a: aPTT tables for Cyno plasma with COMP3Q59

Table 8b: aPTT tables for Cyno plasma with REGN9533

Table 8c: aPTT tables for Cyno plasma with REGN9534

Table 8d: PT tables for Cyno plasma with COMP3Q59

Table 8e: PT tables for Cyno plasma with REGN9533

Table 8f: PT tables for Cyno plasma with REGN9534

Table 9a: aPTT tables for F12 ^hu/hu plasma with COMP3Q59

Table 9b: aPTT tables for F12 ^hu/hu plasma with REGN9533

Table 9c: aPTT tables for F12 ^hu/hu plasma with REGN9534

Table 9d: PT tables for F12 ^hu/hu plasma with COMP3Q59

Table 9e: PT tables for F12 ^hu/hu plasma with REGN9533

Table 9f: PT tables for F12 ^hu/hu plasma with REGN9534

Table 10a: TGA-EA for human plasma with COMP3Q59

Table 10b: TGA-EA for human plasma with REGN9533

Table 10c: TGA-EA for human plasma with REGN9534

Table 10d: TGA-TF for human plasma with COMP3Q59

Table 10e: TGA-TF for human plasma with REGN9533

Table 10f: TGA-TF for human plasma with REGN9534

Table 11a: TGA-EA for Cyno plasma with COMP3Q59

Table 11b: TGA-EA for Cyno plasma with REGN9533

Table 11c: TGA-EA for Cyno plasma with REGN9534

Table 11 d: TGA-TF for Cyno plasma with COMP3Q59

Table 11e: TGA-TF for Cyno plasma with REGN9533

Table 11f: TGA-TF for Cyno plasma with REGN9534

Table 12a: TGA-EA for F12 ^hu/hu mice with COMP3059

Table 12b: TGA-EA for F12 ^hu/hu mice with REGN9533

Table 12c: TGA-EA for F12 ^hu/hu mice with REGN9534

Table 12d: TGA-TF for F12 ^hu/hu mice with COMP3059

Table 12e: TGA-TF for F12 ^hu/hu mice with REGN9533

Table 12f: TGA-TF for F12 ^hu/hu mice with REGN9534

Table 13: TF-induced clot formation and tPA-mediated clot lysis time in human plasma

Table 14: Plasma kallikrein activity in Human Plasma

Table 15: Plasma kallikrein activity in Cyno Plasma

Table 16: Plasma kallikrein activity in Fi2^hu/hu ; Serpingl-/- mouse plasma

[0173] The above results show that the exemplified antibodies inhibit thrombin generation by intrinsic pathway without inhibiting the extrinsic pathway.

Example 6: Generation of YTE variants of anti- FXII monoclonal antibodies

[0174] Fc variants of anti-hFXI l/FXI la monoclonal antibodies were generated through mutations of residues 252, 254 and 256 on the Fc domain to residues Y, T and E, respectively (“YTE” variants). The parental antibody REGN9533 was modified in this manner to yield an exemplary YTE variant: REGN 17653.

[0175] Table 17a sets forth the amino acid sequence identifiers of the heavy and light chain variable regions, CDRs and heavy and light chain constant region of selected YTE variant anti-FXII antibodies of the disclosure.

Table 17a: Amino Acid Sequence Identifiers

[0176] The corresponding nucleic acid sequence identifiers are set forth in Table 17b.

Table 17b: Nucleic Acid Sequence Identifiers

Example 7: Validation of the FcRn^hu/hu B2m^hu/hu mouse model to compare half-lives of YTE variant and parental antibodies

__ _ ___ . _ _ _ hu/hu _ _ hu/hu _ , _

[0177] The suitability of FcRn B2m mice as in vivo models for measurements of differences in half-life of YTE variant antibodies compared to parental antibodies was hu/hu hu/hu hu/hu evaluated. In these experiments, hlgHC-KC (het) x FcRn B2M x F12 mice were dosed intravenously with either anti-SARS-CoV2 parental antibody (REGN10933, hlgG1 isotype), or its YTE variant (REGN13213, hlgG1 isotype) at a single intravenous dose of 10 mg/kg. An anti-Fel D1 monoclonal antibody (hlgG4 isotype) was used as a control. Serum blood collection was performed at days 0 (2 hour post-dose), 1, 2, 3, 4, 7, 14, 21, 30, 45 and 60 post dose. 4 mice were dosed in each test group, and 6 mice were dosed in the control group. The total dosed antibody concentrations in blood plasma were measured by GYROLAB® Immunoassay, with plasma drug concentrations determined after acid dissociation. Acid dissociation was not used for control antibody. To facilitate antibody capture, biotin conjugated anti-idiotype SARS-CoV2 mAbs or biotin conjugated Fel-D1 mAb were used, and detected by Alexa 647-conjugated mouse anti-human lgG1/lgG4 mAb. Antibody concentrations in the plasma sample were determined over 45 days, and pharmacokinetic (PK) parameters were determined.

