Factor XI A2 Domain Binding Antibodies and Methods of Use Thereof

Abstract

The present disclosure provides antibodies that bind to the A2 domain of factor XI (FXI) and methods of using them. According to certain embodiments, the antibodies are antagonistic antibodies that inhibit clot formation via the intrinsic pathway without affecting hemostasis, as shown by the effect of prolonging aPTT without affecting PT. Thus, these antagonistic antibodies can be used to treat blood clotting diseases or disorders or treatment regimens that have clot formation as a risk factor, such as, but not limited to, atrial fibrillation. In certain embodiments, the present disclosure includes antibodies that bind to FXI and mediate clot or thrombus formation. The antibodies of the present disclosure can be fully human non-naturally occurring antibodies.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 270,629, filed October 22, 2021, the contents of which are expressly incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. The XML copy was created on October 19, 2022, is named 118003-01020.XML, and is 45,437 bytes in size.

[0003] The present disclosure relates to antibodies that bind to the apple 2 (A2) domain of factor XI (FXI), compositions comprising these antibodies, and methods of their use. [Background technology]

[0004] The formation of a blood clot (i.e., a thrombus) is initiated through either (a) the contact pathway or (b) the extrinsic pathway. Both pathways converge through a common pathway to activate (c) thrombin, which acts as a serine protease to convert soluble fibrinogen into insoluble chains of fibrin. Cross-linked fibrin proteins, along with aggregated platelets and red blood cells, are the major components of blood clots.

[0005] The extrinsic pathway mediates hemostasis upon vascular injury, where exposed tissue factor (TF) activates factor VII (FVII) to form the FVIIa-TF complex, which in the common pathway activates factor X (FX) to generate prothrombinase, which generates thrombin and subsequent clot formation.

[0006] The contact pathway differs from the extrinsic pathway in that it is less involved in hemostasis but still influences clot formation. Here, coagulation is initiated by intrinsic events such as the release of polyphosphate from platelets or the extrusion of neutrophil extracellular traps (NETs) containing histones and DNA from neutrophils, which activates factor XII (FXII). Activated FXII (i.e., FXIIa) further activates factor XI (FXI) to form FXIa, which leads to the generation of thrombin via the common pathway. Thrombin and platelet-produced polyphosphate also activate FXI in a feed-forward manner to amplify clot formation.

[0007] FXI is the zymogen of the plasma protease FXIa, which sustains thrombin generation via FIX activation. FXI is a 160 kDa disulfide-linked homodimer, with each subunit consisting of, from N- to C-terminus, apple domains A1-A4 and a catalytic domain (CD). A disulfide bond is between the A4 domain of each subunit. FXI subunits are activated by cleavage of one or both of the Arg-Ile bonds located between the A4 and CD domains to form FXIa. In general, cleavage of the Arg-Ile bond is thought to be catalyzed by FXIIa and / or thrombin. Summary of the Invention

[0008] Provided herein are isolated monoclonal antibodies and antigen-binding fragments thereof that bind, e.g., specifically bind, to the A2 domain of factor XI (FXI). In any of the embodiments disclosed herein, the antibodies or antigen-binding fragments thereof may specifically bind to the A2 domain of FXI. The isolated antibodies and antigen-binding fragments of the present disclosure are useful for treating diseases and disorders associated with FXI activity or expression.

[0009] In its broadest aspect, the disclosure provides anti-FXI antibodies that block the activity or activation of FXI and reduce clot formation. These antibodies can be used to prevent, treat, reduce the occurrence, or reduce the adverse effects of clot formation in the bloodstream or tissues in patients in need thereof. Preferably, the anti-FXI antibodies attenuate thrombosis without interfering with hemostasis.

[0010] In certain embodiments, anti-FXI antibodies may be useful in treating a variety of blood clotting disorders or diseases where treatment of the disease involves the use of anticoagulant therapy and where the patient is at risk of bleeding due to the use of anticoagulant therapy. These indications, disorders, or diseases include high-risk atrial fibrillation, primary venous thromboembolism (VTE) prophylaxis, extended VTE treatment, prevention of recurrent ischemia following acute coronary syndromes, end-stage renal disease, medical devices (e.g., mechanical heart valves, ventricular assist devices, small bore grafts, central venous catheters, etc.), extracorporeal circuits, etc.

