Anti-factor XII / XIIa antibodies and uses thereof

Abstract

The present invention provides monoclonal antibodies that bind to Factor XII (FXII) proteins and methods of use thereof. In various embodiments of the invention, the antibodies are fully human antibodies that bind to FXII and an activated form of FXII (FXIIa). In some embodiments, the antibodies of the invention may be used to inhibit or neutralize the activity of FXII, thereby providing a means for the treatment or prevention of diseases, disorders or conditions associated with thrombosis in humans.

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 514,392, filed July 19, 2023, the entire contents of which are expressly incorporated herein by reference.

[0002] Sequence Listing This application contains a sequence list that has been electronically submitted in .XML format and is hereby incorporated herein by reference in its entirety. The .XML copy created on July 18, 2024, is named “118003-11720.xml” and is 56,608 bytes in size. The sequence list contained in this .XML file is part of the specification and is hereby incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to antibodies that specifically bind to coagulation factor XII and antigen-binding fragments of antibodies, as well as therapeutic and diagnostic methods using these antibodies. Background Technology

[0004] Coagulation factors, along with platelets, are essential blood components involved in hemostasis during vascular injury. It is well known that these components can be drivers of thrombosis when their regulation (i.e., production and / or activity) is imbalanced. Thrombotic diseases are thought to originate primarily from the aberrant activation of extrinsic pathways (via tissue factor), but recent studies have focused on… F12 Preclinical studies of defective mice and molecules targeting activated FXII (FXIIa) have shown that the intrinsic coagulation pathway (also known as the contact pathway) 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 inflammation via cleavage of plasma prokallikrein. Therefore, inhibiting FXII activity may lead to a reduction in thrombotic coagulation and inflammation associated with activation of the contact pathway (Schousboe 2007, Biochem. Pharmacol. 75: 1007-13; Danese et al. 2016, Semin. Thromb. Hemostat. 42: 682-8; Weitz 2016, Thromb. Res. 141 Supplement 2: S40-5). Currently used anticoagulants in this field... For example Heparin is an effective anticoagulant, but it also increases the risk of bleeding. Therefore, it is ideal to develop anticoagulants that prevent thrombosis without increasing the risk of bleeding.

[0005] Monoclonal antibodies against FXII are known in the art and have been described in, for example, U.S. Patent / Publication Nos. 4,963,657, 5,500,349, 8,119,137, 8,715,672, 985,6326, 985,6325, 951,8127, 20130095108, 20140072572, 20140072600, and WO2006066878, WO2013014092, EP1830924B1, EP27345522A1 and EP2623110A1.

[0006] A fully human antibody that binds specifically to (activated) FXII protein with high affinity and prevents thrombosis without increasing the risk of bleeding, for the prevention and treatment of various FXII-related diseases. For example It may be important in terms of thrombosis, embolism, and edema. Summary of the Invention

[0007] This disclosure provides antibodies that specifically bind to the activated form of coagulation factor XII protein (FXIIa) and their antigen-binding fragments. In some embodiments, these antibodies can also bind to the proenzyme form of factor XII (FXII). In some embodiments, these antibodies are fully human antibodies that bind to both FXII and FXIIa with high affinity (“dual FXII / FXIIa binding agents”). The antibodies of this disclosure... In particular These antibodies can be used to inhibit or neutralize the activity of FXII proteins. In some embodiments, these antibodies can be used to prevent, treat, or improve at least one symptom or sign of an FXII-related disease or condition in a subject. In some embodiments, these antibodies can be administered prophylactically or therapeutically to subjects who have or are at risk of developing an FXII-related disease or condition. In some preferred embodiments, these antibodies prevent thrombosis without increasing the subject's bleeding risk. Such antibodies can be used as anticoagulant therapy without increasing the bleeding risk when administered to subjects who require it, while reducing the frequency of administration in subjects with an FXII-related disease or condition.

[0008] The antibodies disclosed herein may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect functionality. For example To prolong persistence within the host or eliminate residual effector functions (Reddy) Et al. (J. Immunol. 164:1925-1933, 2000). In some embodiments, these antibodies may be bispecific.

[0009] In a first aspect, this disclosure provides isolated recombinant monoclonal antibodies or antigen-binding fragments thereof that specifically bind to factor XII (FXII) and / or activated factor XII (FXIIa). In some embodiments, these antibodies are fully human monoclonal antibodies. In some embodiments, these antibodies are "dual binders" capable of binding to both FXII and FXIIa.

[0010] Exemplary anti-FXII / FXIIa antibodies of this disclosure are listed in Tables 1 and 2 herein. Table 1 shows the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity-determining region (HCDR) (HCDR1, HCDR2, and HCDR3), and light chain complementarity-determining region (LCDR) (LCDR1, LCDR2, and LCDR3) of the exemplary anti-antibody. Table 2 shows the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary antibody.

[0011] This disclosure provides an antibody or antigen-binding fragment thereof comprising an HCVR containing an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0012] This disclosure also provides an antibody or antigen-binding fragment thereof comprising an LCVR, the LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0013] This disclosure also provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR amino acid sequence pairs (HCVR / LCVR) wherein any of the HCVR amino acid sequences listed in Table 1 is paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, this disclosure provides antibodies or antigen-binding fragments thereof comprising the HCVR / LCVR amino acid sequence pairs contained in any of the exemplary anti-FXII / FXIIa antibodies listed in Table 1. In some embodiments, the HCVR / LCVR amino acid sequence pairs are selected from one of the following: SEQ ID NO: 2 / 22 ( For example REGN9533) or 22 / 30 ( For example REGN9534).

[0014] This disclosure also provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises an amino acid sequence listed in Table 1 having no more than twelve amino acid substitutions, and / or the LCVR comprises an amino acid sequence listed in Table 1 having no more than ten amino acid substitutions. For example, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22, wherein the amino acid sequence has one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid substitutions. In another example, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30, wherein the amino acid sequence has one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions. In one embodiment, this disclosure provides anti-FXII / FXIIa antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22, the amino acid sequence having at least one amino acid substitution, and / or the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30, the amino acid sequence having at least one amino acid substitution.

[0015] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] This disclosure also provides an antibody or antigen-binding fragment 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0021] This disclosure also provides antibodies or antigen-binding fragments thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any one of the HCDR3 amino acid sequences listed in Table 1 paired with any one of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, this disclosure provides antibodies or antigen-binding fragments thereof comprising the HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-FXII antibodies listed in Table 1. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of: SEQ ID NO: 2 / 22 ( For example REGN9533) or 22 / 30 ( For example REGN9534).

[0022] This disclosure also provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises: HCDR1, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid; HCDR2, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid; and HCDR3, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid. In some embodiments, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the LCVR comprises: LCDR1, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid; LCDR2, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid; and LCDR3, which comprises an amino acid sequence differing from the amino acid sequences listed in Table 1 by one amino acid. For example, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises: HCDR1, comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence differing from SEQ ID NO: 4 by one amino acid; HCDR2, comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence differing from SEQ ID NO: 6 by one amino acid; and HCDR3, comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence differing from SEQ ID NO: 8 by one amino acid. In another exemplary embodiment, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, the LCVR comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence differing from SEQ ID NO: 12 by one amino acid; LCDR2 comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by one amino acid; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence differing from SEQ ID NO: 16 by one amino acid.

[0023] As another exemplary example, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the HCVR comprises: HCDR1, comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence differing from SEQ ID NO: 24 by one amino acid; HCDR2, comprising the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence differing from SEQ ID NO: 26 by one amino acid; and HCDR3, comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence differing from SEQ ID NO: 28 by one amino acid. In another exemplary embodiment, this disclosure provides antibodies or antigen-binding fragments thereof comprising HCVR and LCVR, wherein the LCVR comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence differing from SEQ ID NO: 32 by one amino acid; LCDR2 comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by one amino acid; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence differing from SEQ ID NO: 34 by one amino acid.

[0024] This disclosure also provides antibodies or antigen-binding fragments thereof that comprise a set of six CDRs contained within any of the exemplary anti-antibodies listed in Table 1. That is HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3). In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence group is selected from the group consisting of: SEQ ID NO: 4-6-8-12-14-16 ( For example (REGN9533) or 24-26-28-32-14-34 ( For example REGN9534).

[0025] In relevant embodiments, this disclosure provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs contained within an HCVR / LCVR amino acid sequence pair as defined by any of the exemplary antibodies listed in Table 1. That isHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3). For example, this disclosure includes an antibody or antigen-binding fragment thereof comprising the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set contained within the HCVR / LCVR amino acid sequence pair, the amino acid sequence set being selected from the group consisting of: SEQ ID NO: 2 / 10 ( For example REGN9533) or 10 / 30 ( For example Methods and techniques for identifying CDRs within the amino acid sequences of HCVR and LCVR 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 specifications that can be used to identify CDR boundaries include... For example Kabat, Chothia, and AbM definitions exist. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example , Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani Et al. , J. Mol. Biol. 273 :927-948 (1997); and Martin Et al. , Proc. Natl. Acad. Sci. USA 86 :9268-9272 (1989). Public databases can also be used to identify CDR sequences within antibodies.

[0026] In some embodiments, this disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to FXII and / or FXIIa, wherein the antibody or antigen-binding fragment 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) an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22; (ii) an amino acid sequence having at least 90% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22; (iii) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22; or (iv) an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 22 with no more than 12 amino acid substitutions; and the LCVR comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30; (ii) an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30; (iii) an amino acid sequence having at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30; (iv) an amino acid sequence having at least 12 amino acid substitutions; and the LCVR comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and 30; (iv) an amino acid sequence having at least 95% identity with ... and (iv) an amino acid sequence having at least 95% identity with an amino acid sequence The amino acid sequences of the group consisting of SEQ ID NO: 10 and 30 have at least 90% identity; (iii) the amino acid sequences of the group consisting of SEQ ID NO: 10 and 30 have at least 95% identity; or (iv) the amino acid sequences of the group consisting of SEQ ID NO: 10 and 30 have no more than 10 amino acid substitutions.

[0027] This disclosure provides anti-FXII / FXIIa 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 with SEQ ID NO: 18, and the light chain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 20. In another embodiment, the heavy chain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 36, and the light chain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 38. In some embodiments, the heavy chain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 40, and the light chain comprises a sequence having at least 90% sequence identity with SEQ ID NO: 20.

[0028] In some embodiments, this disclosure provides anti-FXII / FXIIa antibodies or antigen-binding fragments thereof comprising an Fc domain containing one or more mutations that alter the function of the Fc region. In some embodiments, the Fc domain contains CH 2 or C H Mutations in region 3 that enhance FcγR binding activity. In some embodiments, the Fc domain comprises at least one mutation of one or more amino acids selected from the group consisting of amino acids at the following 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 of 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 its antigen-binding fragment in plasma compared to an antibody or its antigen-binding fragment 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 its antigen-binding fragment without the mutation.

[0029] This disclosure includes anti-FXII / FXIIa antibodies having modified glycosylation patterns. In some embodiments, modifications may be used, for example, to remove unwanted glycosylation sites, or antibodies lacking the fucose moiety present on the oligosaccharide chain, to improve antibody-dependent cytotoxicity (ADCC) function (see Shield). Et al. (2002) JBC 277:26733). In other applications, galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

[0030] In some embodiments, this disclosure provides antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to FXII. For example, this disclosure includes antibodies and antigen-binding fragments thereof that bind to FXII with higher affinity at neutral pH than at acidic pH. That is (The binding decreases at acidic pH).

[0031] This disclosure also provides antibodies and antigen-binding fragments thereof that compete with antibodies or antigen-binding fragments thereof containing a CDR of HCVR and a CDR of LCVR for specific binding to FXII or FXIIa, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0032] This disclosure also provides antibodies and antigen-binding fragments thereof that cross-compete with reference antibodies or antigen-binding fragments thereof that contain a CDR of HCVR and a CDR of LCVR for binding to FXII / FXIIa, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0033] This disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitopes as a reference antibody or antigen-binding fragment thereof comprising three CDRs of HCVR and three CDRs of LCVR, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0034] In some embodiments, the antibody or its antigen-binding fragment may bind specifically to FXII and / or FXIIa in an agonist manner, i.e., it may enhance or stimulate the binding and / or activity of FXII / FXIIa; in other embodiments, the antibody may bind specifically to FXII and / or FXIIa in an antagonist manner, i.e., it may block the binding and / or activity of FXII.

[0035] This disclosure also provides isolated antibodies and their antigen-binding fragments that block the binding of FXIIa to FXI. In some embodiments, the antibody or its antigen-binding fragment that blocks the binding of FXIIa to FXI may bind to the same epitope on FXIIa as that on FXI, or it may bind to an epitope on FXIIa that is different from that on FXI.

[0036] In some embodiments, the antibody or antigen-binding fragment of this disclosure is bispecific, including having 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 epitope and the second epitope are different and do not overlap.

[0037] In some embodiments, this disclosure provides an isolated antibody or antigen-binding fragment thereof having one or more of the following characteristics: (a) it is a fully human monoclonal antibody; (b) it binds to activated factor XII (FXIIa); and (c) it exhibits a dissociation constant (K0) of less than 1.5 nM at 25°C. D (d) Combined with FXII, as measured by surface plasmon resonance; (d) at 37 °C with a K0 of less than 17 nM. D Combined with FXII, as measured by surface plasmon resonance; (e) at 25°C with a Ki of less than 5 nM, preferably less than 0.9 nM. DCombined with FXIIa, as measured by surface plasmon resonance; (f) at 37°C with a K0 of less than 6.5 nM, preferably less than 2.5 nM. D (g) To bind to FXIIa, as measured by surface plasmon resonance assay; (h) To block thrombin production via the intrinsic pathway at concentrations less than 250 nM, as measured by functional plasma assay; and / or (h) To block thrombin production via the intrinsic pathway without blocking thrombin production via the extrinsic pathway, as measured by functional plasma assay.

[0038] In a second aspect, this disclosure provides nucleic acid molecules encoding anti-FXII / FXIIa antibodies or portions thereof. For example, this disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0039] This disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0040] This disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0041] This disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0042] This disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0043] This disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0044] This disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0045] This disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in some embodiments, the nucleic acid molecule comprises: a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a sequence substantially similar to the polynucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0046] This disclosure also provides a nucleic acid molecule encoding HCVR, wherein the HCVR comprises a set of three CDRs ( That is , HCDR1-HCDR2-HCDR3), where the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined by any of the exemplary antibodies listed in Table 1.