[0178] For quantification of antibody concentrations, the lower limit of quantitation (LLOQ) for SARS-CoV2 antibodies was set at 0.05 pg/ml. For calculations of average concentrations, if more than 50% of values were below limit of quantitation (BLQ), the average was reported as BLQ, whereas if 50% or fewer values were BLQ, averages were calculated with 0 substituted for BLQ. The calculated average was reported if it was greater than LLOQ, whereas the average was reported as BLQ, if it was calculated to be less than or equal to LLOQ. Antibody conentrations were calculated from the immunoassay measurements, and are reported in Tables 18a-18b.

[0179] PK parameters were calculated from the mAb concentration measurements (Table 18c) and confirmed that the SARS-CoV2-YTE variant mAb (REGN13213) had an extended half life compared to the SARS-CoV2 parental mAb (REGN 10933). These observations validated the use of the FcRn^hu/hu B2m^hu/hu mouse model for measuring differences in PK parameters for YTE variants and parental antibodies.

Table 18a: Total concentrations of SARS-CoV2 parental mAb (REGN10933)

Table 18b: Total concentrations of SARS-CoV2-YTE variant mAb (REGN13213)

Table 18c: PK parameters of anti- SARS-CoV2 parental and YTE-varaint antibodies in

—. hu/hu hu/hu

FcRn B2m mice

Example 8: Comparison of PK profiles of REGN17653 and REGN9533

[0180] Having validated the FcRn^hu/hu B2m^hu/hu mouse model as a model to evaluate halflife extension of YTE variant antibodies, the PK profile of an anti-hFXII/FXIIa monoclonal antibody, REGN9533 and its Fc mutated variant (for half-life extension), REGN17653, as compared to an lgG4^p isotype control, REGN1945 was evaluated. These experiments were conducted in mice humanized for F12, FcRn, B2m and expressing fully human IgG, for the purpose of tolerization and reduction of anti-drug antibody responses. Cohorts contained 6-8 mice per tested antibody. Mice dosed with REGN9533, REGN 17653, and REGN1945, received a single intravenous (IV) 10 mg/kg dose. Blood samples were collected at 2 hours and 1 , 2, 3, 4, 7, 14, 21, 30, and 45-days post dosing. Blood was processed into plasma and frozen at -80 °C until analyzed. The functional serum concentrations of REGN9533, REGN 17653 and REGN 1945 were measured using the GyroLab xPlore platform (Gyros).

[0181] Gyros technology uses an affinity flow-through format for automated immunoassays with laser-induced fluorescence detection. Samples are loaded onto a compact disc (CD) which contains multiple radially arranged nanoliter-scale affinity capture columns. Liquid flow is controlled by centrifugal and capillary forces.

[0182] For the measurement of functional REGN9533, REGN 17653 in plasma, an acid dissociation step was performed to ensure that antibodies were not complexed with soluble FXII. This acid dissociation step was not necessary for REGN1945. Post the acid dissociation step, 20 pg/mL of a test article or control article -specific biotinylated capture reagent (anti-REGN9533 mAb, REGN 19721: for REGN9533 and REGN 17653 or Fel Dl.mmH, REGN612: for REGN1945) was added onto a Gyrolab Bioaffy 200 CD containing affinity columns preloaded with streptavidin-coated beads (Dynospheres). The standards used for calibration (REGN9533, REGN 17653) were run at concentrations ranging from 0.3 - 1300 ng/mL or 0.5-2000 ng/mL (REGN 1945). Standards or samples were prepared in 1M Tris-HCI containing normal mouse plasma (REGN9533, REGN 17653) or in phosphate buffered saline (PBS) with 0.5% bovine serum albumin (BSA), containing normal mouse plasma (REGN 1945). Singlets of plasma samples diluted at 1:3020 (REGN9533, REGN 17653) or 1:2000 (REGN 1945) and duplicates of standards were added onto the capture reagent-coated affinity columns at room temperature. Captured antibody was detected using Alexa-647-conjugated mouse anti-human hlgG4 specific monoclonal antibody (REGN1298 at 4 pg/mL) diluted in Rexxip F buffer (Gyros); the resultant fluorescent signal was recorded in response units (RU) by the GyroLab xPlore instrument. The respective assay’s lower limit of quantitation (LLOQ) was defined as the lowest concentration on the standard curve multiplied by the sample dilution factor (1 pg/mL). Sample concentrations were determined by interpolation from a standard curve that was constructed using a 4-parameter logistic curve fit in Gyrolab Evaluator Software. Average concentrations from 2 replicate experiments were used to calculate final concentrations. This assay does not differentiate between bound vs unbound hFXII/FXIIa, and does not detect hFXIIb. Antibody concentrations were evaluated over 45 days, and pharmacokinetic (PK) parameters were determined. Samples from Day 60 were not analyzed since the concentrations of the parental mAb were undetectable by day 45.