[0011] The antibodies of the disclosure may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, e.g., to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0012] Exemplary anti-FXI antibodies of the disclosure are listed herein in Table 1. Table 1 provides amino acid and nucleic acid sequence identifiers for exemplary heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-FXI antibodies. Table 1 also lists nucleic acid sequence identifiers for HCVRs, LCVRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-FXI antibodies.

[0013] The present disclosure provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0014] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0015] The present disclosure also provides antibodies, or antigen-binding fragments thereof, that bind to FXI, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) that comprises any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies, or antigen-binding fragments thereof, that comprise an HCVR / LCVR amino acid sequence pair included within any of the exemplary anti-FXI antibodies listed in Table 1.

[0016] Thus, in a first aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to serum coagulation factor XI (FXI), the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising an amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0017] In one embodiment, the anti-FXI antibody or antigen-binding fragment thereof exhibits one or more properties selected from the group consisting of: (a) an antagonist antibody; (b) a K of less than about 35 pM as measured by surface plasmon resonance at 25° C. or 37° C. D binds to human FXI at (c) a K of less than about 765 pM as measured by surface plasmon resonance at 25° C. or 37° C. D binds to human FXIa at (d) binds to human FXI with a dissociation half-life (t) of greater than about 1,000 minutes as measured by surface plasmon resonance at 25° C. or 37° C.; (e) binds to human FXIa with a dissociation half-life (t) of greater than about 23 minutes as measured by surface plasmon resonance at 25° C. or 37° C.; (f) inhibiting activation of factor Xa (FXa) by FXI by at least about 85% in normal dilute plasma with an IC50 of less than about 39 pM; (g) inhibiting activation of factor Xa (FXa) by FXIa by at least about 25% in normal dilute plasma with an IC50 of less than about 10 nM; (h) preferentially binds to the PKA2 domain (i.e., apple domain 2 or A2) relative to full length FXI, PKA1, PKA3, and PKA4 as determined by label-free biolayer interferometry; (i) specifically binds to an epitope within the FXI PKA2 domain and competes for binding to FXI with an antibody that overlaps with the FXI PKA2 domain; (j) increasing activated partial thromboplastin time (aPTT), a measure of intrinsic clotting time, in primates in vivo by at least two-fold without measurably affecting prothrombin time (PT), a measure of extrinsic clotting time; (k) inhibiting intrinsic pathway peak thrombin activity by 5% to 15% in primates in vivo; (l) prolongs the aPTT in human plasma in vitro by about 2-fold at a concentration of about 33 nM or less without doubling the PT; and / or m) inhibits intrinsic pathway thrombin generation in human plasma in vitro at concentrations of about 31 nM or greater and has no effect on extrinsic pathway thrombin generation up to doses of about 500 nM.

[0018] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof that binds to factor XI (FXI), wherein the antibody or antigen-binding fragment thereof comprises: (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) comprising an amino acid sequence set forth in Table 1; and (b) a CDR of a light chain variable region (LCVR) comprising an amino acid sequence set forth in Table 1.

[0019] In one embodiment, an antibody or antigen-binding fragment thereof that binds to FXI comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the HCVR sequence of SEQ ID NO: 3 or SEQ ID NO: 31, or a substantially similar sequence thereof having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR sequence of SEQ ID NO: 13, or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0020] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises a HCVR having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:31.

[0021] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI further comprises an LCVR having the amino acid sequence of SEQ ID NO:13.

[0022] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises a HCVR having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:31 and a LCVR having the amino acid sequence of SEQ ID NO:13.

[0023] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises the CDRs of the HCVR / LCVR amino acid sequence pair of SEQ ID NO:3 / 13 or SEQ ID NO:31 / 13.

[0024] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0026] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0027] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0028] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0029] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0030] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof comprising an HCDR3 / LCDR3 amino acid sequence pair included in any of the exemplary anti-FXI antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is SEQ ID NO:9 / 19 or SEQ ID NO:37 / 19.