[0047] This disclosure also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs ( That is (LCDR1-LCDR2-LCDR3), where the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined as any of the exemplary antibodies listed in Table 1.

[0048] This disclosure also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In some 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it. In some embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes HCVR and LCVR, wherein both HCVR and LCVR are derived from the same anti-FXII antibody listed in Table 1.

[0049] In a related respect, this disclosure provides recombinant expression vectors capable of expressing polypeptides including heavy or light chain variable regions of antibodies. For example, this disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above. That is The nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences shown in Table 2. In some embodiments, this disclosure provides expression vectors comprising: (a) a nucleic acid molecule containing a nucleic acid sequence encoding an HCVR that binds to an antibody against FXII / 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 containing a nucleic acid sequence encoding an LCVR that binds to an antibody against 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 this disclosure are host cells into which such vectors have been introduced, and methods for producing antibodies or portions thereof by culturing host cells under conditions that allow for the production of antibodies or antibody fragments, and for recovering antibodies and antibody fragments thus produced. In some embodiments, these host cells comprise mammalian cells or prokaryotic cells. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells or Escherichia coli (E. coli). Escherichia coli ( E. coliCells. In some embodiments, this disclosure provides methods for producing antibodies or antigen-binding fragments thereof, the methods comprising: introducing an expression vector into a host cell, the expression vector comprising a nucleic acid sequence encoding an HCVR and / or LCVR of the antibody or an antigen-binding fragment thereof, the nucleic acid sequence being operatively linked to a promoter; culturing the host cell under conditions favorable to the expression of the nucleic acid sequence; and isolating the antibody or antigen-binding fragment thereof from the culture medium and / or the host cell. The isolated antibody or antigen-binding fragment thereof may be purified using any method known in the art.

[0050] In a third aspect, this disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to FXII / FXIIa, and a pharmaceutically acceptable carrier. In a related aspect, this disclosure features a composition that is a combination of an anti-FXII / FXIIa antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent advantageously combined with the anti-FXII / FXIIa antibody. Exemplary agents that can be advantageously combined with the anti-FXII / FXIIa antibody include, but are not limited to: other agents that bind and / or inhibit FXII activity (including other antibodies or antigen-binding fragments thereof), and / or agents that do not directly bind to FXII but still treat or improve at least one symptom or sign of FXII-related diseases or conditions. Further combination therapies and co-preparations involving the anti-FXII antibody of this disclosure are disclosed elsewhere herein.

[0051] In a fourth aspect, this disclosure provides methods for treating a subject with an FXII-related disease or condition using an anti-FXII / FXIIa antibody or an antigen-binding portion of an antibody of this disclosure, wherein the treatment method comprises administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising an antibody or an antigen-binding fragment of an antibody of this disclosure. The treated condition is any disease or condition that is improved, alleviated, suppressed, or prevented by inhibiting FXII activity. In some embodiments, this disclosure provides methods for preventing or treating FXII-related diseases or conditions, comprising administering to a subject in need a therapeutically effective amount of an anti-FXII / FXIIa antibody of this disclosure or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof may be administered prophylactically or therapeutically to a subject who has or is at risk of developing an FXII-related disease or condition. In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure is administered to a subject in need in combination with a second therapeutic agent. The second therapeutic agent may be selected from the group consisting of: anticoagulants, direct thrombin inhibitors, thrombolytic agents, fibrinolytic agents, antiplatelet agents, anti-inflammatory agents, antihypertensive agents, secondary anti-FXII antibodies, lipid-lowering agents, mechanical thrombectomy, catheter-guided thrombolysis, compression stockings, surgery, and any other drugs or therapies known in the art. In some embodiments, the second therapeutic agent may be an agent for counteracting or mitigating any possible side effects associated with the antibody or its antigen-binding fragment of the present disclosure (if such side effects occur). The antibody or its fragment may be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or its fragment may be administered at a dose of about 0.1 mg / kg of subject body weight to about 100 mg / kg of subject body weight. In some embodiments, the antibody of the present disclosure may be administered at one or more doses comprising about 10 mg to about 600 mg.

[0052] This disclosure also includes the use of the anti-FXII / FXIIa antibody or antigen-binding fragment thereof in the manufacture of a medicament for treating a disease or condition that would benefit from blocking FXII / FXIIa binding and / or activity.

[0053] Other embodiments will become apparent from the following detailed description. Attached Figure Description

[0054] Figure 1A The changes in mAb and hFXII / FXIIa concentrations over time are shown after each individual mouse was administered a single dose of 10 mg / kg of the anti-hFXII / FXIIa parental antibody (REGN9533). Figure 1BThe changes in mAb concentration and hFXII / FXIIa concentration over time are shown after each individual mouse was administered a single dose of 10 mg / kg of the anti-hFXII / FXIIa YTE variant antibody (REGN17653). Figure 1C The changes in mAb concentration and hFXII / FXIIa concentration over time are shown after each individual mouse was given a single dose of 10 mg / kg of the anti-Fel D1 control antibody (REGN1945). Detailed Implementation

[0055] Before describing the methods of this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference in their entirety.

[0057] Definition The term "FXII," also known as "Factor XII," refers to coagulation factor XII, also called Hagmann factor. The FXII protein, composed of 596 amino acids, is the zymogen form of factor XIIa. It is a β-globulin plasma serine protease composed of an N-terminal heavy chain and a C-terminal light chain. Its heavy chain contains two fibronectin-type domains (type I and type II), two epidermal growth factor-like domains, a Klinger domain, and a proline-rich region, while its light chain contains the protease domain. Exposure of blood to negatively charged substances or artificial surfaces triggers thrombin production and fibrin formation via a series of reactions called contact activation. Factor XII activates factor XI and prokallikrein in the coagulation cascade. Factor XII itself can be activated into factor XIIa by plasma kallikrein, platelet or bacterial polyphosphates, extracellular DNA or RNA, heparin released from mast cells, amyloid peptides, misfolded protein aggregates, and negatively charged surfaces such as glass. This is the starting point of the endogenous pathway. The amino acid sequence of the full-length FXII protein is exemplified by the amino acid sequence provided in UniProtKB / Swiss-Prot accession number P00748.3. The amino acid sequence of the full-length FXII protein is also shown in SEQ ID NO: 65 herein. The term “FXII” includes recombinant FXII protein or fragments thereof. The term also includes FXII protein or fragments thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signaling sequence such as ROR1.

[0058] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule that comprises four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds. That is "Whole antibody molecule" and its multimer ( For example IgM or its antigen-binding fragment. Each heavy chain contains a heavy chain variable region (“HCVR” or “V”). H ") and heavy-chain constant regions (containing domain C) H 1. C H 2 and C H 3). Each light chain contains a light chain variable region (“LCVR” or “V”). L ") and light chain constant region (C L V H and V L The region can be further subdivided into highly variable regions known as complementarity-determining regions (CDRs), interspersed with more conservative regions known as framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, the FRs of the antibody (or its antigen-binding fragment) may be identical to human germline sequences, or may be natural or artificially modified. The common amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0059] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies in which one or two CDRs can be omitted for binding have been described in the scientific literature. Padlan Et al. (1995 FASEB J. 9:133-139) Based on published crystal structure analysis, the contact region between the antibody and its antigen was analyzed, concluding that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies with one or two CDRs lacking amino acids that contact the antigen (see also Vajdos). Et al. 2002 J Mol Biol 320:415-428).

[0060] Based on previous research (e.g., residues H60-H65 in CDRH2 are generally not essential), CDR residues that do not contact the antigen can be identified from regions of the Kabat CDR located outside the Chothia CDR using molecular modeling and / or empirical methods. If the CDR or its residues are omitted, they are typically substituted with amino acids occupying corresponding positions in another human antibody sequence or in consensus sequences of such sequences. Substitution positions within the CDR and the amino acids to be substituted can also be selected empirically. Empirical substitutions can be either conserved or non-conserved.

[0061] Compared to the corresponding germline sequences, the fully human anti-FXII monoclonal antibody disclosed herein may include one or more amino acid substitutions, insertions, and / or deletions in the frame and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. This disclosure includes antibodies derived from any amino acid sequence disclosed herein, and their antigen-binding fragments, wherein one or more amino acids in one or more frame and / or CDR regions are mutated to corresponding residues in the germline sequence of the derived antibody, or mutated to corresponding residues in another human germline sequence, or mutated to conserved amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, those skilled in the art can readily generate numerous antibody and antigen-binding fragments comprising one or more single germline mutations or combinations thereof. In some embodiments, V H and / or V L All framework and / or CDR residues within the domain are mutated back to residues found in the original germline sequence of the derived antibody. In other embodiments, only certain residues are mutated back to the original germline sequence. For example Mutated residues found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only in CDR1, CDR2, or CDR3. In other embodiments, one or more of the frame and / or CDR residues are mutated into different germline sequences. That is The corresponding residues of the germline sequence (different from the germline sequence of the originally derived antibody). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations within the framework and / or CDR region. For example In this process, certain individual residues are mutated to corresponding residues in a specific germline sequence, while other residues different from the original germline sequence are maintained or mutated to corresponding residues in different germline sequences. Once obtained, one or more desired properties of antibody and antigen-binding fragments containing one or more germline mutations can be readily tested, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibody and antigen-binding fragments obtained in this general manner are covered within this disclosure.

[0062] This disclosure also includes fully human anti-FXII monoclonal antibodies comprising variants of any one of the disclosed HCVR, LCVR, and / or CDR amino acid sequences having one or more conserved substitutions. For example, this disclosure includes anti-FXII antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, relative to any one of the disclosed HCVR, LCVR, and / or CDR amino acid sequences, having For example , with 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., of the conserved amino acid substitutions.

[0063] As used herein, the term "human antibody" is intended to comprise antibodies having variable and constant regions derived from human immunoglobulin sequences. The human mAbs of this disclosure may comprise amino acid residues not encoded by human immunoglobulin sequences ( For example ,pass In vitro Random or site-specific mutagenesis formed or through In vivo Mutations introduced by somatic mutations), such as in CDR, and particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include those derived from another mammalian species ( For example The term refers to mAbs whose CDR sequences (in mice) have been grafted onto human FR sequences. This term includes antibodies recombined in or generated in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated or generated from human subjects.

[0064] As used herein, the term "recombination" refers to techniques or methods known in the art as recombinant DNA technologies (including...). For example The antibodies or antigen-binding fragments thereof disclosed herein are produced, expressed, isolated, or obtained through DNA splicing and transgenic expression. This term refers to antibodies produced, expressed, isolated, or obtained in non-human mammals (including transgenic non-human mammals). For example Transgenic mice) or cells ( For example Antibodies expressed in CHO cell expression systems or isolated from recombinant human antibody libraries.

[0065] The terms "specific binding" or "specific binding with..." refer to the formation of a complex between an antibody or its antigen-binding fragment and an antigen that is relatively stable under physiological conditions. Specific binding can consist of at least approximately 1 x 10-1 -8 Characterized by M or a smaller equilibrium dissociation constant ( For example Smaller K D(Indicating 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, etc. As described herein, antibodies have been tested using surface plasmon resonance (SPL). For example The antibody was identified by BIACORE™ as specifically binding to FXII. Furthermore, as used herein, multispecific antibodies that bind to one domain of FXII and one or more other antigens, or bispecific antibodies that bind to two different regions of FXII, are still considered to be “specifically binding” antibodies.

[0066] The term "high-affinity" antibody refers to a mAb that has binding affinity for FXII, expressed as K. D This indicates that at least 10 -8 M; Preferred 10 -9 M; more preferably 10 -10 M, or even better 10 -11 M, such as through surface plasmon resonance. For example Measured by BIACORE™ or solution affinity ELISA.

[0067] The terms "slowed rate," "Koff," or "kd" refer to the rate constant of the antibody dissociating from FXII, which is 1 x 10⁻⁶. -3 s -1 Or smaller, preferably 1 x 10 -4 s -1 Or even smaller, such as through surface plasmon resonance, For example BIACORE™ is confirmed.

[0068] As used herein, the term “half-life” for an antibody refers to the time required for a given amount of antibody to be cleared from the blood or for half to be eliminated from the body of a subject. In some embodiments, the anti-hFXII / FXIIa mAb of this disclosure has a half-life of at least 0.5 days, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 15 days, or 20 days.

[0069] As used herein, the terms “antigen-binding portion” and “antigen-binding fragment” of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms “antigen-binding fragment” or “antibody fragment” of an antibody refer to one or more fragments of an antibody that retain the ability to bind to the FXII protein.

[0070] In specific embodiments, the antibodies or antibody fragments disclosed herein may be conjugated to portions such as ligands or therapeutic portions (“immunoconjugates”), second anti-FXII antibodies, or any other therapeutic portion that can be used to treat FXII-related diseases or conditions.

[0071] As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) with different antigen specificities. For example The isolated antibodies or fragments thereof that specifically bind to FXII and / or FXIIa are substantially free of antibodies that specifically bind to antigens other than FXII or FXIIa.

[0072] As used herein, "blocking antibody" or "neutralizing antibody" (or "antibody that neutralizes FXII activity" or "antagonistic antibody") refers to an antibody that, upon binding to FXII / FXIIa, inhibits at least one biological activity of FXII. For example, the antibodies of this disclosure can prevent or block coagulation via an endogenous pathway.

[0073] As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0074] As used in this article, the term "K" D "It is intended to refer to the equilibrium dissociation constant of a specific antibody-antigen interaction."

[0075] The term "epitope" refers to an antigenic determinant cluster that interacts with a specific antigen-binding site in a variable region of an antibody molecule, known as a complementary site. A single antigen may have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and may have different biological effects. The term "epitope" can also refer to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are typically a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may comprise determinants grouped as chemically active surfaces of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural properties and / or specific charge properties.

[0076] As used herein, the term "cross-competition" refers to an antibody or its antigen-binding fragment binding to an antigen and inhibiting or blocking the binding of another antibody or its antigen-binding fragment. The term also includes bidirectional competition between two antibodies. That isThe first antibody binds to and blocks the binding of the second antibody, and vice versa. In some embodiments, the first and second antibodies may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that the binding of one inhibits or blocks the binding of the second antibody. For example , Via (Stereoscopic hindrance). Cross-competition between antibodies can be measured by methods known in the art (e.g., by real-time label-free biolayer interferometry). Cross-competition between two antibodies can be expressed as the binding of the second antibody being lower than the background signal caused by self-binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can also be expressed as, for example, the % binding of the second antibody being less than the baseline self-background binding (where the first and second antibodies are the same antibody).