[0183] For quantification of antibody and protein concentrations, the lower limit of quantitation (LLOQ) for anti-hFXI l/FXI la and isotype-control antibodies was set at 1 pg/ml, and LLOQ for hFXII/FXIIa levels was set at 0.2 pg/ml. The baseline protein concentration was determined to be 11.8 pg/ml from pre-bleed samples For calculation of average concentrations, if more than 50% of values were below limit of quantitation (BLQ), the average was reported as BLQ, whereas if 50% or fewer values were BLQ, averages were calculated with 0 substituted for BLQ. The calculated average was reported if it was greater than LLOQ, whereas the average was reported as BLQ, if it was calculated to be less than or equal to LLOQ.. FIGs. 1A-1C show the time course of mAb concentrations and hFXII/FXIIa concentrations measured for each individual mouse in the 3 treatment groups during the course of this study. Average concentrations were calculated from these mesaurements for each treatment group, and are listed as a function of time in Tables 19a- 19f.

[0184] The calculated average concentrations reveal that concentrations of the REGN 17653 antibody decreased steadily over time in a linear fashion until Day 30. After Day 30, 5 out of 6 mice in the isotype control group, and 6 out of 7 mice in the REGN 17653 group exhibited rapid clearance, resulting in the majority of mice being BLQ at Day 45, although one mouse in each treatment group had a measurable mAB concentration at Day 45.

[0185] The average FXII/FXIIa protein concentrations show that peak total hFXII/FXIIa concentrations increased 20-fold over baseline at Day 7 for REGN17653 (anti-hFXII/FXIIa-YTE mAb), compared to 7-fold at Day 3 for REGN9533 (parental Anti-h FXI l/FXI la mAb). The isotype control showed an initial increase of ~3-fold over baseline and concentrations remained around that level throughout the study. hFXII/FXIIa concentrations were observed to return to around the same concentrations as isotype control after Day 21 for REGN 17653 and after Day 7 for REGN9533.

[0186] Next, antibody concentrations corresponding to peak hFXII/FXIIa levels were calculated for each treatment group and are listed in Table 20. Here, antibody concentrations were converted to units of nM based on the FXI I molecular weight of 80 kDa, with the baseline antibody level of 11.8 pg/ml corresponding to a concentration of 133 nM. The data reveal that the average concentration of parental REGN9533 was 536 nM at peak hFXII/FXIIa levels of 953 nM on Day 3, while the concentration of YTE-variant REGN17653 was 528 nM at peak hFXII/FXIIa levels of 2559 nM on Day 7.

[0187] Finally, PK parameters were calculated from the mAb concentration measurements for each treatment group and are listed in Table 21. A comparison of the PK profiles revealed that the YTE-variant REGN 17653 exhibits longer half-life, higher drug exposure and slower clearance rate as compared to its parental mAb REGN9533 and control mAb. [0188] Collectively, the mouse studies reveal that a YTE variant of an anti- hFXII/FXIIa monoclonal antibody has an extended half-life compared to the parental antibody. Table 19a: Total concentrations of anti-hFXI l/FXI la parental mAb (REGN9533)

Table 19b: Total concentrations of anti-hFXI l/FXIIa YTE variant mAb (REGN17653)

Table 19c: Total concentrations of anti-Fel D1 control mAb (REGN1945)

Table 19d: Total hFXII/FXIIa concentrations in anti-hFXI l/FXI la parental mAb (REGN9533) treatment group

Table 19e: Total hFXII/FXIIa concentrations in anti-hFXI l/FXI la YTE variant mAb (REGN17653) treatment group

Table 19f: Total hFXII/FXI la concentrations in anti-Fel D1 control mAb (REGN1945) group

Table 20: Average Molar Concentrations of anti-hFXI l/FXI la mAbs and isotype control with corresponding hFXII/FXI la Concentrations in hlgHC-KC (het) x FcRnh^u/hu B2m^hu/hu x F12 ^hu/hu mice

Table 21: PK parameters of REGN 17653 and REGN9533 mAbs in hlgHC-KC (het) x

FcRnh^u/hu B2m^hu/hu x F12 ^hu/hu mice

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

INFORMAL SEQUENCE LISTING

SEQ ID NO: 1.