[0031] The disclosure also provides antibodies or antigen-binding fragments thereof that bind FXI comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) included in any of the exemplary anti-FXI antibodies listed in Table 1. In certain embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 amino acid sequence set is SEQ ID NOs:5, 7, 9, 15, 17, and 19, or SEQ ID NOs:33, 35, 37, 15, 17, and 19.

[0032] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. 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 are also available to identify CDR sequences within antibodies.

[0033] In one embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:5; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 7; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 9; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 15; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 17, and (f) an isolated antibody or antigen-binding fragment thereof that binds to FXI, comprising an LCDR3 domain having the amino acid sequence of SEQ ID NO:19.

[0034] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises the set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of SEQ ID NOs: 5-7-9-15-17-19.

[0035] In one embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof, comprising: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 33; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 35; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 37; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 15; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 17, and (f) an isolated antibody or antigen-binding fragment thereof that binds to FXI, comprising an LCDR3 domain having the amino acid sequence of SEQ ID NO:19.

[0036] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises the set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of SEQ ID NOs: 33-35-37-15-17-19.

[0037] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises an antibody or antigen-binding fragment thereof that competes with a reference antibody for binding to FXI, wherein the reference antibody preferentially binds to Apple 2 (A2 or PKA2) of FXI.

[0038] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises an antibody or antigen-binding fragment thereof that binds to the same epitope as a reference antibody, wherein the reference antibody preferentially binds to Apple 2 (A2 or PKA2) of FXI.

[0039] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 1,000 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0040] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 500 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0041] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 250 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0042] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 100 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0043] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 50 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0044] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 25 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXI.

[0045] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a dissociation half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0046] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 20 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0047] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 60 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0048] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 2 hours as measured by surface plasmon resonance at 25°C or 37°C.

[0049] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 5 hours as measured by surface plasmon resonance at 25°C or 37°C.

[0050] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 10 hours as measured by surface plasmon resonance at 25°C or 37°C.

[0051] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 15 hours as measured by surface plasmon resonance at 25°C or 37°C.

[0052] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 16 hours as measured by surface plasmon resonance at 25°C or 37°C.

[0053] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 1,000 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0054] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 100 nM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXIa.

[0055] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 10 nM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXIa.

[0056] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 1,000 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXIa.

[0057] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 500 pM as measured by surface plasmon resonance at 25° C. or 37° C. D It binds to human FXIa.

[0058] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a dissociation half-life (t1 / 2) of greater than about 5 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0059] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a t1 / 2 of greater than about 10 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0060] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t1 / 2 of greater than about 15 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0061] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a t1 / 2 of greater than about 20 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0062] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a t1 / 2 of greater than about 25 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0063] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI inhibits activation of factor Xa (FXa) by FXI in normal dilution serum by at least about 85% with an IC50 of less than about 39 pM.

[0064] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds FXI inhibits activation of factor Xa (FXa) by FXIa by at least about 25% in normal dilution serum with an IC50 of less than about 10 nM.

[0065] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds FXI preferentially binds to the PKA2 domain (i.e., apple domain 2 or A2) relative to full length FXI, PKA1, PKA3, and PKA4 as determined by label-free biolayer interferometry.

[0066] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds FXI specifically binds to an epitope within FXI PKA2 and competes for binding to FXI with an antibody that overlaps with FXI PKA2.

[0067] In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds to FXI increases activated partial thromboplastin time (aPTT), a measure of intrinsic clotting time, in primates in vivo by at least two-fold, without measurably affecting prothrombin time (PT), a measure of extrinsic clotting time.

[0068] In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds FXI inhibits in vivo intrinsic pathway peak thrombin activity in a primate by about 5%-15%, about 1%-20%, about 0.5%-25%, about 3%-5%, about 4%-6%, about 5%-7%, about 6%-8%, about 7%-9%, about 8%-10%, about 9%-11%, about 10%-12%, about 11%-13%, about 12%-14%, about 13%-15%, about 14%-16%, about 15%-17%, about 16%-18%, about 17%-19%, or about 18%-20%.