[0077] As discussed below, when referring to nucleic acids or fragments thereof, the term "substantially identical" or "substantially the same" means that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, the nucleotide bases have at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP. In some cases, a nucleic acid molecule having substantial identity with a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0078] When applied to peptides, the terms "substantially similar" or "substantially similar" mean that, when optimally aligned using default gap weights, such as through procedures like GAP or BESTFIT, two peptide sequences share at least 90% sequence identity, and more preferably at least 95%, 98%, or 99% sequence identity. Preferably, the different residue positions are due to conserved amino acid substitutions. "Conserved amino acid substitution" is where amino acid residues are replaced with substances having similar chemical properties (…). For example An amino acid substitution is the substitution of another amino acid residue on the side chain (R group) of a protein, where the substitution is either charge- or hydrophobic. Generally, conserved amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conserved substitution, the percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. Methods for making this adjustment are well known to those skilled in the art. See also For examplePearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups with side chains having 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: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are found in Gonnet. Et al. Any variation of the PAM250 log-likelihood matrix with positive values ​​as disclosed in (1992) Science 256: 1443 45, which is incorporated herein by reference. The “moderately conservative” substitution is any variation of the PAM250 log-likelihood matrix with non-negative values.

[0079] Sequence analysis software is typically used to measure the sequence similarity of peptides. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications (including conserved amino acid substitutions) to match similar sequences. For example, GCG 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 peptides (such as homologous peptides from different biological species) or between wild-type proteins and their mutants. See also For example GCG version 6.1. Peptide sequences can also be compared using FASTA with default or recommended parameters; the procedure in GCG version 6.1. FASTA ( For example FASTA2 and FASTA3 provide a comparison of the best overlap between the query sequence and the search sequence, as well as the percentage of sequence identity (Pearson (2000)). Ibid When comparing the sequences of this disclosure with databases containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST using default parameters, particularly BLASTP or TBLASTN. See also For example Altschul Et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0080] The phrase “therapeutic effective amount” refers to the amount that produces the desired effect to be achieved by its application. The exact amount will depend on the purpose of treatment and will be determined by a person skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0081] As used herein, the term "subject" refers to a person in need of improvement, prevention, and / or treatment of FXII-related conditions. Animals, preferably mammals, more preferably humans, that have a disease or condition (such as thrombosis or embolism). This term includes human subjects who have or are at risk of having such a disease or condition.

[0082] As used herein, the term "treatment" ("treat", "treating", or "treatment") means a reduction or improvement in the severity of at least one symptom or sign of an FXII-related disease or condition resulting from the administration of a therapeutic agent, such as an antibody disclosed herein, to a subject in need. This term includes suppression of disease progression or worsening of symptoms / signs. It also includes a positive prognosis for the disease, i.e., the subject may recover from or experience a reduction in disease following the administration of a therapeutic agent, such as an antibody disclosed herein. The therapeutic agent may be administered to the subject at a therapeutic dose.

[0083] The term “prevent” (“preventing” or “prevention”) means the suppression of the manifestation of FXII-related disease or condition or any symptoms or signs of such disease or condition after administration of the antibody disclosed herein.

[0084] Antigen-binding fragments of antibodies As used herein, unless otherwise specified, the term "antibody" should be understood to encompass antibody molecules comprising two immunoglobulin heavy chains and two immunoglobulin light chains. That is "Whole antibody molecule") and its antigen-binding fragment. As used herein, the terms "antigen-binding portion" and "antigen-binding fragment" of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the terms "antigen-binding fragment" or "antibody fragment" of an antibody refer to one or more fragments of an antibody that retain the ability to specifically bind to the FXII protein. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing a CDR, or isolated CDRs. In some embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. The antigen-binding fragment of an antibody may beFor example Derivation from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and (optionally) constant domains of the antibody. Such DNA is known and / or can be readily derived from... For example Commercial sources, DNA library (including, For example DNA can be obtained (phage-antibody libraries) or synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into suitable conformations, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0085] 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 composed of analogous antibody hypervariable regions ( For example The amino acid residues of the isolated complementarity-determining region (CDR), such as the CDR3 peptide, or the restricted FR3-CDR3-FR4 peptide. Other engineered molecules, as used herein, include domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-transplanted antibodies, binary antibodies, ternary antibodies, quaternary antibodies, microantibodies, and nanobodies. For example Monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapies (SMIPs), and shark variable IgNAR domains are also included in the description of "antigen-binding fragments".

[0086] Antibody antigen-binding fragments typically contain at least one variable domain. Variable domains can have any size or amino acid composition and will generally include at least one CDR adjacent to or within a frame having one or more frame sequences. L V associated with the structural domain H In the antigen-binding fragment of the domain, V H and V L Domains can be arranged in any suitable arrangement relative to each other. For example, variable regions can be dimers and contain V. H - V H V H -V L or V L - V L Dimer. Alternatively, the antigen-binding fragment of the antibody may contain monomer V. H or V L Structural domain.

[0087] In some embodiments, the antigen-binding fragment of the 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 can be found within the antigen-binding fragment of the antibody disclosed herein include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v)V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3;(xi) V L -C H 1-C H 2;(xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of variable and constant structural domains, including any of the exemplary configurations listed above, the variable and constant structural domains may be directly connected to each other or connected via full or partial hinge areas or joint areas. The hinge area may consist of at least two ( For example Composed of 5, 10, 15, 20, 40, 60 or more amino acids, this results in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies disclosed herein may include those listed above and / or with one or more monomers V. H or V L structural domain ( For exampleHomodimers or heterodimers (or other polymers) of any variable and constant domain configuration that are non-covalently associated via disulfide bonds.

[0088] Like whole antibody molecules, antigen-binding fragments can be monospecific or multispecific. For example (Bispecific). A multispecific antigen-binding fragment of an antibody will typically include at least two distinct variable domains, each capable of specifically binding to a single antigen or to a different epitope on the same antigen. Any multispecific antibody form containing the exemplary bispecific antibody forms disclosed herein can be used in the context of the antigen-binding fragments of the antibodies of this disclosure using conventional techniques available in the art.

[0089] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of this disclosure to prepare human antibodies that specifically bind to FXII and / or FXIIa.

[0090] An immunogen containing any of the following components can be used to generate antibodies against the FXII and / or FXIIa proteins. In some embodiments, the antibodies of this disclosure are obtained from mice immunized with full-length natural FXII or FXIIa protein (see, for example, UniProtKB / Swiss-Prot accession number P00748.3) or with DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof can be produced using standard biochemical techniques, modified, and used as an immunogen.

[0091] In some embodiments, the immunogen may be expressed on Escherichia coli Recombinant FXII protein or fragments thereof in any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0092] Using the VELOCIMMUNE® technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, a high-affinity chimeric antibody containing human variable regions and mouse constant regions is initially isolated. The VELOCIMMUNE® technology involves producing transgenic mice with a genome comprising human heavy and light chain variable regions operatively linked to endogenous mouse constant region loci, such that the mouse produces antibodies comprising human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operatively linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0093] Typically, VELOCIMMUNE® mice are attacked with a target antigen, and lymphocytes (such as B cells) are recovered from the antibody-expressing mice. These lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, and these hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding variable regions of the heavy and light chains can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or variable domains of the light and heavy chains can be directly isolated from antigen-specific lymphocytes.

[0094] First, high-affinity chimeric antibodies containing both human variable regions and mouse constant regions are isolated. As described in the Experimental Section below, the antibodies are characterized and screened to obtain desired properties, including affinity, selectivity, epitopes, etc. The mouse constant regions are then replaced with the desired human constant regions to generate the fully human antibodies of this disclosure, such as wild-type or modified IgG1 or IgG4. While the selected constant regions may vary depending on the specific application, high-affinity antigen binding and target-specific properties are present in the variable regions.

[0095] bioequivalent The anti-FXII antibodies and antibody fragments disclosed herein encompass proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to FXII proteins. Such variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids compared to the parental sequence, but exhibit substantially equivalent biological activity to the described antibodies. Similarly, the DNA sequences encoding antibodies disclosed herein encompass sequences that, when compared to the disclosed sequences, include one or more additions, deletions, or substitutions of nucleotides, but encode antibodies or antibody fragments substantially bioequivalent to the antibodies or antibody fragments disclosed herein.

[0096] For example, if two antigen-binding proteins or antibodies are drug equivalents or drug substitutes that do not show significant differences in absorption rate and extent when administered at the same molar dose (single or multiple doses) under similar experimental conditions, they are considered bioequivalent. If some antibodies are equivalent in extent of absorption but not in rate of absorption, they are considered equivalents or drug substitutes, and can still be considered bioequivalent because such differences in absorption rate are intentional and reflected in the labeling. For example Long-term use is not necessary to achieve effective drug concentrations in the body and is considered medically irrelevant for the specific drug product being studied.

[0097] In one embodiment, the two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or potency.

[0098] In one embodiment, two antigen-binding proteins are bioequivalent compared to continuous therapy where there is no switching between the reference product and the biological product, if the patient can switch between the reference product and the biological product once or multiple times without an expected increased risk of side effects (including clinically significant changes in immunogenicity or reduced efficacy).

[0099] In one embodiment, the two antigen-binding proteins are bioequivalent if they both function through one or more co-operating mechanisms targeting one or more conditions of use (provided those mechanisms are known).

[0100] Bioequivalence can be achieved through In vivo and / or In vitro Methods for proof. Bioequivalence measures include... For example (a) In humans or other mammals In vivo The test measures the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids as a function of time; (b) has been used in human trials. In vivoBioavailability data are relevant and can be reasonably predicted. In vitro (c) Tests conducted in humans or other mammals. In vivo The test, in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) the safety, efficacy, bioavailability or bioequivalence of the antibody is determined in a well-controlled clinical trial.

[0101] Bioequivalent variants of the antibodies disclosed herein can be constructed, for example, by various substitutions of residues or sequences, or by deletion of terminal or internal residues or sequences required for non-biological activity. For example, cysteine ​​residues essential for non-biological activity can be deleted or substituted with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antibodies can include antibody variants comprising amino acid changes that modify the glycosylation properties of the antibody. For example Mutations that eliminate or remove glycosylation.

[0102] Anti-FXII antibodies including Fc variants According to certain embodiments of this disclosure, an anti-FXII antibody comprising an Fc domain is provided, the Fc domain including For example One or more mutations that enhance or weaken antibody binding to the FcRn receptor at acidic pH compared to neutral pH. For example, this disclosure includes C in the Fc domain. H Zone 2 or C H Region 3 contains mutated anti-FXII antibodies, where the mutation increases the Fc domain in an acidic environment ( For example The affinity of the antibody for FcRn (in the endosome, at a pH ranging from about 5.5 to about 6.0). When administered to animals, such mutations can lead to an increase in the serum half-life of the antibody. Non-limiting examples of such Fc modifications include... For example Modifications in the following locations: 250 ( For example (E or Q); 250 and 428 ( For example , L or F); 252 ( For example L / Y / F / W or T), 254 ( For example (S or T) and 256 ( For example S / R / Q / E / D or T); or modifications at the following locations: 428 and / or 433 ( For example H / L / R / S / P / Q or K) and / or 434 ( For example A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or modifications at the following locations: 250 and / or 428; or modifications at the following locations: 307 or 308 ( For example308F, V308F) and 434. In one embodiment, the modification includes 428L ( For example M428L) and / or 434S ( For example Modified with N434S; 428L, 259I ( ​ V259I) and 308F ( ​ Modified by V308F; 433K ( ​ H433K) and 434 ( ​ Modified by ,434Y; 252, 254 and 256 ( ​ Modifications for 252Y, 254T, and 256E; modifications for 250Q and 428L ( ​ T250Q and M428L); and 307 and / or 308 modifications ( ​ , 308F or 308P). In yet another embodiment, the modification includes 265A ( ​ (D265A) and / or 297A ( ​ , N297A) modification. In one embodiment, the mutation includes 252Y, 254T, and 256E modifications (“YTE” variant). In one embodiment, the mutation in the Fc domain compared to the unmodified antibody ( ​ (252Y, 254T, and 256E) can prolong the half-life of the antibody in plasma. In one embodiment, mutations in the Fc domain (252Y, 254T, and 256E) compared to unmodified antibodies can prolong the half-life of the antibody in plasma. ​ (252Y, 254T and 256E) can reduce the clearance rate of antibodies in plasma.

[0103] For example, this disclosure includes anti-FXII antibodies comprising an Fc domain containing one or more pairs or one or more groups of mutations selected from the group consisting of: 250Q and 248L ( ​ T250Q and M248L); 252Y, 254T and 256E ( ​ M252Y, S254T and T256E; 428L and 434S ( ​ M428L and N434S); 257I and 311I ( ​ P257I and Q311I); 257I and 434H ( ​ P257I and N434H); 376V and 434H ( ​ D376V and N434H); 307A, 380A and 434A ( ​ T307A, E380A, and N434A); and 433K and 434F ( ​(H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations within the antibody variable domains disclosed herein are envisioned within the scope of this disclosure.

[0104] This disclosure also includes chimeric heavy chain constancy (C H Anti-FXII antibody in region ) where the chimeric C H The region includes C derived from more than one immunoglobulin isotype. H The region segment. For example, the antibody disclosed herein may include a chimeric C H The region includes C molecules derived from human IgG1, human IgG2, or human IgG4. H 2. Part or all of the structural domain, which is related to the C molecule derived from human IgG1, human IgG2, or human IgG4. H 3. Domain binding. According to some embodiments, the antibody of this disclosure includes a chimeric C-domain having a chimeric hinge region. H The region. For example, the chimeric hinge may include an “upper hinge” amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4, which is combined with a “lower hinge” sequence (amino acid residues at positions 228 to 236 according to EU numbering) derived from the hinge region of human IgG1, human IgG2, or human IgG4. According to some embodiments, the chimeric hinge region includes amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. In some embodiments, it includes chimeric C as described herein. H Antibodies in this region can exhibit modified Fc effector functions that do not adversely affect the therapeutic or pharmacokinetic properties of the antibody. ​ U.S. Patent Application Publication 2014 / 0243504, the contents of which are incorporated herein by reference in their entirety.