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGCACTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTGGTAGTGGTGGTAAC

ACATACTACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGA ACTCGCTGTATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGT GCGAGCTTCATACCAGCTGCCATAAGAGGGGGCGACTGGATCGACCCCTGGGGCCAG GGAACCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 2.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTY YADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVT VSS;

SEQ ID NO: 3.

GGA TTC ACC TTT AGC AGC TAT GCC;

SEQ ID NO: 4.

G F T F S S Y A;

SEQ ID NO: 5.

ATT GGT GGT AGT GGT GGT AAC ACA;

SEQ ID NO: 6.

I G G S G G N T;

SEQ ID NO: 7.

GCG AGC TTC ATA CCA GCT GCC ATA AGA GGG GGC GAC TGG ATC GAC CCC;

SEQ ID NO: 9.

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCGAGTCAGGGCATAAGAAATTATTTAGCCTGGTATCAACAGAAA CCAGGGAAGATTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCC ATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG CAGCCTGAAGATATTGCAACTTATTACTGTCAATACTATAACAGTGCCCCGCTCACTTTC GGCGGAGGGACCAAGGTGGAGATCAAA;

SEQ ID NO: 10.

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKIPKLLIYAASTLQSGVPSRF SGSGSGTDFTLTISSLQPEDIATYYCQYYNSAPLTFGGGTKVEIK;

SEQ ID NO: 11.

CAG GGC ATA AGA AAT TAT;

SEQ ID NO: 12.

Q G I R N Y;

SEQ ID NO: 13.

GCT GCA TCC; SEQ ID NO: 14.

A A S;

SEQ ID NO: 15.

CAA TAG TAT AAC AGT GCC CCG CTC ACT;

SEQ ID NO: 16.

Q Y Y N S A P L T;

SEQ ID NO: 17.

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGCACTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTGGTAGTGGTGGTAAC ACATACTACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGA

ACTCGCTGTATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGT GCGAGCTTCATACCAGCTGCCATAAGAGGGGGCGACTGGATCGACCCCTGGGGCCAG GGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG

CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGG

ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG

TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAG CCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCAC

CCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACC

CAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGT GAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA

TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAG

CGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGT CTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG

CCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAAC

CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT

GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGG AGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACA

GAAGTCCCTCTCCCTGTCTCTGGGTAAATGA;

SEQ ID NO: 18.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTY

YADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVT

VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS

VFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG N VFSCSVM H EALH N H YTQKSLSLSLG K*;

SEQ ID NO: 19.

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCGAGTCAGGGCATAAGAAATTATTTAGCCTGGTATCAACAGAAA

CCAGGGAAGATTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCC

ATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG

CAGCCTGAAGATATTGCAACTTATTACTGTCAATACTATAACAGTGCCCCGCTCACTTTC GGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGG GTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGA AGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAG;

SEQ ID NO: 20.

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKIPKLLIYAASTLQSGVPSRF SGSGSGTDFTLTISSLQPEDIATYYCQYYNSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*;

SEQ ID NO: 21.

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCA

CTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGA

TCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAAGACATACTACAGGTCCAA

GTGGTATAATGATTATACAAAATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATC CAAGAACCACTTCTCCCTGCAACTGAACTCTATGACTCCCGAGGACACGGCTGTGTATT ACTGTGCAAGAGAGGTTAGTGGGAGGTACAACTGGTTCGACTCCTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCA;

SEQ ID NO: 22.

QVQLQQSG PG LVKPSQTLSLTCAI SG DSVSSNSAAWN Wl RQSPSRG LEWLG KTYYRSKW YNDYTKSVKSRITINPDTSKNHFSLQLNSMTPEDTAVYYCAREVSGRYNWFDSWGQGTLV TVSS;

SEQ ID NO: 23.

GGG GAC AGT GTC TCT AGC AAC AGT GCT GCT;

SEQ ID NO: 24.

G D S V S S N S A A;

SEQ ID NO: 25.

ACA TAG TAG AGG TGC AAG TGG TAT AAT;

SEQ ID NO: 26.

T Y Y R S K W Y N;

SEQ ID NO: 27.