[0069] In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds FXI extends the aPTT in human plasma in vitro by about 2-fold at a concentration of about 100 pM to 100 nM, about 1 nM to 50 nM, about 5 nM to 40 nM, about 10 nM to 35 nM, 60 nM or less, 55 nM or less, 50 nM or less, 45 nM or less, 40 nM or less, 39 nM or less, 38 nM or less, 37 nM or less, 36 nM or less, 35 nM or less, 34 nM or less, 33 nM or less, 32 nM or less, 31 nM or less, 30 nM or less, 25 nM, or 20 nM or less, wherein the anti-FXI extends the aPTT by about 2-fold without doubling the PT.

[0070] In one embodiment, an isolated antibody or antigen-binding fragment thereof that binds FXI inhibits intrinsic pathway thrombin generation in human plasma in vitro at a concentration of about 10 nM-100 nM, about 15 nM-500 nM, about 20 nM-60 nM, about 25 nM-50 nM, 25 nM or more, 26 nM or more, 27 nM or more, 28 nM or more, 29 nM or more, 30 nM or more, 31 nM or more, 32 nM or more, 33 nM or more, 34 nM or more, 35 nM or more, 36 nM or more, 37 nM or more, 38 nM or more, 39 nM or more, or 40 nM or more, where anti-FXI inhibits intrinsic pathway thrombin generation without effect on extrinsic pathway thrombin generation at doses up to about 500 nM.

[0071] In a second aspect, the disclosure provides nucleic acid molecules encoding anti-FXI antibodies or portions thereof. For example, the disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0072] The disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0073] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 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 thereto.

[0074] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 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 thereto.

[0075] The present disclosure also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 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 thereto.

[0076] The present disclosure also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 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 thereto.

[0077] The present disclosure also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 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 thereto.

[0078] The present disclosure also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 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 thereto.

[0079] The present disclosure also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), and the amino acid sequence sets of HCDR1, HCDR2, HCDR3 are as defined by any of the exemplary anti-FXI antibodies listed in Table 1.

[0080] The present disclosure also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), and the amino acid sequence sets of LCDR1, LCDR2, LCDR3 are as defined by any of the exemplary anti-FXI antibodies listed in Table 1.

[0081] The disclosure also provides nucleic acid molecules encoding both a HCVR and a LCVR, where the HCVR comprises any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect of the disclosure, the nucleic acid molecule encodes a HCVR and a LCVR, where the HCVR and LCVR are both derived from the same anti-FXI antibody listed in Table 1.

[0082] In a third aspect, the disclosure provides a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-FXI antibody. For example, the disclosure includes a recombinant expression vector comprising any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Table 1. Also included within the scope of the disclosure are host cells into which such vectors have been introduced, as well as methods of producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0083] The present disclosure includes anti-FXI antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be made to modify complement-dependent cytotoxicity (CDC).

[0084] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising at least one antibody, or antigen-binding fragment thereof, of the present disclosure that specifically binds to FXI, and a pharma- ceutically acceptable carrier.

[0085] In a related aspect, the disclosure features a composition that is a combination of an anti-FXI antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-FXI antibody. The second therapeutic agent may be useful in alleviating at least one symptom of a disease or disorder.

[0086] In a fifth aspect, the present disclosure provides a method for enhancing biological activity mediated by FXI, the method comprising contacting FXI with a biologically effective amount of an antagonist anti-FXI antibody of Table 1, or contacting FXI with a pharmaceutical composition comprising a biologically effective amount of an antagonist anti-FXI antibody of Table 1.

[0087] In certain embodiments, the biological activity is blood clotting or blood clotting as a result of the intrinsic clotting pathway, but not as a result of the extrinsic (i.e., for example, tissue factor-induced) pathway, and blood clotting or blood clotting as a result of the intrinsic clotting pathway, and not as a result of the extrinsic pathway, is inhibited or otherwise reduced upon contact of FXI or FXIa with an antagonist anti-FXI antibody.