[0105] Biological characteristics of antibodies Typically, the antibodies of this disclosure exert their effects by binding to the FXII protein and preventing its cleavage into FXIIa and FXIIb. In some embodiments, the antibody binds to the activated form of factor XII (FXIIa) (“dual FXII / FXIIa binders”). For example, this disclosure includes antibodies and antigen-binding fragments of antibodies at a Kc of less than 20 nM. D Combining FXII protein ( ​ (at 25°C or 37°C), as measured by surface plasmon resonance. ​The assay format defined in the examples herein is used. In some embodiments, the antibody or its antigen-binding fragment is in a Kc concentration of less than about 20 nM, less than about 17 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than 0.5 nM, or less than 0.3 nM. D Combined with FXII, such as measurements via surface plasmon resonance, ​ Use the measurement format defined in the examples in this article, or a similar measurement.

[0106] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to human FXII protein with a dissociation half-life (t½) greater than about 1 minute, as measured by surface plasmon resonance at 25°C or 37°C. ​ Use the assay format defined in the examples herein, or a substantially similar assay. In some embodiments, the antibody or antigen-binding fragment of this disclosure binds to the FXII protein at t½ times 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 37°C. ​ Use the measurement format defined in Example 3 of this paper, or a similar measurement.

[0107] This disclosure includes antibodies and antigen-binding fragments of antibodies, wherein the antibodies and antigen-binding fragments of antibodies have a Kc of less than 9 nM. D Combining FXIIa protein ( ​ (at 25°C or 37°C), as measured by surface plasmon resonance. ​ The assay format defined in Example 3 of this document is used. In some embodiments, the antibody or its antigen-binding fragment is in a Kc concentration of less than about 9 nM, less than about 5 nM, less than about 2 nM, less than about 1 nM, less than about 500 pM, or less than 350 pM. D Combined with FXIIa, such as measurements via surface plasmon resonance, ​ Use the measurement format defined in the examples in this article, or a substantially similar measurement.

[0108] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to human FXIIa protein with a dissociation half-life (t½) greater than about 1 minute, as measured by surface plasmon resonance at 25°C or 37°C. ​Use the assay format defined in the examples herein, or a substantially similar assay. In some embodiments, the antibody or antigen-binding fragments of this disclosure bind to the FXIIa protein at t½ times 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 37°C. ​ Use the measurement format defined in the examples in this article, or a similar measurement.

[0109] This disclosure includes antibodies and antigen-binding fragments of antibodies, wherein the antibodies and antigen-binding fragments of antibodies have a Kc of less than 5 nM. D Combining FXIIab protein ( ​ (at 25°C or 37°C), as measured by surface plasmon resonance. ​ ​ The assay format defined in Example 3 of this document is used. In some embodiments, the antibody or its antigen-binding fragment is in a Kc concentration of less than about 5 nM, less than about 3 mM, less than about 1 nM, less than about 500 pM, or less than 300 pM. D Combined with FXIIab, such as measurements via surface plasmon resonance, ​ Use the measurement format defined in the examples in this article, or a substantially similar measurement.

[0110] This disclosure also includes antibodies and antigen-binding fragments thereof that bind to human FXIIab protein with a dissociation half-life (t½) greater than about 1 minute, as measured by surface plasmon resonance at 25°C or 37°C. ​ Use the assay format defined in the examples herein, or a substantially similar assay. In some embodiments, the antibody or antigen-binding fragment of this disclosure binds to the FXIIa protein at t½ times 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 37°C. ​ Use the measurement format defined in the examples in this article, or a similar measurement.

[0111] This disclosure also includes antibodies and antigen-binding fragments thereof that, at concentrations below 250 nM, below 200 nM, or below 150 nM, inhibit thrombin production via an intrinsic pathway, such as... ​ The assay is performed using the assay format described in the examples herein, or a substantially similar assay. In some embodiments, this disclosure includes antibodies and antigen-binding fragments thereof that inhibit thrombin production via the endogenous pathway without blocking thrombin production via the extrinsic pathway, such as...​ Use the measurement format defined in the examples in this article, or a basically similar measurement.

[0112] In one embodiment, this disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the FXII protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) it is a fully human monoclonal antibody; (b) it binds to activated factor XII (FXIIa); (c) it exhibits a dissociation constant (K0) of less than 1.5 nM at 25 °C. D (d) Combined with FXII, as measured by surface plasmon resonance; (d) at 37 °C with a K0 of less than 17 nM. D Combined with FXII, as measured by surface plasmon resonance; (e) at 25°C with a Ki of less than 5 nM, preferably less than 0.9 nM. D Combined with FXIIa, as measured by surface plasmon resonance; (f) at 37°C with a K0 of less than 6.5 nM, preferably less than 2.5 nM. D (g) In combination with FXIIa, as measured by surface plasmon resonance assay; and (h) Inhibit thrombin production via the intrinsic pathway at concentrations less than 250 nM, as measured by functional plasma assay; and (h) Inhibit thrombin production via the intrinsic pathway without inhibiting thrombin production via the extrinsic pathway, as measured by functional plasma assay.

[0113] The antibodies disclosed herein may possess one or more of the biological characteristics described above, or any combination thereof. Other biological characteristics of the antibodies disclosed herein, including the working examples herein, will be apparent to those skilled in the art upon reading this disclosure.

[0114] Epitope plotting and related techniques This disclosure includes anti-FXII / FXIIa antibodies that interact with one or more amino acids found in one or more regions of the FXII / FXIIa protein molecule (including the heavy and light chains). The epitopes to which the antibodies bind can consist of three or more amino acids located within any of the aforementioned domains of the FXII protein molecule. ​ A single continuous sequence of amino acids consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more. ​ Linear epitopes within the structural domains. Alternatively, epitopes can be located in one or both of the aforementioned structural domains of the protein molecule. ​ It consists of multiple non-continuous amino acids (or amino acid sequences) within a conformational epitope.

[0115] Various techniques known to those skilled in the art can be used to determine whether an antibody “interacts with one or more amino acids” within a peptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays, such as those described in *Antibody*, Harlow and Lane (Cold Spring Harbor Press, Cold Spring, NY). Other methods include alanine scanning mutation analysis and peptide blotting analysis (Reineke (2004) Methods Mol. Biol. 248: 443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. Additionally, 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 amino acids within a peptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, hydrogen / deuterium exchange methods involve deuterating the protein of interest and then binding the antibody to the deuterated protein. Next, the protein / antibody complex was transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex underwent hydrogen-deuterium reverse exchange at a slower rate than those within amino acids not part of the interface. Therefore, the amino acids forming part of the protein / antibody interface retain deuterium and thus exhibit a relatively higher quality compared to amino acids not included in the interface. After dissociation of the antibody, the target protein was subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterated residues corresponding to the specific amino acids interacting with the antibody. ​ ​ , 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 that responds to B cells and / or T cells. B cell epitopes can be formed from either consecutive amino acids or non-consecutive amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically contain at least three, and more usually at least five or eight to ten amino acids in a unique spatial conformation.

[0117] Modification-assisted analysis (MAP), also known as antigen structure-based antibody analysis (ASAP), is a method for classifying a large number of monoclonal antibodies (mAbs) against the same antigen based on the similarity of their binding characteristics to chemically or enzymatically modified antigen surfaces (see US 2004 / 0101920, which is hereby incorporated herein by reference in its entirety). Each category can reflect a unique epitope that is significantly different from or partially overlaps with the epitope represented by another category. This technique allows for rapid filtering of genetically identical antibodies, thus focusing characterization on genetically distinct antibodies. When applied to hybridoma screening, MAP can help identify rare hybridoma clones that produce mAbs with the desired characteristics. MAP can be used to sort the antibodies of this disclosure into groups of antibodies that bind to different epitopes.

[0118] In some embodiments, this disclosure includes anti-FXII antibodies and antigen-binding fragments thereof that interact with one or more epitopes in the heavy and / or light chains of FXIIa. The one or more epitopes may be composed of three or more epitopes located in the heavy and / or light chains of FXIIa. ​ An epitope consists of one or more consecutive sequences of amino acids (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). Alternatively, an epitope may consist of multiple non-consecutive amino acids (or amino acid sequences) located within FXIIa.

[0119] This disclosure includes anti-FXII antibodies that bind to the same epitope or a portion of an epitope as any of the specific exemplary antibodies listed in Table 1. Similarly, this disclosure also includes anti-FXII antibodies that compete with any of the specific exemplary antibodies listed in Table 1 for binding to the FXII protein or fragments thereof. For example, this disclosure includes anti-FXII antibodies that cross-compete with one or more antibodies listed in Table 1 for binding to the FXII protein.

[0120] Using conventional methods known in the art, it can be readily determined whether an antibody binds to the same epitope as a reference anti-FXII antibody or competes with it for binding. For example, to determine whether a test antibody binds to the same epitope as the reference anti-FXII antibody of this disclosure, the reference antibody is bound to an FXII protein or peptide under saturation conditions. Next, the ability of the test antibody to bind to the FXII protein molecule is evaluated. If the test antibody can bind to FXII after 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 cannot bind to the anti-FXII protein after saturation binding with the reference anti-FXII protein, then the test antibody can bind to the same epitope as the reference anti-FXII antibody of this disclosure.

[0121] To determine whether an antibody competes with a reference anti-FXII antibody for binding, the binding method described above is performed bidirectionally: In a first orientation, the reference antibody is allowed to bind to the FXII protein under saturation conditions, and then the binding of the test antibody to the FXII molecule is evaluated. In a second orientation, the test antibody is allowed to bind to the FXII molecule under saturation conditions, and then the binding of the reference antibody to the FXII molecule is evaluated. If, in both orientations, only the first (saturated) antibody is able to bind to the FXII molecule, then it can be concluded that the test antibody and the reference antibody compete for binding to FXII. As will be understood by those skilled in the art, the antibody that competes for binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but the binding of the reference antibody can be spatially blocked by binding to overlapping or adjacent epitopes.

[0122] If two antibodies competitively inhibit (block) the binding of another antibody to an antigen, then both antibodies bind to the same or overlapping epitopes. In other words, an excess of 1, 5, 10, 20, or 100 times that of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see [reference]). ​ Junghans ​ (Cancer Res. 1990 50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have overlapping epitopes.

[0123] Then other routine experiments can be conducted. ​ (Peptide mutation and binding analysis) to confirm whether the observed lack of binding to the test antibody is actually due to binding to the same epitope as the reference antibody or whether it is caused by steric hindrance (or another phenomenon). This sorting experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0124] Immunoconjugates This disclosure covers human anti-FXII monoclonal antibodies (“immunoconjugates”) conjugated to a therapeutic portion for the treatment of FXII-related diseases or conditions. ​(Thrombosis). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. An antibody may be linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any location along the length of the molecule capable of binding its target. Examples of immunoconjugates include antibody-pharmaceutical conjugates and antibody-toxin fusion proteins. In one embodiment, the agent may be a second, different antibody against the FXII protein. The type of therapeutic moiety that may be conjugated to an anti-FXII antibody will be considered, taking 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.

[0125] Multispecific antibodies The antibodies disclosed herein can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific to different epitopes of a single target polypeptide, or may contain antigen-binding domains specific to more than one target polypeptide. See also ​ Tutt ​ , 1991, J. Immunol.147:60-69; Kufer ​ , 2004,Trends Biotechnol. 22:238-244.

[0126] Any multispecific antigen-binding molecule or variant thereof disclosed herein can be used with standard molecular biology techniques ( ​ The technology (using recombinant DNA and protein expression techniques) is constructed as is known to those skilled in the art.

[0127] In some embodiments, FXII-specific antibodies are produced in a bispecific form (“bispecific”), wherein variable regions that bind to different domains of the FXII protein are linked together to confer bidomain specificity in a single binding molecule. Properly designed bispecific antibodies can enhance the overall inhibitory potency of the FXII protein by increasing both specificity and binding affinity. ​ Variable regions (fragments of the N-terminal domain) with specific binding properties, or variable regions capable of binding to different regions within the same domain, are paired onto a structural scaffold, allowing each region to simultaneously bind to different epitopes or to different regions within the same domain. For example, in bispecific antibodies, a heavy chain variable region (VN) from a binding agent specific to one domain is used. H ) and light chain variable regions (V) from a series of binders specific to the second structural domain L Recombination to identify non-homologous V LPartners, these partners can be with the original V H Pairing without breaking the V H The original specificity. In this way, a single V L Excerpt ( ​ V L 1) Can be used with two different V H structural domain ( ​ V H 1 and V H 2) Combining to produce two combined "arms" (V) H 1-V L 1 and V H 2-V L 1) Composition of bispecific antibodies. Using a single V L Fragments reduce system complexity and thus simplify and improve the efficiency of cloning, expression, and purification processes for the production of bispecific antibodies (see, for example, USSN13 / 022759 and US2010 / 0331527).

[0128] Alternatively, antibodies binding to more than one domain and a second target (such as, but not limited to, a second different anti-FXII antibody) can be prepared in a bispecific form using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions binding to different regions can be linked together with variable regions binding to relevant sites on, for example, the extracellular domain of FXII, to confer dual antigen specificity in a single binding molecule. Appropriately designed bispecific antibodies of this nature have dual functionality. Combining variable regions specific for the extracellular domain with variable regions specific for the exterior of the extracellular domain and pairing them on a structural scaffold that allows each variable region to bind to a different antigen.

[0129] Exemplary bispecific antibody forms that may be used in the context of this disclosure relate to a first immunoglobulin (Ig) C. H 3 structural domains and second Ig C H 3. Use of structural domains, where the first and second Ig C H The three domains differ from each other by at least one amino acid, and this at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to bispecific antibodies lacking amino acid differences. In one embodiment, the first IgC H 3. The domain binds to protein A and the second Ig C. H Domain 3 contains mutations that reduce or eliminate protein A binding, such as the H95R modification (via IMGT exon numbering; H435R via EU numbering). Second C H 3 may further include Y96F modification (via IMGT; Y436F via EU). Second CH Other possible modifications in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I (via IMGT; D356E, L358M, N384S, K392N, V397M, and V422I, via EU), in the case of IgG1 antibodies; N44S, K52N, and V82I (via IMGT; N384S, K392N, and V422I, via EU), in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (via IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I, via EU), in the case of IgG4 antibodies. Variations of the bispecific antibody forms described above are contemplated within the scope of this disclosure.