GCA AGA GAG GTT AGT GGG AGG TAG AAC TGG TTC GAC TCC;

SEQ ID NO: 28.

A R E V S G R Y N W F D S;

SEQ ID NO: 29.

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGACCATTAACAGTTACTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCGAAGTGGGGTCC CATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTACAGAACCTTCACTTTCGGC GGAGGGACCAAGGTGGAGATCAAA;

SEQ ID NO: 30.

DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWYQQKPGKAPKLLIYAASSLRSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQNYRTFTFGGGTKVEIK;

SEQ ID NO: 31.

CAG ACC ATT AAC AGT TAC;

SEQ ID NO: 32.

Q T I N S Y;

SEQ ID NO: 33.

CAA CAG AAT TAC AGA ACC TTC ACT;

SEQ ID NO: 34.

Q Q N Y R T F T;

SEQ ID NO: 35.

CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCA

CTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGA

TCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAAGACATACTACAGGTCCAA

GTGGTATAATGATTATACAAAATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATC

CAAGAACCACTTCTCCCTGCAACTGAACTCTATGACTCCCGAGGACACGGCTGTGTATT

ACTGTGCAAGAGAGGTTAGTGGGAGGTACAACTGGTTCGACTCCTGGGGCCAGGGAA

CCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCC

CTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTA

CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA

CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC

GTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCA

GCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTG

CCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAG

GACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGC

CAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT

GCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTC

CTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCA

ACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG

AGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGT

CAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGA

GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA

CGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGG GAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGT CCCTCTCCCTGTCTCTGGGTAAATGA;

SEQ ID NO: 36.

QVQLQQSG PG LVKPSQTLSLTCAI SG DSVSSNSAAWN Wl RQSPSRG LEWLG KTYYRSKW

YNDYTKSVKSRITINPDTSKNHFSLQLNSMTPEDTAVYYCAREVSGRYNWFDSWGQGTLV

TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

PSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK*;

SEQ ID NO: 37.

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCA

CCATCACTTGCCGGGCAAGTCAGACCATTAACAGTTACTTAAATTGGTATCAGCAGAAA

CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCGAAGTGGGGTCC CATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG

CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTACAGAACCTTCACTTTCGGC

GGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCC

CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC

TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA

ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCA

GCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT

CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTT

AG;

SEQ ID NO: 38.

DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWYQQKPGKAPKLLIYAASSLRSGVPSRF

SGSGSGTDFTLTISSLQPEDFATYYCQQNYRTFTFGGGTKVEIKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKHKVYACEVTHQGLSSPVTKSFNRGEC*;

SEQ ID NO: 39.

GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAG

ACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGCACTGGGTCCGC

CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTGGTAGTGGTGGTAAC

ACATACTACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGA

ACTCGCTGTATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGT

GCGAGCTTCATACCAGCTGCCATAAGAGGGGGCGACTGGATCGACCCCTGGGGCCAG

GGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG

CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGG

ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG

TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT

GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAG

CCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCAC

CCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACC

CAAGGACACTCTCTACATCACCCGGGAGCCTGAGGTCACGTGCGTGGTGGTGGACGT

GAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA

TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAG

CGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGT

CTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG

CCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAAC

CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT

GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT

CCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGG

AGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACA

GAAGTCCCTCTCCCTGTCTCTGGGTAAATGA

SEQ ID NO: 40.

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTY

YADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVT

VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS

VFLFPPKPKDTLYITREPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

RWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN

QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK

Claims

CLAIMS What is claimed is:

1. An antibody, or antigen-binding fragment thereof, that binds to human Factor XII (FXI I) , wherein the antibody, or antigen-binding fragment thereof, comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein the HCDR1 has an amino acid sequence selected from SEQ ID NOs: 4 and 24, the HCR2 has an amino acid sequence selected from SEQ ID NOs: 6 and 26, and the HCDR3 has an amino acid sequence selected from SEQ ID NOs: 8 and 28, wherein the LCDR1 has an amino acid sequence selected from SEQ ID NOs: 12 and 32, the LCR2 has an amino acid sequence of SEQ ID NO: 14, and the LCDR3 has an amino acid sequence selected from SEQ ID NOs: 16 and 34.

2. The antibody, or antigen binding portion thereof, of claim 1 , wherein:

(a) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4;

(b) the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6;

(c) the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 8;

(d) the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 12;

(e) the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 14; and

(f) the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 16; or wherein:

(a) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 24;

(b) the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 26;

(c) the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 28;

(d) the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 32;

(f) the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 34.