[0088] In a sixth aspect, the present disclosure provides a therapeutic method for treating a disease or disorder associated with FXI activity or expression, or at least one symptom associated with the disease or disorder, using an anti-FXI antibody or antigen-binding portion of the antibody of the present disclosure. The therapeutic method according to this aspect of the present disclosure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of the antibody of the present disclosure. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, inhibited, or prevented by targeting FXI and / or inactivating FXI-mediated blood coagulation.

[0089] In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method of treating pathological intrinsic coagulation without adversely affecting hemostasis. In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method of treating pathological intrinsic coagulation without adversely affecting hemostasis. In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method of treating pathological intrinsic coagulation without adversely affecting hemostasis. In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method of treating pathological intrinsic coagulation without adversely affecting hemostasis. In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method of treating pathological intrinsic coagulation without adversely affecting hemostasis. the use of estrogen as a stimulant for the treatment of a disease, disorder, coagulation side effect, indirect coagulation effect, such as any one of the following: supplemental use of estrogen including estrogen-releasing hormone (ESHH) (pill), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other diseases leading to reduced activity, heparin-induced thrombocytopenia (reduction of platelets in the blood due to heparin or low molecular weight heparin preparations), prolonged air travel, antiphospholipid syndrome, deep vein thrombosis or pulmonary embolism, myeloproliferative disorders such as polycythemia vera or essential thrombocytosis, paroxysmal nocturnal hemoglobinuria, inflammatory bowel syndrome, HIV / AIDS, nephrotic syndrome, COVID-19 infection or spike protein immune effect.

[0090] A seventh aspect of the present disclosure provides a method of preventing thrombosis in a subject without adversely affecting hemostasis, the method comprising administering to the subject a therapeutically effective amount of a FXI antagonist antibody of Table 1, or a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof.

[0091] In one embodiment, the above-mentioned method can be achieved by administering to a subject in need thereof an antagonist anti-FXI antibody, or an antigen-binding fragment thereof, comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in Table 1, or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0092] In one embodiment, the method of the disclosure may be achieved by administering an antagonist FXI antibody of the disclosure, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the HCVR sequence of SEQ ID NO: 3 or SEQ ID NO: 31, or a substantially similar sequence thereof having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LCVR sequence of SEQ ID NO: 13, or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0093] In one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:31.

[0094] In one embodiment, the antibody or antigen-binding fragment thereof comprises a LCVR having the amino acid sequence of SEQ ID NO:13.

[0095] In one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 3 and a LCVR having the amino acid sequence of SEQ ID NO: 13. In one embodiment, the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 31 and a LCVR having the amino acid sequence of SEQ ID NO: 13.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof comprises the CDRs of the HCVR / LCVR amino acid sequence pair of SEQ ID NO:3 / 13 or SEQ ID NO:31 / 13.

[0097] In one embodiment, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:3 / 13 or SEQ ID NO:31 / 13.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:5; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 7; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 9; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 15; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 17, and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 19.

[0099] In one embodiment, the antibody or antigen-binding fragment thereof comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 5-7-9-15-17-19.

[0100] In one embodiment, the antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 33; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 35; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 37; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 15; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 17, and (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO: 19.

[0101] In one embodiment, the antibody or antigen-binding fragment thereof comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 33-35-37-15-17-19.

[0102] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to human FXI.

[0103] In one embodiment, the disease or disorder treated with an anti-FXI antibody of the present disclosure is thrombosis and any complications resulting from thrombosis.

[0104] Any disease or disorder associated with FXI activity or expression is envisioned to be suitable for treatment with the antibodies of the present disclosure. These disorders may include, but are not limited to, any disease or condition in which harmful blood clot formation is a risk, particularly conditions in which intrinsic coagulation and hemostasis are a risk to the patient.