[0130] Other exemplary forms of bispecificity that may be used in the context of this disclosure include, but are not limited to, those that are not specifically used in this disclosure. ​ Based on scFv or binary antibody bispecific form, IgG-scFv fusion, dual variable domain (DVD)-Ig, quadroma, nob-into-holes, common light chain ( ​ (including common light chains with nodular entry pores, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual-action Fab(DAF)-IgG, and Mab 2 Bispecific form ( ​ Klein ​ 2012, mAbs 4:6, 1-11, and the references cited therein, for review of the aforementioned forms. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugations. ​ This process utilizes non-natural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which are then self-assembled into multimeric complexes with defined composition, valence, and geometry. ​ Kazane ​ ​ , ​ .[ ​ : ​ ]).

[0131] Therapeutic agents and preparations This disclosure provides therapeutic compositions comprising the anti-FXII antibody or its antigen-binding fragment thereof. The therapeutic compositions according to this disclosure are to be administered together with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. A variety of suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See also Powell. ​ “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0132] The dosage of the antibody can vary depending on the age and body size of the subject to be administered, the target disease, symptoms, route of administration, etc. When the antibody of this disclosure is used to treat or prevent a disease in an adult patient, it is generally advantageous to administer a single dose of about 0.1 mg / kg to about 100 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. In some embodiments, the antibody or antigen-binding fragment of this disclosure may be administered at an initial dose of at least about 0.1 mg to about 800 mg, about 1 mg to about 600 mg, about 5 mg to about 500 mg, or about 10 mg to about 400 mg. In some embodiments, a second or multiple subsequent doses of the antibody or its antigen-binding fragment may be administered after the initial dose, in amounts that may be substantially the same as or less than the initial dose, wherein the intervals between subsequent doses are at least 1 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 compositions disclosed herein. ​ Encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see [link to documentation]). ​ Wu ​(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 can be administered via any convenient route, such as by infusion or bolus, or by absorption through the epithelial or mucosal lining of the skin. ​ (Oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in combination with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles (specifically, liposomes) (see, for example, Langer (1990) Science 249:1527-1533).

[0134] This paper also envisions the use of nanoparticles to deliver the antibodies disclosed herein. Antibody-conjugated nanoparticles could be used for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and their preparation and use are described in detail in Arruebo, M. ​ The reference 2009 (“Antibody-conjugated nanoparticles for biomedical applications”, J. Nanomat. Vol. 2009, Article ID 439389, 24 pages, doi:10.1155 / 2009 / 439389) is incorporated herein by reference. Nanoparticles can be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described, for example, in US 8257740 or US 8246995, each of which is incorporated herein by reference in its entirety.

[0135] In some cases, a controlled-release system can be used to deliver a drug composition. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled-release system can be placed near the target of the composition, thus requiring only a portion of the systemic dose.

[0136] Injectable formulations may include dosage forms for intravenous injection, subcutaneous injection, intracranial injection, intraperitoneal injection, intramuscular injection, infusion, etc. These injectable formulations may be prepared using publicly known methods.

[0137] The disclosed pharmaceutical compositions can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, regarding subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the disclosed pharmaceutical compositions. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0138] Advantageously, the above-described pharmaceutical compositions for oral or parenteral use are formulated into dosage forms in unit doses suitable for containing a specific amount of the active ingredient. Such single-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained in each unit dose dosage form is typically from about 5 mg to 500 mg; particularly in the injectable form, the antibody is preferably contained in the form of from about 5 mg to about 300 mg, and for other dosage forms, it is contained in the form of from about 10 mg to about 300 mg.

[0139] Therapeutic uses of antibodies The antibodies disclosed herein may be used to treat and / or prevent coagulation (including thrombosis and embolism) or edema ( ​ This disclosure pertains to diseases, conditions, or illnesses associated with hereditary angioedema, and / or to the relief of at least one symptom associated with such disease, condition, or illness. In some embodiments, the antibody or antigen-binding fragment thereof may be administered in a therapeutic dose to a patient suffering from a disease, condition, or illness associated with coagulation or edema. In some embodiments, the antibody or antigen-binding fragment thereof of this disclosure is administered to a subject in need to prevent thrombosis without increasing the risk of bleeding.

[0140] In some embodiments, the antibodies disclosed herein may be used to treat or prevent at least one symptom or sign of an FXII-related disease or condition selected from the group consisting of: venous thrombosis, arterial thrombosis, device thrombosis, thromboembolism, hereditary angioedema, stroke, thrombotic tendency, myocardial ischemia, atherosclerotic plaque rupture, use of mechanical valve prostheses, use of blood-contact medical devices, use of blood-contact extracorporeal circulation circuits, venous thromboembolism, pulmonary embolism, deep vein thrombosis, portal vein thrombosis, Budd-Chiari syndrome, Paget-Schroth disease, renal vein thrombosis, cerebral venous sinus thrombosis, jugular vein thrombosis, cavernous sinus thrombosis, hepatic artery thrombosis, limb ischemia, and myocardial infarction.

[0141] This document also envisions the prophylactic use of one or more antibodies of this disclosure in subjects at risk of thrombosis, such as those using extracorporeal membrane oxygenation (ECMO) machines. The antibodies of this disclosure can be used to prevent thrombotic blockage of the oxygenator and tubing in the extracorporeal circulation circuit.

[0142] In another embodiment of this disclosure, the antibody of the present invention is used to prepare a pharmaceutical composition or medicament for treating a patient suffering from the diseases, conditions, or illnesses disclosed herein. In another embodiment of this disclosure, the antibody of the present invention may be used in combination with any other agent or therapy known to those skilled in the art for treating or improving the diseases, conditions, or illnesses disclosed herein.

[0143] Combination therapy Combination therapies may include the antibodies of this disclosure as well as any additional therapeutic agents that can be used advantageously in combination with the antibodies of this disclosure or with biologically active fragments of the antibodies of this disclosure. The antibodies of this disclosure may be synergistically combined with one or more drugs or therapies for treating diseases or conditions associated with thrombosis, particularly for preventing thrombosis or treating subjects at risk of thrombosis due to an underlying disease, condition, or ailment (described elsewhere herein). In some embodiments, the antibodies of this disclosure may be used in combination with a second therapeutic agent to relieve one or more symptoms of said disease or ailment.

[0144] Depending on the disease, symptom, or condition, the antibodies disclosed herein can be used in combination with one or more other therapeutic agents, including but not limited to: anticoagulants ( ​ Warfarin, heparin, phenylindanone, fondaparinux sodium, ileparin), thrombin inhibitors ( ​ Argatroban, Lepirudine, Bivalirudin or Dabigatran etexilate), thrombolytic drugs, antiplatelet drugs ( ​ Aspirin), antihypertensive drugs ( ​ Angiotensin-converting enzyme inhibitors, beta-blockers, calcium channel blockers, and immunosuppressants ( ​ Vincristine, cyclosporine A or methotrexate), fibrinolytic agents, cholesterol-lowering agents ( ​ Statins or PCSK9 inhibitors, such as alikumab, and anti-inflammatory drugs ( ​ Treatment options include corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDs), secondary anti-FXII antibodies, mechanical thrombectomy, catheter-guided thrombolysis, and surgery.

[0145] As used herein, the term "combination" means that an additional therapeutically active component may be administered before, simultaneously with, or after the administration of the anti-FXII antibody of this disclosure. The term "combination" also includes the sequential or simultaneous administration of the anti-FXII antibody and a second therapeutic agent.

[0146] Additional therapeutically active components may be administered to the subject prior to administration of the anti-FXII antibody of this disclosure. For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less than 30 minutes before administration of the second component, the first component may be considered administered “before” the second component. In other embodiments, additional therapeutically active components may be administered to the subject after administration of the anti-FXII antibody of this disclosure. For example, if the first component is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or longer after administration of the second component, the first component may be considered administered “after” the second component. In other embodiments, additional therapeutically active components may be administered to the subject concurrently with administration of the anti-FXII antibody of this disclosure. For the purposes of this disclosure, “concurrent” administration includes... ​ The anti-FXII antibody and the additional therapeutically active component were administered to the subject in a single dosage form, or in separate dosage forms administered to the subject within approximately 30 minutes or less of each other. If administered in different dosage forms, each dosage form could be administered via the same route. ​ Anti-FXII antibodies and other therapeutically active ingredients can be administered intravenously, etc.; alternatively, each dosage form can be administered via different routes of administration. ​ Anti-FXII antibodies can be administered intravenously, and other therapeutically active ingredients can be administered orally. In any case, for the purposes of this disclosure, administration of a component in a single dosage form, via the same route in a separate dosage form, or via different routes in a separate dosage form is considered "simultaneous administration." For the purposes of this disclosure, administration of anti-FXII antibodies "before," "simultaneously with," or "after" administration of other therapeutically active ingredients (as defined above herein) is considered administration of anti-FXII antibodies "in combination" with other therapeutically active ingredients.

[0147] This disclosure includes pharmaceutical compositions wherein the anti-FXII antibody of this disclosure is co-formulated with one or more additional therapeutically active components as described elsewhere herein.

[0148] Diagnostic uses of antibodies The antibodies disclosed herein can be used to detect and / or measure FXII in samples. ​ For diagnostic purposes. Some embodiments envision the use of one or more antibodies of this disclosure in assays for detecting FXII-related diseases or conditions. Exemplary diagnostic assays for FXII may include... ​The sample obtained from the patient is contacted with the anti-FXII antibody of this disclosure, wherein the anti-FXII antibody is labeled with a detectable tag or reporter molecule or used as a capture ligand to selectively isolate FXII from the patient sample. Alternatively, unlabeled anti-FXII antibodies may be combined with secondary antibodies that are themselves detectably labeled for diagnostic applications. The detectable tag or reporter molecule may be a radioisotope, such as... 3 H, 14 C 32 P, 35 S or 125 I; a fluorescent or chemiluminescent component, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-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 this disclosure include any tissue or fluid sample obtainable from a patient that contains a detectable amount of FXII protein or fragments thereof under normal or pathological conditions. Typically, the measurement will be taken from a healthy patient ( ​ The baseline or standard level of FXII is initially established by measuring the level of FXII protein in specific samples obtained from patients (who do not have FXII-related diseases). This baseline level of FXII can then be compared with the FXII levels measured in samples obtained from individuals suspected of having FXII-related conditions or exhibiting symptoms associated with such conditions.

[0150] Antibodies specific to the FXII protein may not contain an additional label or portion, or these antibodies may contain an N-terminal or C-terminal label or portion. In one embodiment, the label or portion is biotin. In a binding assay, the position of the label (if present) can determine the orientation of the peptide relative to the surface on which it binds. For example, if the surface is coated with avidin, a peptide containing N-terminal biotin will be oriented such that the C-terminal portion of the peptide is distal to the surface.

[0151] Example The following examples are provided to offer a complete disclosure and description to those skilled in the art regarding how to prepare and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure that the figures used ( ​ The accuracy of quantities, temperatures, etc., is guaranteed, but some experimental errors and biases should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is atmospheric pressure or close to atmospheric pressure.

[0152] Example 1: Generating human antibodies against factor XII / activated factor XII (FXII / FXIIa) proteins VELOCIMMUNE contains DNA encoding the variable regions of the human immunoglobulin heavy chain and κ light chain. ® Human antibodies against FXII / FXIIa proteins were generated in mice. 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 fusing with myeloma cells, as described in U.S. Patent 7,582,298, which is specifically incorporated herein by reference in its entirety. Using this method, several fully human anti-FXII antibodies were obtained. ​ (Antibodies with human variable domains and human constant domains); exemplary antibodies produced in this manner are named REGN9533 and REGN9534.

[0154] The biological characteristics of an exemplary antibody produced according to the method shown below are described in detail in the example shown below.

[0155] Example 2: Amino acid sequence and nucleotide sequence Table 1 shows the heavy chain variable region and light chain variable region of the selected anti-FXII antibody of this disclosure, as well as the amino acid sequence identifier of the CDR.

[0156] Table 1: Amino Acid Sequence Identifiers The corresponding nucleic acid sequence identifiers are shown in Table 2.

[0157] Table 2: Nucleic Acid Sequence Identifiers The antibodies mentioned herein typically possess a fully human variable region, but their constant region may be a human constant region or a mouse constant region. As will be understood by those skilled in the art, antibodies with a specific Fc isotype can be converted into antibodies with different Fc isotypes. ​Antibodies containing mouse IgG1 Fc can be converted to antibodies containing human IgG4, etc.; however, the variable domains (containing CDRs) indicated by the numerical identifiers shown in Table 2 will remain the same, and the expected antigen-binding properties are the same or substantially similar regardless of the nature of the Fc domain. In some embodiments, selected antibodies containing mouse IgG1 Fc are converted to antibodies containing human IgG4 Fc. In one embodiment, the IgG4 Fc domain contains two or more amino acid changes disclosed in US20100331527. In one embodiment, human IgG4 Fc contains a serine-to-proline mutation (S108P) in the hinge region to promote dimer stability.

[0158] The control construct used in the following examples For comparative purposes, the experiments disclosed in this paper include the following control construct (anti-FXII antibody): ​ 1 A monoclonal antibody targeting human FXII / FXIIa, which has the V of antibody “3F7” according to EP20110175105 (CSL Behring GmbH). H / V L sequence; and " ​ "A human monoclonal antibody against human FXII, which has the V according to antibody "15H8" in WO 2014 / 089493 (Vanderbilt Univ. / Aronora.") H / V L sequence.

[0159] Example 3: Biacore binding kinetics of anti-FXII monoclonal antibodies with different FXII reagents measured at 25°C. The equilibrium dissociation constant (KD) for the binding of different FXII reagents to purified anti-FXII monoclonal antibodies was determined using a Biacore 8K biosensor based on real-time surface plasmon resonance. All binding studies were performed at 25 °C in a running buffer of 10 mM HEPES, 300 mM NaCl, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-P). The surface of the Biacore CM5 sensor chip was first derivatized with a monoclonal mouse anti-human Fc antibody (REGN2567) via amine conjugation to capture the anti-FXII monoclonal antibody and an irrelevant isotype control. Binding studies were conducted for human FXII, FXIIa, and FXIIab. First, different concentrations of hFXII, hFXIIa, and hFXIIab (30 nM to 1.11 nM; 3-fold serial dilutions) were prepared in HBS-P running buffer and injected onto the surface of anti-FXII monoclonal antibody captured by anti-human Fc at a flow rate of 30 µL / min for 3 min, while monitoring the dissociation of the monoclonal antibody-bound FXII reagent in HBS-P running buffer for 8 min. The association rate (ka) and dissociation rate (kd) were determined by fitting a real-time binding sensor map to a 1:1 binding model with mass transfer constraints using Biacore Insight evaluation software. The binding-dissociation equilibrium constant (KD) and dissociation half-life (t½) were calculated based on the following kinetic rates: The binding kinetics parameters of hFXIIa, hFXIIab, or hFXII with the different anti-FXII monoclonal antibodies of the present invention at 25°C are shown in Tables 3 to 5.