3. The antibody, or antigen binding portion thereof, of any one of the previous claims, wherein: the heavy chain variable region comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 2 and the light chain variable region comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 10; or the heavy chain variable region comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 22 and the light chain variable region comprises a sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 30.

4. The antibody, or antigen binding portion thereof, of any one of the previous claims, wherein: the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; or the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 22 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 30.

5. The antibody, or antigen-binding portion thereof, of any one of the previous claims, comprising a heavy chain (HC) and a light chain (LC), wherein: the heavy chain comprises a sequence having at least 90% identity to SEQ ID NO: 18, and the light chain comprises a sequence having at least 90% identity to SEQ ID NO: 20; the heavy chain comprises a sequence having at least 90% identity to SEQ ID NO: 36, and the light chain comprises a sequence having at least 90% identity to SEQ ID NO: 38; or the heavy chain comprises a sequence having at lest 90% identity to SEQ ID NO: 40, and the light chain comprises a sequence having at least 90% identity to SEQ ID NO: 20.

6. The antibody, or antigen-binding portion thereof, of any one of the previous claims, comprising a heavy chain (HC) and a light chain (LC), wherein: the heavy chain comprises a sequence of SEQ ID NO: 18, and the light chain comprises a sequence of SEQ ID NO: 20; or the heavy chain comprises a sequence of SEQ ID NO: 36, and the light chain comprises a sequence of SEQ ID NO: 38.

7. The antibody, or antigen-binding portion thereof, of any one of claims 1-5, comprising a heavy chain (HC) and a light chain (LC), wherein: the heavy chain comprises a sequence of SEQ ID NO: 40, and the light chain comprises a sequence of SEQ ID NO: 20.

8. An antibody, or antigen-binding fragment thereof, that binds to human FXII/FXIIa, wherein the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs (HCDR1 , HCDR2 and HCDR3) contained within a HCVR and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a LCVR;

(a) wherein the HCVR comprises an amino acid sequence having at least 90% identity SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10; or

(b) wherein the HCVR comprises an amino acid sequence having at least 90% identity SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 30.

9. The antibody, or antigen-binding fragment thereof, of claim 8,

(a) wherein the HCVR comprises an amino acid sequence having at least 95% identity SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10; or

(b) wherein the HCVR comprises an amino acid sequence having at least 95% identity SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 30.

10. The antibody, or antigen-binding fragment thereof, of claim 8 or claim 9,

(a) wherein the HCVR comprises an amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO: 10; or

(b) wherein the HCVR comprises an amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO: 30.

11. The antibody, or antigen-binding fragment thereof, of any one of claims 8-10,

(a) wherein the HCVR comprises an amino acid sequence of SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence of SEQ ID NO: 10; or

(b) wherein the HCVR comprises an amino acid sequence of SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence of SEQ ID NO: 30.

12. The antibody, or antigen-binding fragment thereof, of any one of claims 8-11 , wherein: (a) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4;

(b) the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 6;

(c) the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 8;

(d) the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 12;

(a) the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 24;

(b) the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 26;

(c) the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 28;

(d) the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 32;

(f) the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 34.

13. The antibody, or antigen-binding fragment thereof, of any one of claims 8-12, comprising a heavy chain (HC) and a light chain (LC), wherein: the heavy chain comprises a sequence of SEQ ID NO: 18, and the light chain comprises a sequence of SEQ ID NO: 20; or the heavy chain comprises a sequence of SEQ ID NO: 36, and the light chain comprises a sequence of SEQ ID NO: 38.

14. The antibody, or antigen-binding fragment thereof, of any one of claims 8-12, comprising a heavy chain (HC) and a light chain (LC), wherein: the heavy chain comprises a sequence of SEQ ID NO: 40, and the light chain comprises a sequence of SEQ ID NO: 20.

15. An antibody, or antigen-binding fragment thereof, that binds to human Factor XI I (FXI I) , wherein the antibody, or antigen-binding fragment thereof, comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein

(a) the HCDR1 comprises an amino acid sequence G-F-T-F-S-S-Y-A (SEQ ID NO: 4);

(b) the HCDR2 comprises an amino acid sequence l-G-G-S-G-G-N-T (SEQ ID NO: 6); (c) the HCDR3 comprises an amino acid sequence A-S-F-l-P-A-A-l-R-G-G-D-W-l-D-P (SEQ ID NO: 8);

(d) the LCDR1 comprises an amino acid sequence Q-G-l-R-N-Y (SEQ ID NO: 12);

(e) the LCDR2 comprises an amino acid sequence A-A-S (SEQ ID NO: 14); and

(f) the LCDR3 comprises an amino acid sequence Q-Y-Y-N-S-A-P-L-T (SEQ ID NO: 16).