[0105] Other embodiments will become apparent from review of the following detailed description. [Brief description of the drawings]

[0106] [Figure 1] Line graphs depicting fold change in aPTT as a function of log antibody concentration are plotted. Filled pyramids depict either an isotype control antibody (top apex) or the FXI A2 antibody of the present invention, REGN9933 (bottom apex, shown as H4H29790P2). Filled circles depict comparison antibodies binding to FXI A2. [Diagram 2] A line graph is plotted showing thrombin activity as a function of concentration of isotype control antibody (REGN1945) over time. [Diagram 3] 1 depicts a line graph showing thrombin activity as a function of concentration of a subject antibody (REGN9933) that binds to FXI A2 over time. [Figure 4]A line graph is plotted showing thrombin activity as a function of concentration of a control antibody (COMP3448) that binds to FXI A2 over time. [Diagram 5] A line graph is plotted showing thrombin activity as a function of concentration of isotype control antibody (REGN1945) over time. [Figure 6] 1 depicts a line graph showing thrombin activity as a function of concentration of a subject antibody (REGN9933) that binds to FXI A2 over time. [Figure 7] A line graph is plotted showing thrombin activity as a function of concentration of a control antibody (COMP3448) that binds to FXI A2 over time. [Figure 8A] A line graph depicting the fold change in activated partial thromboplastin time (aPTT) as a function of log concentration of antibody is plotted. The darker shades depict isotype control. The lighter shades depict the subject antibody REGN9933, which binds to FXI A2. For the aPTT clotting assay, human donor plasma was incubated with two-fold serial dilutions of either REGN9933 or IgG4P isotype control from 19 nM to 1200 nM, followed by the addition of ellagic acid (EA), a specific activator of the intrinsic coagulation pathway. [Figure 8B] A line graph showing the fold change in prothrombin time (PT) as a function of the log concentration of antibody is plotted. In the PT clotting assay, human donor plasma was incubated with either REGN9933 or IgG4P isotype control at 600 nM and 1200 nM, followed by the addition of tissue factor (TF), a specific activator of the extrinsic coagulation pathway. Figure 8B shows that the lines for REGN9933 and IgG4P isotype control in the PT clotting assay overlap, and therefore cannot be distinguished. The change in aPTT and PT compared to baseline (i.e., non-antibody control) is determined, and the average change of duplicate samples is plotted for each antibody concentration tested. [Figure 9A] 1 depicts a line graph showing ellagic acid (EA)-mediated thrombin activity as a function of concentration of a subject antibody binding to FXI A2 over time. [Figure 9B] A line graph showing EA-mediated thrombin activity as a function of isotype control antibody concentration over time is plotted. [Figure 9C] 1 depicts a line graph showing tissue factor (TF)-mediated thrombin activity as a function of concentration of a subject antibody, REGN9933, that binds to FXI A2 over time. [Figure 9D] Line graphs are plotted showing TF-mediated thrombin activity as a function of the concentration of isotype control antibody over time. Human donor plasma was incubated with two-fold serial dilutions of either the subject mAbs or IgG4P isotype control from 16 nM to 500 nM, followed by the addition of (Figures 9A and 9B) ellagic acid (EA), a specific activator of the intrinsic coagulation pathway, or (Figures 9B and 9C) tissue factor (TF), a specific activator of the extrinsic coagulation pathway. The measured real-time thrombin concentration values ​​were plotted against time to obtain thrombogram profiles for each antibody concentration tested as well as the no-antibody control (i.e., PBS). [Figure 10]REGN9933 is associated with increased levels of total FXI in plasma. Female cynomolgus monkeys were administered a single intravenous (IV) slow bolus injection of vehicle (control, n=3), 1 mg / kg REGN9933 (n=3), 3 mg / kg REGN9933 (n=3), or 10 mg / kg REGN9933 (n=3) and monitored for 8 weeks after dosing. Blood samples were collected from all animals pre-dose and 5 minutes, 6 hours, days 2, 3, 4, 6, 8, 11, 15, 22, 29, 36, 43, 50, and 57 after dosing. Pre-dose measurements served as the baseline for each animal (FIGS. 10A and 10B). Figure 10A plots a line graph showing total REGN9933 in serum measured by enzyme-linked immunosorbent assay (ELISA) using a mouse anti-human IgG Fc monoclonal antibody (mAb). Figure 10 2.1.1 Drug Total plots a line graph showing total FXI in plasma measured using ELISA using a goat anti-human FXI mAb, where fold change in total FXI (relative to baseline) is reported. Data are expressed as group mean ± standard error of the mean (SEM). [Figure 11] REGN9933 prolongs aPTT but not PT, with a dose-dependent effect on duration. Female cynomolgus monkeys were administered a single intravenous (IV) slow bolus injection of vehicle (control, n=3), 1 mg / kg REGN9933 (n=3), 3 mg / kg REGN9933 (n=3), or 10 mg / kg REGN9933 (n=3) and monitored for 8 weeks after dosing. Blood samples were collected from all animals pre-dose and 5 minutes, 6 hours, 2 days, 3 days, 4 days, 6 days, 8 days, 11 days, 15 days, 22 days, 29 days, 36 days, 43 days, 50 days, and 57 days after dosing. Pre-dose measurements served as the baseline for each animal. (Figures 11A and 11B). Figure 11A depicts a line graph showing EA-mediated aPTT as a function of fold change in aPTT (relative to baseline) over time. Figure 11B depicts a line graph showing TF-mediated PT as a function of fold change in PT (relative to baseline) over time. Data are presented as group mean ± standard error of the mean (SEM). [Figure 12] REGN9933 reduces the endogenous thrombin p generating capacity of the intrinsic and extrinsic coagulation pathways and has a dose-dependent effect on the duration of activity in the intrinsic pathway. Female cynomolgus monkeys were administered a single intravenous (IV) slow bolus injection of vehicle (control, n=3), 1 mg / kg REGN9933 (n=3), 3 mg / kg REGN9933 (n=3), or 10 mg / kg REGN9933 (n=3) and monitored for 8 weeks after dosing. Blood samples were collected from all animals pre-dose and 5 minutes, 6 hours, 2 days, 3 days, 4 days, 6 days, 8 days, 11 days, 15 days, 22 days, 29 days, 36 days, 43 days, 50 days, and 57 days after dosing. The pre-dose measurements served as the baseline for each animal. Thrombin generation assays (TGA) were performed on plasma samples using ellagic acid (EA) and tissue factor (TF) as activators of the intrinsic and extrinsic coagulation pathways, respectively (Figures 12A and 12B). Figure 12A depicts a line graph showing EA-mediated aPTT as a function of fold change in endogenous thrombin potential (ETP) over time (relative to baseline). Figure 12B depicts a line graph showing TF-mediated PT as a function of fold change in ETP over time (relative to baseline). Data are presented as group mean ± standard error of the mean (SEM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] Before describing the present disclosure, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, since such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.As used herein, the term "about" when used in relation to a specific recited numerical value means that the value may vary from the recited value by 1% or less.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0109] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0110] definition Expressions such as "FXI", also known as "coagulation factor XI" or "Factor XI", refer to the human plasma serine protease (unless specified to be from another species) comprising the amino acid sequence set forth in amino acid residues 19-625 of Accession Number NP_000119.1 (SEQ ID NO: 21). Human FXI comprising a myc-myc-hexahistidine tag is set forth as SEQ ID NO: 22 (amino acid residues 1-607 are human FXI and amino acid residues 608-635 are the myc-myc-hexahistidine tag).