[0160] At 25°C, anti-FXII monoclonal antibodies bound to hFXIIa with KD values ​​ranging from 322 pM to 2.09 nM, as shown in Table 3. At 25°C, anti-FXII monoclonal antibodies bound to hFXIIab with KD values ​​ranging from 264 pM to 2.59 nM, and REGN4026 did not bind to hFXIIab, as shown in Table 4. At 25°C, anti-FXII monoclonal antibodies bound to hFXII with KD values ​​ranging from 233 pM to 27.5 nM, and REGN3059 did not bind to hFXII, as shown in Table 5.

[0161] Table 3: Binding kinetic parameters of hFXIIa to FXII monoclonal antibody at 25℃.

[0162] Table 4: Binding kinetic parameters of hFXIIab to FXII monoclonal antibody at 25℃.

[0163] NB = No binding (approximately twice as high as the REGN1945 isotype control RU binding rate) Table 5: Binding kinetic parameters of hFXII to FXII monoclonal antibody at 25℃.

[0164] NB = No binding (approximately twice as high as the REGN1945 isotype control RU binding rate) Example 4: Using the pre-complex method to study Octet cross-competition among different anti-FXII monoclonal antibodies Binding competition between anti-FXII monoclonal antibodies was determined using real-time label-free biolayer interferometry on the OctetHTX biosensor (Pall ForteBio Corp.). The entire experiment was conducted at 25°C with the plate shaken at 1000 rpm in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant Tween-20, and 0.1 mg / mL BSA (HBS-P buffer). To assess whether the two antibodies could competitively bind to the corresponding epitopes on human FXIIa (ERL), anti-human FXII monoclonal antibodies at a wavelength of approximately 1.38 nm to 2.32 nm were first captured onto the tip of the Octet biosensor (Pall ForteBio Corp., #18-5060) coated with anti-hFc antibody by immersing the tip in a well containing a solution of 50 µg / mL anti-human FXII monoclonal antibody (hereinafter referred to as mAb-1) for 5 minutes. The biosensor tip was then saturated with an unrelated IgG4 isotype control monoclonal antibody (hereinafter referred to as the blocking mAb) by immersing it in wells containing 200 µg / mL blocking mAb solution for 10 minutes. The biosensor tip was then subsequently immersed in wells containing a co-complex solution of 25 nM hFXIIa and 1 µM secondary anti-human FXII monoclonal antibody (hereinafter referred to as mAb-2), which had been pre-incubated for 2 hours. The biosensor tip was washed in HBS-P buffer between each step of the experiment. Real-time binding responses were monitored during the experiment, and the binding response at the end of each step was recorded. Background binding of the response to the binding of the pre-complexed human FXII mAb-2 to the captured mAb-1 was corrected for, compared, and the competitive / non-competitive behavior of different anti-FXII monoclonal antibodies was determined.

[0165] Table 6 clearly defines the competitive relationship between antibodies in two directions, regardless of the binding order.

[0166] Table 6: Cross-competition between anti-hFXII antibody and human FXII binding.

[0167] Example 5: Using humans, cynomolgus monkeys, and F12 hu / hu REGN9533 and REGN9534 assays performed on mouse plasma showed positive results in coagulation assays (aPTT, PT), thrombin production assay (TGA), clot lysis assay, and plasma kallikrein activity assay. ​ Functional characterization ​ aPTT was determined on a Diagnostica Stago STart4 hemostasis analyzer as follows: A total of 50 μL of pooled normal human plasma was added to a cuvette at 37°C. After 1 minute, 5 μL of a 2x serially diluted test product (antibody or small molecule inhibitor) dissolved in PBS was added to the cuvette and incubated for 5 minutes. Then, 50 μL of APPT-XL ellagic acid (Thermo Scientific) was added and incubated for 300 seconds, followed by the addition of 50 μL of 20 mM calcium chloride (Thermo Scientific) to initiate the reaction. The measured clotting time at the test product concentration was normalized relative to baseline (drug-free) plasma clotting time and plotted against the logarithmic molar concentration of the test product. The results were analyzed using Prism 5 software (GraphPad) with nonlinear regression (4-parameter logic) to obtain the doubling time concentration (C0). 2xt ).

[0168] ​ PT was determined on a Diagnostica Stago STart4 hemostasis analyzer as follows. A total of 50 μL of pooled normal human plasma was added to a cuvette at 37°C. After 1 minute, 5 μL of a 2x serially diluted test product (antibody or small molecule inhibitor) dissolved in PBS was added to the cuvette and incubated for 5 minutes. Then, 100 μL of tissue factor (TriniCLOT PT Excel, Diagnostica Stago) was added to initiate the reaction. The measured clotting time at the test product concentration was normalized relative to baseline (drug-free) plasma clotting time and plotted against the logarithmic molar concentration of the test product. The results were analyzed using Prism 5 software (GraphPad) with nonlinear regression (4-parameter logic) to obtain the doubling time concentration (C0). 2xt ).

[0169] ​ Thrombin generation curves were determined on an automated thrombography system calibrated by Diagnostica Stago as follows: A total of 55 μL of pooled normal human plasma was added to the wells of a microplate at 37°C. Then, 5 μL of a 2x serially diluted test sample (antibody or small molecule inhibitor) dissolved in PBS was added to the wells and incubated for 30 minutes. 15 μL of APPT-XL ellagic acid (Thermo Fisher Scientific) was diluted with MP reagent and added to the wells, and incubated for 30 minutes. Immediately before 90 minutes of continuous microplate reading, 15 μL of Fluo Flu Cal substrate (Diagnostica Stago) was added. The measured real-time thrombin concentrations were plotted against time to generate thrombography curves for each test sample concentration used.

[0170] ​ Thrombin generation plots were determined on an automated thrombosis plot calibrated by Diagnostica Stago as follows: A total of 55 μL of pooled normal human plasma was added to the wells of a microplate at 37°C. Then, 5 μL of a 2x serially diluted test product (antibody or small molecule inhibitor) dissolved in PBS was added to the wells and incubated for 30 minutes. 15 μL of tissue factor PPP reagent (Diagnostica Stago) was added to the wells and incubated for 30 minutes. Then, immediately before 90 minutes of continuous microplate reading, 15 μL of Fluo Flu Cal substrate (Diagnostica Stago) was added. The measured real-time thrombin concentration values ​​were plotted against time to generate thrombosis plot curves for each test product concentration used.

[0171] ​ Clot dissolution was measured at 37°C using a microplate reader capable of kinetic readings at OD405 nm for 3 hours. A total of 50 μL of pooled normal human plasma was added to each well of a 96-well microplate at 37°C. 5 μL of a 2x serially diluted test product (antibody or small molecule inhibitor) dissolved in PBS was added to each well, and the plate was incubated at 37°C for 30 minutes. Then, 50 μL of activation mixture (final ellagic acid concentration of 62.5 nM or final tissue factor concentration of 0.125 pM with tPA of 50 ng / mL, totaling 100 μL / well) was added to each well, and the plate was immediately read every 30 seconds for 180 minutes using a Molecular Devices i3 system in OD405 nM kinetic mode. Clot dissolution time (CLT) was defined as the time between the 50% clot formation curve and the 50% clot dissolution curve.

[0172] ​ At 37°C, it is able to OD 405nm Plasma kallikrein activity was measured on a microplate reader in kinetic mode for 1 hour. Plasma samples were diluted with 20 mM HBS buffer (500-fold dilution for humans, 250-fold dilution for cynomolgus monkeys, or 500-fold dilution for mice). 100 μL of diluted plasma and 20 μL of mAb were added to each well and incubated at 37°C for 30 minutes, followed by activation with dextran sulfate (DXS; 5 μL of 100 μg / ml DXS) for 10 minutes. 25 μL of 1 mM kallikrein substrate (final concentration for assay) was added to each well, and the sample was immediately read on a microplate reader to measure Vmax (the maximum change in OD at 405 nm per minute). All Vmax readings were corrected by subtracting Vmax from the blank control (HBS assay buffer) and then plotted against the logarithmic molar concentration of the test sample. The results were analyzed using Prism 8 software (GraphPad) with nonlinear regression (4-parameter logic) to obtain the IC50 (half-maximal inhibitory concentration).

[0173] ​ Dose-response curves were generated to determine the effects of anti-FXII / FXIIa on aPTT and PT in combined normal “healthy” human plasma. The control, COMP3059, is an anti-FXIIa antibody that, at a dose of 600 nM, prolonged aPTT to three times the baseline without affecting PT (Tables 7a and 7d). Regeneron’s anti-FXII / FXIIa mAbs (REGN9533 and REGN9534) prolonged aPTT from baseline to approximately seven times and six times, respectively, at a 600 nM dose, without increasing PT (Tables 7b–7c, 7e–7f). The efficacy of the drugs in inhibiting coagulation activity was indexed by arbitrary “doubling time” concentrations (the drug concentration required to prolong coagulation time more than twice the baseline value). The doubling times of Regeneron's FXII / FXIIa mAbs REGN9533 and REGN9534 were approximately 125 nM and 200 nM, respectively, similar to COMP3059, which also required 200 nM. At the highest concentration tested (600 nM), neither of Regeneron's anti-FXII / FXIIa mAbs doubled the PT clotting time; the same was true for COMP3059. In plasma from concomitant normal "healthy" cynomolgus monkeys, anti-FXII / FXIIa mAbs were less effective at doses up to 600 nM, as REGN9533 prolonged aPTT to approximately 3.2 times from baseline, while REGN9534 prolonged it to approximately 1.9 times, and COMP3059 prolonged it to approximately 1.5 times (Tables 8a to 8f). The aPTT doubling time for REGN9533 was 250 nM, while the aPTT doubling times for REGN9534 and COMP3059 were greater than 600 nM. All mAbs showed no change in PT at doses up to 600 nM and did not reach the required doubling time (Tables 8d to 8f). This was observed in the combined normal "healthy" data. F12 hu / hu In mouse plasma, the efficacy of anti-FXII / FXIIa mAbs decreased at doses up to 600 nM because REGN9533 and REGN9534 prolonged the aPTT to approximately 2.X times from baseline, while COMP3059 prolonged it to approximately 1.9 times (Tables 9a to 9f). The aPTT doubling time for REGN9533 was approximately 250 nM, and for REGN9534 it was 300 nM, but for COMP3059 it was approximately 2000 nM (Tables 9a to 9c). At doses up to 600 nM, all mAbs showed no change in PT and did not reach the required doubling time.

[0174] The efficacy of anti-FXII / FXIIa mAbs in inhibiting thrombin production (i.e., the prolongation of thrombin detection time = lag time, the reduction in peak thrombin and the decrease in total thrombin production = intrinsic thrombin potential) was evaluated when plasma was triggered by ellagic acid or tissue factor. In pooled normal “healthy” human plasma, both Regeneron FXII / FXIIa mAbs required a drug concentration of at least 125 nM to begin affecting ellagic acid-activated thrombin production, but at doses ≥ 250 nM, Regeneron's anti-FXII / FXIIa mAbs completely inhibited thrombin production (Tables 10a to 10f), indicating that Regeneron's mAbs inhibited the intrinsic coagulation pathway, thereby preventing downstream thrombin production. COMP3059 showed a dose-dependent effect on thrombin production, with almost complete inhibition at 500 nM. Tissue factor-triggered thrombin production was minimally affected by Regeneron's anti-FXII / FXIIa mAb or COMP3059 (less than 30% of baseline thrombin production). Tables 10a to 10f summarize the concentrations of test products required to double the lag time and halve the peak thrombin and total thrombin production when clotting is activated by ellagic acid or tissue factor. In pooled normal “healthy” cynomolgus monkey plasma, Regeneron's FXII / FXIIa mAb and COMP3059 showed poor inhibition of ellagic acid-triggered thrombin production, even at maximum doses 10 times those used in human plasma. REGN9533 and REGN9534 reduced thrombin production to approximately 60% of baseline, while COMP3059 reduced it by 50% (Tables 11a to 11f). All anti-FXII / FXIIa mAbs had no significant effect on tissue factor-activated thrombin production. Tables 11a to 11f summarize the concentrations of anti-FXII / FXIIa required to double the lag time and halve the peak thrombin time and total thrombin production when clotting in cynomolgus monkey plasma is activated by ellagic acid or tissue factor. (In combined normal "healthy" samples...) F12 hu / hu In mouse plasma, both Regeneron's FXII / FXIIa mAb and COMP3059 effectively inhibited thrombin production induced by ellagic acid at doses up to 500 nM. REGN9533 and REGN9534 reduced thrombin production to approximately 25% of baseline, while COMP3059 reduced it by approximately 30% (Table 12). All anti-FXII / FXIIa mAbs had no significant effect on thrombin production activated by tissue factor. Tables 12a to 12f summarize the effects of thrombin production induced by tissue factor. F12 hu / huThe concentration of anti-FXII / FXIIa required to double the lag time and halve the peak thrombin and total thrombin production in mouse plasma when clotting is activated by ellagic acid or tissue factor.

[0175] In turbidity assays evaluating coagulation and clot dissolution times, the ability of anti-FXII / FXIIa mAb to affect fibrinolysis was assessed. In the presence of X pM tissue factor (TF), all anti-FXII / FXIIa mAbs and the control isotype antibody (REGN1945) produced similar coagulation characteristics, as shown by similar clot formation times of 4 minutes (Table 13), indicating that anti-FXII / FXIIa mAb does not affect extrinsic pathway-mediated coagulation. In the presence of tissue plasminogen activator (tPA 50 ng / mL), after TF-induced coagulation, REGN9533, REGN9534, and REGN1945 showed similar clot dissolution times (i.e., decreased light signal) of 120 to 125 minutes, while COMP3059 showed a slightly longer clot dissolution time of 144 minutes (Table 13).