16. An antibody, or antigen-binding fragment thereof, that binds to Factor XII (FXI I) , wherein the antibody, or antigen-binding fragment thereof, comprises three heavy chain complementarity determining regions (CDRs) (HCDR1 , HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1 , LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein

(a) the HCDR1 comprises an amino acid sequence G-D-S-V-S-S-N-S-A-A (SEQ ID NO: 24);

(b) the HCDR2 comprises an amino acid sequence T-Y-Y-R-S-K-W-Y-N (SEQ ID NO: 26);

(c) the HCDR3 comprises an amino acid sequence A-R-E-V-S-G-R-Y-N-W-F-D-S (SEQ ID NO: 28);

(d) the LCDR1 comprises an amino acid sequence Q-T-l-N-S-Y (SEQ ID NO: 32);

(e) the LCDR2 comprises an amino acid sequence A-A-S (SEQ ID NO: 14); and

(f) the LCDR3 comprises an amino acid sequence Q-Q-N-Y-R-T-F-T (SEQ ID NO: 34).

17. The antibody, or antigen-binding fragment thereof, of any one of the previous claims, comprising an Fc domain comprising one or more mutations that alter Fc region function.

18. The antibody, or antigen-binding fragment thereof, of claim 15, wherein the Fc domain comprises a mutation in the CH2 or a CH3 region that increase FcyR binding activity.

19. The antibody, or antigen-binding fragment thereof, of claim 17 or claim 18, wherein the Fc domain comprises at least one mutation in one or more amino acids selected from the group consisting of amino acids at positions 248, 250, 252, 254, 256. 257, 307, 311, 376, 380, 428, 433 and 434.

20. The antibody, or antigen-binding fragment thereof, of claim 19, wherein the Fc domain comprises at least one mutation in one or more amino acids at positions 252, 254 and 256.

21. The antibody, or antigen-binding fragment thereof, of claim 19 or claim 20, wherein the at least one mutation in the Fc domain comprises: mutating position 252 to Y mutating position 254 to T; and/or mutating position 256 to E.

22. The antibody, or antigen-binding fragment thereof, of any one of claims 17-21, wherein the at least one mutation in the Fc domain increases the half-life of the antibody, or antigen-binding fragment thereof, in blood plasma compared to an antibody, or an antigenbinding fragment thereof, without the mutation.

23. The antibody, or antigen-binding fragment thereof, of claim 22, wherein the half-life is increased by at least 1.2-fold, preferably at least 1.5-fold, compared to an antibody, or an antigen-binding fragment thereof, without the mutation.

24. The antibody, or antigen-binding fragment thereof, of any one of the previous claims, comprising a modified glycosylation pattern, wherein the modification increases antibody dependent cellular cytotoxicity (ADCC) function and/or alters complement dependent cytotoxicity (CDC) activity.

25. The antibody, or antigen-binding fragment thereof, of any one of the previous claims, wherein the antibody or antigen-binding fragment thereof, is a monoclonal antibody, a bispecific antibody, a multi-specific antibody, or an antigen-binding fragment thereof.

26. The antibody, or antigen-binding fragment thereof, of claim 25, wherein the multispecific antibody binds different epitopes of FXII or contains antigen-binding domains specific for FXII and one or more additional target polypeptides.

27. The antibody, or antigen-binding fragment thereof, of any one of the previous claims, comprising one or more of the following characteristics:

(a) is a fully human monoclonal antibody;

(b) binds to activated Factor XI I (FXIIa, FXIIab);

(c) binds to FXII with a dissociation constant (KD) of less than 1.5nM at 25 °C;

(d) binds to FXII with a KD of less than 17nM at 37 °C;

(e) binds to FXIIa with a KD of less than 5nM, preferably less than 0.9nM, at 25 °C; (f) binds to FXIIa with a KD of less than 6.5nM, preferably less than 2.5nM, at 37 °C;

(g) binds to FXIIab with a KD of less than 5nM, preferably less than 0.7nM, at 25 °C;

(h) blocks thrombin generation by intrinsic pathway at a concentration of less than 250nM; and

(I) blocks thrombin generation by intrinsic pathway without blocking thrombin generation by extrinsic pathway; or any combinations thereof of (a)-(l).

28. The antibody, or the antigen binding fragment thereof, of any one of the previous claims, wherein the antibody is a humanized antibody or a chimeric antibody.