[0176] The ability of mAbs to inhibit FXIIa-mediated plasma kallikrein activity triggered by dextran sulfate (DXS) was also evaluated. In pooled normal “healthy” human plasma activated with dextran sulfate (DXS), REGN9533 and REGN9534 showed an IC50 of approximately 0.35 nM. 50 Inhibition of plasma kallikrein activity (Table 14). REGN1945 showed no effect, while COMP3059 showed an IC50 increase. 50 The value was 0.49 nM. Table 14 lists the inhibition rates at the mAb concentrations used. In pooled normal “healthy” cynomolgus monkey plasma activated with DXS, REGN9533 and REGN9534 were less potent than human plasma in inhibiting plasma kallikrein activity, with an IC50 of 0.49 nM. 50 These are approximately 1.5 nM and 2.5 nM, respectively (Table 15). REGN1945 showed no effect, while the COMP3059 IC... 50 The value was 9.2 nM. Table 15 lists the inhibition rates at the mAb concentrations used. (The text then abruptly shifts to discussing the combined normal "healthy" cells activated with DXS.) F12 hu / hu ; ​ - / - In mouse plasma, REGN9533 and REGN9534 were less effective than human plasma in inhibiting plasma kallikrein activity, with lower IC50 values. 50 These are approximately 0.7 nM and 1.6 nM, respectively (Table 16). REGN1945 showed no effect, while COMP3059's IC... 50The value was 13.7 nM. Table 16 lists the inhibition rates at the mAb concentrations used.

[0177] Regeneron's anti-FXII / FXIIa mAb REGN9533 and REGN9534 showed better results in humans than in cynomolgus monkeys or... F12 hu / hu In mouse plasma, these mAbs more effectively inhibited intrinsic coagulation pathway activity (i.e., aPTT and TGA-EA). They had no effect on extrinsic coagulation pathway activity (PT and TGA-TF). In the presence of tPA, these mAbs did not affect clot dissolution time. Furthermore, REGN9533 and REGN9534 were found to effectively inhibit plasma kallikrein activation induced by dextran sulfate (i.e., plasma kallikrein activity).

[0178] Table 7a: aPTT table of human plasma with COMP3059 Table 7b: aPTT table for human plasma with REGN9533 Table 7c: aPTT table for human plasma with REGN9534 Table 7d: PT Table of Human Plasma with COMP3059 Table 7e: PT Table of Human Plasma with REGN9533 Table 7f: PT Table of Human Plasma with REGN9534 Table 8a: aPTT table of cynomolgus monkey plasma with COMP3059 Table 8b: aPTT table of cynomolgus plasma with REGN9533 Table 8c: aPTT table of cynomolgus monkey plasma with REGN9534 Table 8d: PT table of cynomolgus monkey plasma with COMP3059 Table 8e: PT table of cynomolgus monkey plasma with REGN9533 Table 8f: PT table of cynomolgus monkey plasma with REGN9534 Table 9a: Products with COMP3059 F12 hu / hu aPTT expression in plasma Table 9b: Table with REGN9533 F12 hu / hu aPTT expression in plasma Table 9c: Table with REGN9534 F12 hu / hu aPTT expression in plasma Table 9d: Table with COMP3059 F12 hu / hu PT levels in plasma Table 9e: Table with REGN9533 F12 hu / hu PT levels in plasma Table 9f: Table with REGN9534 F12 hu / hu PT levels in plasma Table 10a: TGA-EA in human plasma with COMP3059 Table 10b: TGA-EA in human plasma with REGN9533 Table 10c: TGA-EA in human plasma with REGN9534 Table 10d: TGA-TF in human plasma with COMP3059 Table 10e: TGA-TF in human plasma with REGN9533 Table 10f: TGA-TF in human plasma with REGN9534 Table 11a: TGA-EA from cynomolgus monkey plasma containing COMP3059 Table 11b: TGA-EA from cynomolgus monkey plasma containing REGN9533 Table 11c: TGA-EA from cynomolgus monkey plasma containing REGN9534 Table 11d: TGA-TF containing cynomolgus monkey plasma with COMP3059 Table 11e: TGA-TF from cynomolgus monkey plasma containing REGN9533 Table 11f: TGA-TF from cynomolgus monkey plasma containing REGN9534 Table 12a: Products with COMP3059 F12 hu / hu ​ TGA-EA Table 12b: Table with REGN9533 F12 hu / hu ​ TGA-EA Table 12c: Table with REGN9534 F12 hu / hu ​ TGA-EA Table 12d: Table with COMP3059 F12 hu / hu ​ TGA-TF Table 12e: Table with REGN9533 F12 hu / hu ​ TGA-TF Table 12f: Table with REGN9534 F12 hu / hu ​ TGA-TF Table 13: TF-induced clot formation and tPA-mediated clot dissolution time in human plasma Table 14: Plasma kallikrein activity in human plasma Table 15: Plasma kinin-releasing enzyme activity in cynomolgus monkey plasma Table 16: F12 hu / hu Plasma kinin-releasing enzyme activity; ​ mouse plasma The results above indicate that the antibodies described above inhibit thrombin production through the endogenous pathway, but not through the exogenous pathway.

[0179] Example 6: Generation of YTE variants of anti-FXII monoclonal antibody By mutating residues 252, 254, and 256 on the Fc domain to residues Y, T, and E, respectively, an Fc variant of the anti-hFXII / FXIIa monoclonal antibody (“YTE variant”) is generated. Modifying the parental antibody REGN9533 in this manner yields an exemplary YTE variant: REGN17653.

[0180] Table 17a shows the amino acid sequence identifiers of the heavy and light chain variable regions, CDRs, and heavy and light chain constant regions of the YTE variant anti-FXII antibody selected in this disclosure.

[0181] Table 17a: Amino acid sequence identifiers The corresponding nucleic acid sequence identifiers are shown in Table 17b.

[0182] Table 17b: Nucleic Acid Sequence Identifiers Example 7: Verifying FcRn hu / hu B2m hu / hu Mouse models were used to compare the half-lives of YTE variant antibodies and parental antibodies. FcRn was reviewed hu / hu B2m hu / hu mice as ​ The model is applicable for measuring the difference in half-life between YTE variant antibodies and parental antibodies. In these experiments, hIgHC-KC (het) x FcRn hu / hu B2M hu / hu x F12 hu / huMice were administered intravenously with either the parental SARS-CoV2 antibody (REGN10933, hIgG1 isotype) or its YTE variant (REGN13213, hIgG1 isotype) at a single intravenous dose of 10 mg / kg. An anti-Fel D1 monoclonal antibody (hIgG4 isotype) was used as a control. Serum samples were collected on days 0 (2 hours post-administration), 1, 2, 3, 4, 7, 14, 21, 30, 45, and 60 post-administration. Four mice were administered the antibody to each test group, and six mice were administered the antibody to the control group. The results were obtained via GYROLAB. ® Immunoassays were used to measure total drug-administered antibody concentrations in plasma, followed by plasma drug concentrations after acid dissociation. Acid dissociation was not used for control antibodies. For antibody capture, biotin-conjugated anti-idiotypic SARS-CoV2 mAb or biotin-conjugated Fel-D1 mAb were used, and detection was performed using Alexa 647-conjugated mouse anti-human IgG1 / IgG4 mAb. Antibody concentrations in plasma samples were measured over 45 days, and pharmacokinetic (PK) parameters were determined.

[0183] To quantify antibody concentrations, the lower limit of quantification (LLOQ) for SARS-CoV2 antibodies was set at 0.05 µg / ml. When calculating the average concentration, if more than 50% of the values ​​were below the limit of quantification (BLQ), the average was reported as BLQ; if 50% or fewer of the values ​​were BLQ, 0 was used instead of BLQ in the average calculation. If the calculated average was greater than the LLOQ, it was reported as the calculated average; if the calculated average was less than or equal to the LLOQ, it was reported as BLQ. Antibody concentrations were calculated based on immunoassay results and are listed in Tables 18a and 18b.

[0184] PK parameters were calculated based on mAb concentration measurements (Table 18c), confirming that the SARS-CoV2-YTE variant mAb (REGN13213) has a longer half-life compared to the parental SARS-CoV2 mAb (REGN10933). These observations validated the use of FcRn hu / hu B2m hu / hu A mouse model was used to measure the differences in PK parameters between the YTE variant and the parental antibody.

[0185] Table 18a: Total concentration of SARS-CoV2 parental mAb (REGN10933) Table 18b: Total concentration of SARS-CoV2-YTE variant mAb (REGN13213) Table 18c: Anti-SARS-CoV2 parental antibodies and YTE variant antibodies in FcRn hu / hu B2m hu / hu PK parameters in mice Example 8: Comparison of pharmacograms for REGN17653 and REGN9533 After verifying FcRn hu / hu B2m hu / hu After using the mouse model as a model to evaluate the prolonged half-life of YTE variant antibodies, it was compared with IgG4. P The pharmacokinetic (PK) profiles of the anti-hFXII / FXIIa monoclonal antibody REGN9533 and its Fc mutant variant (for extended half-life) REGN17653 were evaluated compared to the isotype control REGN1945. These experiments were performed in F12, FcRn, B2m humanized mice fully expressing human IgG to improve tolerance and reduce anti-drug antibody responses. Each antibody cohort consisted of 6 to 8 mice. Mice administered REGN9533, REGN17653, and REGN1945 received a single intravenous (IV) dose of 10 mg / kg. Blood samples were collected at 2 hours and on days 1, 2, 3, 4, 7, 14, 21, 30, and 45 post-administration. Blood was processed, separated into plasma, and frozen at -80°C until analysis. Functional serum concentrations of REGN9533, REGN17653, and REGN1945 were measured using the GyroLab xPlore platform (Gyros).

[0186] Gyros' technology employs an affinity flow format for automated immunoassays with laser-induced fluorescence detection. Samples are loaded onto an optical disc (CD) containing multiple radially arranged nanoliter-scale affinity trap columns. Liquid flow is controlled by centrifugal and capillary forces.

[0187] To measure functional REGN9533 and REGN17653 in plasma, an acid dissociation step is performed to ensure that the antibody does not complex with soluble FXII. REGN1945 does not require an acid dissociation step. After the acid dissociation step, 20 μg / mL of the test or control product-specific biotinylated capture reagent (anti-REGN9533 mAb, REGN19721: for REGN9533 and REGN17653, or Fel D1.mmH, REGN612: for REGN1945) is added to a Gyrolab Bioaffy 200 CD (Dynospheres) containing an affinity column pre-loaded with streptavidin-coated beads. The concentration range of the standards used for calibration (REGN9533, REGN17653) is 0.3 ng / mL to 1300 ng / mL or 0.5 ng / mL to 2000 ng / mL (REGN1945). Standards or samples were prepared in 1M Tris-HCl (REGN9533, REGN17653) containing normal mouse plasma or in phosphate-buffered saline (PBS) (with 0.5% bovine serum albumin (BSA)) containing normal mouse plasma (REGN1945). Single plasma samples diluted 1:3020 (REGN9533, REGN17653) or 1:2000 (REGN1945) and replicates of standards were added to an affinity column coated with a capture reagent at room temperature. The captured antibody was detected using Alexa-647-conjugated mouse anti-human hIgG4 specific monoclonal antibody (4 μg / mL REGN1298) diluted in Rexxip F buffer (Gyros); the resulting fluorescence signal was recorded in response units (RU) using a GyroLab xPlore instrument. The corresponding limit of quantitation (LLOQ) was defined as the lowest concentration on the standard curve multiplied by the sample dilution factor (1 µg / mL). Sample concentrations were determined by standard curve interpolation, which was constructed using a 4-parameter logistic curve fitted in Gyrolab Evaluator software. The final concentration was calculated using the average concentration from two replicate experiments. This assay could not distinguish between bound and free hFXII / FXIIa, nor could it detect hFXIIb. Antibody concentrations were evaluated over 45 days, and pharmacokinetic (PK) parameters were determined. Samples from day 60 were not analyzed because the concentration of the parental mAb was undetectable at day 45.

[0188] To quantify antibody and protein concentrations, the lower limit of quantification (LLOQ) for anti-hFXII / FXIIa antibody and allotype control antibody was set at 1 µg / ml, and the LLOQ for hFXII / FXIIa levels was set at 0.2 µg / ml. Based on pre-collection blood samples, the baseline protein concentration was 11.8 µg / ml. When calculating the average concentration, if more than 50% of the values ​​were below the limit of quantification (BLQ), the average was reported as BLQ; if 50% or fewer of the values ​​were BLQ, 0 was used instead of BLQ in the average calculation. If the calculated average was greater than the LLOQ, the calculated average was reported; if the calculated average was less than or equal to the LLOQ, the average was reported as BLQ. ​ The changes in mAb concentration and hFXII / FXIIa concentration over time for each individual mouse in the three treatment groups are shown. The mean concentrations for each treatment group were calculated based on these measurements and are presented as a function of time in Tables 19a to 19f.

[0189] Calculated average concentrations revealed that the concentration of REGN17653 antibody decreased linearly and steadily over time until day 30. After day 30, rapid clearance was observed in 5 out of 6 mice in the isotype control group and 6 out of 7 mice in the REGN17653 group, resulting in most mice being BLQ by day 45, although mAB concentration was still detectable in one mouse in each treatment group by day 45.

[0190] Mean FXII / FXIIa protein concentrations showed that REGN17653 (anti-hFXII / FXIIa-YTE mAb) peaked at day 7 with a 20-fold increase in total hFXII / FXIIa concentration compared to baseline, while REGN9533 (parental anti-hFXII / FXIIa mAb) peaked at day 3 with a 7-fold increase in total hFXII / FXIIa concentration compared to baseline. The isotype control showed an initial increase of approximately 3-fold compared to baseline, and concentrations remained near this level throughout the study. hFXII / FXIIa concentrations of REGN17653 were observed to return to approximately the same levels as the isotype control after day 21, and those of REGN9533 after day 7.

[0191] Next, the antibody concentrations corresponding to the peak hFXII / FXIIa levels in each treatment group were calculated and are listed in Table 20. Here, antibody concentrations are converted to nM units based on the FXII molecular weight of 80 kDa, where a baseline antibody level of 11.8 µg / ml corresponds to a concentration of 133 nM. The data revealed that on day 3, at a peak hFXII / FXIIa level of 953 nM, the average concentration of the parental REGN9533 was 536 nM, while on day 7, at a peak hFXII / FXIIa level of 2559 nM, the concentration of the YTE variant REGN17653 was 528 nM.

[0192] Finally, PK parameters were calculated based on mAb concentration measurements in each treatment group and are listed in Table 21. PK plot comparisons revealed that, compared with the parental mAb REGN9533 and the control mAb, the YTE variant REGN17653 exhibited a longer half-life, higher drug exposure, and slower clearance.

[0193] In summary, mouse studies revealed that the YTE variant of the anti-hFXII / FXIIa monoclonal antibody has a longer half-life compared to the parental antibody.