29. The antibody, or the antigen binding fragment thereof, of any one of the previous claims, wherein the antibody, or the antigen binding fragment thereof is chemically or biologically conjugated to a therapeutic moiety.

30. The antibody, or the antigen binding fragment thereof, of claim 29, wherein the conjugated moiety comprises a radioactive agent, a cytokine, an interferon, a target or reporter moiety, an enzyme, a second different antibody, a peptide or protein, or a therapeutic agent.

31. An antibody or antigen-binding fragment thereof that competes for binding to Factor XII with an antibody or antigen-binding fragment thereof of any one of the previous claims.

32. An antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof of any one of the previous claims.

33. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to Factor Xll/Xlla according to any one of the previous claims and a pharmaceutically acceptable carrier or diluent.

34. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a HCVR of the antibody or antigen-binding fragment thereof, as set forth in any one of claims 1-32.

35. An isolated polynucleotide molecule comprising a polynucleotide sequence that encodes a LCVR of the antibody or antigen-binding fragment thereof, as set forth in any one of claims 1-32.

36. A vector comprising the polynucleotide sequence of claim 34, the polynucleotide sequence of claim 35, or both the polynucleotide sequences of claim 34 and claim 35.

37. A cell expressing the vector of claim 36.

38. A method of producing an anti-FXI l/FXI la antibody or antigen-binding fragment thereof, comprising growing the cell of claim 37 under conditions permitting production of the antibody or antigen-binding fragment thereof, and recovering the antibody or fragment so produced.

39. The method of claim 38, further comprising formulating the antibody or antigenbinding fragment thereof as a pharmaceutical composition comprising an acceptable carrier.

40. A method of preventing the formation of a thrombus or treating a subject at risk of thrombus formation, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-32 to a subject in need thereof.

41. The method of claim 40, wherein the subject has a disease, disorder or condition selected from the group consisting of venous thrombosis, arterial thrombosis, device thrombosis, thromboembolism, hereditary angioedema, stroke, thrombophilia, cardiac ischemia, atherosclerotic plaque rupture, use of mechanical valve prostheses, use of bloodcontacting medical devices, use of blood-contacting extracorporeal circuits, venous thromboembolism, pulmonary embolism, deep vein thrombosis, portal vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, renal vein thrombosis, cerebral venous sinus thrombosis, jugular vein thrombosis, cavernous sinus thrombosis, hepatic artery thrombosis, limb ischemia and myocardial infarction.

42. The method of claims 40 or 41 , wherein the pharmaceutical composition is administered prophylactically or therapeutically to the subject in need thereof.

43. The method of any one of claims 40-42, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

44. The method of claim 43, wherein the second therapeutic agent is selected from the group consisting of an anti-coagulant, a direct thrombin inhibitor, a thrombolytic drug, a fibrinolytic drug, an anti-platelet drug, an anti-inflammatory drug, an anti-hypertensive drug, a second anti-FXII antibody, a lipid-lowering drug, mechanical clot retrieval, catheter-guided thrombolysis, compression stockings, and surgery.

45. The method of any one of claims 40-44, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

46. The method of any one of claims 40-45, wherein the pharmaceutical composition is administered at a dose of about 0.1 mg/kg of body weight to about 100 mg/kg of body weight of the subject.

47. The method of any one of claims 40-46, wherein the pharmaceutical composition is administered at one or more doses comprising between about 10 mg to about 600 mg to the subject.

48. The method of any one of claims 40-47, wherein the pharmaceutical composition is administered for more than one dose, wherein the subsequent doses are approximately the same or less than that of the initial dose, wherein the subsequent dose are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

49 A method of preventing the formation of a thrombus or treating a subject at risk of thrombus formation, the method comprising administering a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-32 prior to, concurrent with, or after one or more additional therapeutic agents to a subject in need thereof.

50. The method of claim 49, wherein the one or more additional therapeutic agents are selected from an anti-coagulant, a thrombin inhibitor, a thrombolytic drug, an anti-platelet drug, an antihypertensive, an immunosuppressive agent, a fibrinolytic agent, a cholesterol- lowering agent, an anti-inflammatory drug, a second anti-FXII antibody, mechanical clot retrieval, catheter-guided thrombolysis and surgery.

51. A method of diagnosing or detecting a FXII-associated-disease or disorder, the method comprising contacting a sample of a subject with one or more antibodies or antigenbinding fragment thereof of any one of claims 1-32, wherein the antibody or antigen-binding fragment thereof is detectably labeled.