[0194] Table 19a: Total concentration of anti-hFXII / FXIIa parental mAb (REGN9533) Table 19b: Total concentration of anti-hFXII / FXIIa YTE variant mAb (REGN17653) Table 19c: Total concentration of anti-Fel D1 control mAb (REGN1945) Table 19d: Total concentration of hFXII / FXIIa in the anti-hFXII / FXIIa parental mAb (REGN9533) treatment group Table 19e: Total concentrations of hFXII / FXIIa in the anti-hFXII / FXIIa YTE variant mAb (REGN17653) treatment group Table 19f: Total concentrations of hFXII / FXIIa in the anti-Fel D1 control mAb (REGN1945) treatment group Table 20: In hIgHC-KC (het) x FcRnh u / hu B2mhu / hu x F12 hu / hu In mice, the mean molar concentrations of anti-hFXII / FXIIamAb and isotype controls, and their corresponding hFXII / FXIIa concentrations. Table 21: In hIgHC-KC (het) x FcRnh u / hu B2m hu / hu x F12 hu / hu PK parameters of REGN17653 and REGN9533 mAb in mice This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention will become apparent to those skilled in the art from the foregoing description, in addition to those described herein. Such modifications are intended to fall within the scope of the appended claims.

[0195] Informal sequence list SEQ ID NO: 1. GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTGGTAGTGGTGGTAACACATACTACGCAGA CTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTATTTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAGCTTCATACCAGCTGCCATAAGAGGGGGCGACTGGATCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA; SEQ ID NO: 2. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVTVSS; SEQ ID NO: 3. GGA TTC ACC TTT AGC AGC TAT GCC; SEQ ID NO: 4. GFTFSSYA; SEQ ID NO: 5. ATT GGT GGT AGT GGT GGT AAC ACA; SEQ ID NO: 6. IGGSGGNT; SEQ ID NO: 7. GCG AGC TTC ATA CCA GCT GCC ATA AGA GGG GGC GAC TGG ATC GAC CCC; SEQ ID NO: 8. ASFIPAAIRGGDWIDP; SEQ ID NO: 9. GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATAAGAAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAGATTCCTAAGCTCCTGATCTATGCTGCATCCACTTT GCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACTTATTACTGTCAATACTATAACAGTGCCCCGCTCACTTTCGGCGGGAGGGACCAAGGTGGAGATCAAA; SEQ ID NO: 10. DIQMTQSPSSLSSASVGDRVTITCRASQGIRNYLAWYQQKPGKIPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQYYNSAPLTFGGGTKVEIK; 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 TAC TAT AAC AGT GCC CCG CTC ACT; SEQ ID NO: 16. Q Y Y N S A P L T; SEQ ID NO: 17. SEQ ID NO: 18. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK ; SEQ ID NO: 19. GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATAAGAAATTATTTAGCCTGGTATCAACAGAAACCAGGGAAGATTCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACTTATTACTGTCAATACTATAACAGTGCCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG; SEQ ID NO: 20. DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKIPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQYYNSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ; SEQ ID NO: 21. CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCATCGAGAGGCCTTGAGTGGCTGGGAAAGACATACTACAGGTCCAAGTGGTATAATGA TTATACAAAATCTGTGAAAAGTCGAATAACCATCAACCCAGACACATCCAAGAACCACTTCTCCCTGCAACTGAACTCTATGACTCCCGAGGACACGGCTGTGTATTACTGTGCAAGAGAGGTTAGTGGGGAGGTACAACTGGTTCGACTCCTGGGGCCAGGGAACCTGGTCACCGTCTCCTCA; SEQ ID NO: 22. QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSSNSAAWNWIRQSPSRGLEWLGKTYYRSKWYNDYTKSVKSRITINPDTSKNHFSLQLNSMTPEDTAVYYCAREVSGRYNWFDSWGQGTLVTVSS; SEQ ID NO: 23. GGG GAC AGT GTC TCT AGC AAC AGT GCT GCT; SEQ ID NO: 24. GDSVSSNSAA; SEQ ID NO: 25. ACA TAC TAC AGG TCC AAG TGG TAT AAT; SEQ ID NO: 26. TYYRSKWYN; SEQ ID NO: 27. GCA AGA GAG GTT AGT GGG AGG TAC AAC TGG TTC GAC TCC; SEQ ID NO: 28. AREVSGRYNWFDS; SEQ ID NO: 29. GACATCCAGATGACCCAGTCTCCATCCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAACAGTTACTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGT TTGCGAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTACAGAACCTTCACTTTCGGCGGAGGGACCAAAGGTGGAGATCAAA; SEQ ID NO: 30. DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWYQQKPGKAPKLLIAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNYRTFTFGGGTKVEIK; SEQ ID NO: 31. CAG ACC ATT AAC AGT TAC; SEQ ID NO: 32. QTINSY; SEQ ID NO: 33. CAA CAG AAT TAC AGA ACC TTC ACT; SEQ ID NO: 34. QQNYRTFT; SEQ ID NO: 35. SEQ ID NO: 36. QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRSKWYNDYTKSVKSRITINPDTSKNHFSLQLNSMTPEDTAVYYCAREVSGRYNWFDSWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK ; SEQ ID NO: 37. GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAACAGTTACTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCGAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTACAGAACCTTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG; SEQ ID NO: 38. DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWYQQKPGKAPKLLIYAASSLRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYRTFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ; SEQ ID NO: 39. SEQ ID NO: 40. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSGIGGSGGNTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCASFIPAAIRGGDWIDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to human factor XII (FXII), wherein said antibody or antigen-binding fragment 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 said HCDR1 has an amino acid sequence selected from SEQ ID NO: 4 and 24, said HCDR2 has an amino acid sequence selected from SEQ ID NO: 6 and 26, and said HCDR3 has an amino acid sequence selected from SEQ ID NO: 8 and 28, wherein said LCDR1 has an amino acid sequence selected from SEQ ID NO: 12 and 32, said LCR2 has an amino acid sequence selected from SEQ ID NO: 14, and said LCDR3 has an amino acid sequence selected from SEQ ID NO: 16 and 34.

2. The antibody or its antigen-binding portion according to claim 1, wherein: (a) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 4; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6; (c) The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 8; (d) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14; and (f) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16; or in: (a) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 24; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26; (c) The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 28; (d) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 32; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14; and (f) The LCDR2 contains the amino acid sequence shown in SEQ ID NO:

34.

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

30.

4. The antibody or its antigen-binding portion according to any one of the preceding 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 contains the amino acid sequence of SEQ ID NO: 22, and the light chain variable region contains the amino acid sequence of SEQ ID NO:

30.

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

20.

6. The antibody or antigen-binding moiety thereof according to any one of the preceding claims, wherein the antibody or antigen-binding moiety comprises a heavy chain (HC) and a light chain (LC), wherein: The heavy chain comprises the sequence of SEQ ID NO: 18, and the light chain comprises the sequence of SEQ ID NO: 20; or The heavy chain contains the sequence of SEQ ID NO: 36, and the light chain contains the sequence of SEQ ID NO:

38.

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

20.

8. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to human FXII / FXIIa, wherein said antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained in an HCVR and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in an LCVR. (a) wherein the HCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 10; or (b) wherein the HCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

30.

9. The antibody or its antigen-binding fragment according to claim 8, (a) wherein the HCVR comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 2; and wherein the LCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 10; or (b) wherein the HCVR comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 22; and wherein the LCVR comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

30.

10. The antibody or antigen-binding fragment thereof according to 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 is contained in the amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO: 22; and wherein the LCVR is contained in the amino acid sequence having no more than 12 amino acid substitutions in SEQ ID NO:

30.

11. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 10, (a) wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 2; and wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 10; or (b) wherein the HCVR comprises the amino acid sequence of SEQ ID NO: 22; and wherein the LCVR comprises the amino acid sequence of SEQ ID NO:

30.

12. The antibody or antigen-binding fragment thereof according to any one of claims 8 to 11, wherein: (a) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 4; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6; (c) The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 8; (d) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 12; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14; and (f) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 16; or in: (a) The HCDR1 contains the amino acid sequence shown in SEQ ID NO: 24; (b) The HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26; (c) The HCDR3 contains the amino acid sequence shown in SEQ ID NO: 28; (d) The LCDR1 contains the amino acid sequence shown in SEQ ID NO: 32; (e) The LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 14; and (f) The LCDR2 contains the amino acid sequence shown in SEQ ID NO:

34.

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

38.

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

20.

15. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to human factor XII (FXII), wherein said antibody or antigen-binding fragment 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 contains the amino acid sequence GFTFSSYA (SEQ ID NO: 4); (b) The HCDR2 contains the amino acid sequence IGSGGNT (SEQ ID NO: 6); (c) The HCDR3 contains the amino acid sequence ASFIPAAIRGGDWIDP (SEQ ID NO: 8); (d) The LCDR1 contains the amino acid sequence QGIRNY (SEQ ID NO: 12); (e) The LCDR2 contains the amino acid sequence AAS (SEQ ID NO: 14); and (f) The LCDR3 contains the amino acid sequence QYYNSAPLT (SEQ ID NO: 16).

16. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to factor XII (FXII), wherein said antibody or antigen-binding fragment 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 contains the amino acid sequence GDSVSSNSAA (SEQ ID NO: 24); (b) The HCDR2 contains the amino acid sequence TYYRSKWYN (SEQ ID NO: 26); (c) The HCDR3 contains the amino acid sequence AREVSGRYNWFDS (SEQ ID NO: 28); (d) The LCDR1 contains the amino acid sequence QTINSY (SEQ ID NO: 32); (e) The LCDR2 contains the amino acid sequence AAS (SEQ ID NO: 14); and (f) The LCDR3 contains the amino acid sequence QQNYRTFT (SEQ ID NO: 34).

17. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises an Fc domain, the Fc domain comprising one or more mutations that alter the function of the Fc region.

18. The antibody or antigen-binding fragment thereof according to claim 15, wherein the Fc domain is contained in C H 2 or C H Mutations in region 3 that enhance FcγR binding activity.

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

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

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

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

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

24. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises a modified glycosylation pattern, wherein the modification enhances antibody-dependent cytotoxicity (ADCC) function and / or alters complement-dependent cytotoxicity (CDC) activity.

25. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a bispecific antibody, a multispecific antibody, or an antigen-binding fragment thereof.

26. The antibody or antigen-binding fragment thereof according to claim 25, wherein the multispecific antibody binds to different epitopes of FXII, or comprises an antigen-binding domain specific to FXII and one or more other target peptides.

27. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises one or more of the following features: (a) is a fully human monoclonal antibody; (b) Binds to activated factors XII (FXIIa, FXIIab); (c) It binds to FXII at 25 °C with a dissociation constant (KD) of less than 1.5 nM; (d) Binding with FXII at 37°C with KD less than 17 nM; (e) KD is combined with FXIIa at 25°C in a concentration of less than 5 nM, preferably less than 0.9 nM; (f) KD is combined with FXIIa at 37°C in a concentration of less than 6.5 nM, preferably less than 2.5 nM; (g) KD is combined with FXIIab at 25°C in a concentration of less than 5 nM, preferably less than 0.7 nM; (h) Blocking thrombin generation via the intrinsic pathway at concentrations less than 250 nM; and (I) Block thrombin generation via the intrinsic pathway, but not via the extrinsic pathway; Or any combination thereof from (a) to (I).

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

29. An antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is chemically or biologically conjugated to the therapeutic portion.

30. The antibody or antigen-binding fragment thereof according to claim 29, wherein the conjugated portion 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 an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof competing with an antibody or antigen-binding fragment thereof according to any one of the preceding claims for binding to factor XII.

32. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof binding to the same epitope as an antibody or antigen-binding fragment thereof according to any one of the preceding claims.

33. A pharmaceutical composition comprising a separated antibody or antigen-binding fragment thereof that binds to factor XII / XIIa according to any one of the preceding claims, and a pharmaceutically acceptable carrier or diluent.

34. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 32.

35. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding an LCVR of an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 32.

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

37. A cell expressing the vector according to claim 36.

38. A method for producing an anti-FXII / FXIIa antibody or an antigen-binding fragment thereof, the method comprising growing cells according to claim 37 under conditions permissible for producing the antibody or an antigen-binding fragment thereof, and recovering the antibody or fragment thus produced.

39. The method of claim 38, further comprising formulating the antibody or an antigen-binding fragment thereof into a pharmaceutical composition comprising an acceptable carrier.

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

41. The method of claim 40, wherein the subject suffers from a disease, condition, or illness selected from the group consisting of: venous thrombosis, arterial thrombosis, device thrombosis, thromboembolism, hereditary angioedema, stroke, thrombotic tendency, myocardial ischemia, atherosclerotic plaque rupture, use of mechanical valve prostheses, use of blood-contact medical devices, use of blood-contact extracorporeal circulation circuits, venous thromboembolism, pulmonary embolism, deep vein thrombosis, portal vein thrombosis, Budd-Chiari syndrome, Paget-Schroth 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 according to claim 40 or 41, wherein the pharmaceutical composition is administered to the subject in need in a preventative or therapeutic manner.

43. The method according to any one of claims 40 to 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: anticoagulants, direct thrombin inhibitors, thrombolytic drugs, fibrinolytic drugs, antiplatelet drugs, anti-inflammatory drugs, antihypertensive drugs, secondary anti-FXII antibodies, lipid-lowering drugs, mechanical thrombectomy, catheter-guided thrombolysis, compression stockings, and surgery.

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

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

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

48. The method according to any one of claims 40 to 47, wherein the pharmaceutical composition is administered in more than one dose, wherein subsequent doses are substantially the same as or less than the initial dose, wherein the interval between the subsequent doses is at least 1 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 for preventing or treating a subject at risk of thrombosis, the method comprising administering a pharmaceutical composition to the subject in need before, simultaneously with, or after one or more other therapeutic agents, the pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 32.

50. The method of claim 49, wherein the one or more additional therapeutic agents are selected from: anticoagulants, thrombin inhibitors, thrombolytic drugs, antiplatelet drugs, antihypertensive drugs, immunosuppressants, fibrinolytics, cholesterol-lowering agents, anti-inflammatory drugs, secondary anti-FXII antibodies, mechanical thrombectomy, catheter-guided thrombolysis, and surgery.

51. A method for diagnosing or detecting FXII-related diseases or conditions, the method comprising contacting a sample of a subject with one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 32, wherein the antibodies or antigen-binding fragments thereof are detectably labeled.

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