[0111] In certain examples, cell lines expressing FXI protein, subunits of FXI protein, and chimeric proteins comprising one or more FXI subunits, a tag sequence, and a plasma kallikrein protein sequence were prepared. For example, SEQ ID NO:23 (construct hFXI_PKA1) is a chimera comprising, at amino acids 1-85, the apple 1 domain of human kallikrein B1 (PKA1) (amino acids G20-C104 of human kallikrein B1 [SEQ ID NO:28]), amino acids 86-606, amino acids H105-V625 of human FXI (hFXI), and, at amino acids 607-634, a myc-myc-hexadistidine tag.

[0112] For example, SEQ ID NO: 24 (construct hFXI_PKA2) is a chimera that contains, at amino acids 1-90, amino acids E19-S108 of hFXI, at amino acids 91-174, the apple 2 domain of hKLKB1 (PKA2, also referred to as "A2") (amino acids C111-C193 SEQ ID NO: 28), at amino acids 175-605, amino acids A195-V625 of hFXI, and, at amino acids 606-633, a myc-myc-hexadistidine tag.

[0113] For example, SEQ ID NO: 25 (construct hFXI_PKA3) is a chimera comprising, at amino acids 1-180, amino acids E19-L198 of hFXI, at amino acids 181-264, the apple 3 domain (PKA3) of hKLKB1 (amino acids C201-C284 SEQ ID NO: 28), at amino acids 265-605, amino acids H285-V625 of hFXI, and, at amino acids 606-633, a myc-myc-hexadistidine tag.

[0114] For example, SEQ ID NO: 26 (construct hFXI_PKA4) is a chimera comprising, at amino acids 1 to 271, amino acids E19 to V289 of hFXI, at amino acids 272 to 355, the apple 4 domain of hKLKB1 (PKA4) (amino acids C292 to C375 SEQ ID NO: 28), at amino acids 356 to 605, amino acids M376 to V625 of hFXI, and, at amino acids 606 to 633, a myc-myc-hexadistidine tag.

[0115] For example, SEQ ID NO: 27 (construct hKLKB1.mmh) is a chimera that comprises, at amino acids 1-619, amino acids G20-A638 of hKLKB1, and, at amino acids 620-647, a myc-myc-hexadistidine tag.

[0116] As used herein, the term "anti-FXI antibody" includes both monovalent antibodies having a single specificity, and bispecific antibodies comprising a first arm that binds to FXI and a second arm that binds to a second (target) antigen, wherein the anti-FXI arm comprises any of the HCVR / LCVR or CDR sequences set forth in Table 1 herein. The term "anti-FXI antibody" also includes antibody-drug conjugates (ADCs) comprising an anti-FXI antibody or an antigen-binding portion thereof conjugated to a drug or toxin (i.e., a cytotoxic agent). The term "anti-FXI antibody" also includes antibody-radionuclide conjugates (ARCs) comprising an anti-FXI antibody or an antigen-binding portion thereof conjugated to a radionuclide.

[0117] As used herein, the term "anti-FXI antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with FXI or a portion of FXI, or the apple 2 domain of FXI or an epitope within the apple 2 domain of FXI. The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or VVR). H The heavy chain constant region is made up of three domains: H 1. C H 2, and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-FXI antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the aligned analysis of two or more CDRs.

[0118] As used herein, the term "antibody" also includes antigen-binding fragments of full-length antibody molecules. "Antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms, as used herein, include any enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from full-length antibody molecules using any suitable standard techniques, such as, for example, proteolytic digestion techniques or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable domains and, optionally, antibody constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated using chemical or molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, introduce codons, create cysteine ​​residues, modify, add or delete amino acids.

[0119] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0120] An antigen-binding fragment of an antibody will typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L V bound to the domain H In an antibody-binding fragment having a domain, H Domain and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric V H Domain or V L It may include a domain.

[0121] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a VH -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 arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains may be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or to one or more monomeric V H Domain or V LThe domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above in non-covalent association (e.g., by disulfide bond(s)).

[0122] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0123] In certain instances, it may be desirable to antagonize FXI, for example, to inhibit the formation of blood clots. However, the antibodies of the present disclosure function as antagonist antibodies that function as inhibitors of FXI or FXIa activity and simultaneously function as inhibitors of intrinsic pathway thrombosis / blood clot formation. The antibodies of the present disclosure may function by preventing the interaction between FXI and its upstream activator coagulation factor XII (FXII) and / or coagulation factor II (FII or thrombin). The antibodies of the present disclosure may also function by preventing the interaction between FXI and its downstream target coagulation factor IX (FIX). The antibodies of the present disclosure may also function by sequestering FXI from the patient's bloodstream.

[0124] The term "human antibody," as used herein, is intended to include non-naturally occurring human antibodies. The term includes antibodies that are recombinantly produced in a non-human mammal or in the cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject.

[0125] The antibodies of the present disclosure may in some embodiments be recombinant and / or non-naturally occurring human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. In certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) to thereby improve the V of the recombinant antibody. H Area and V L The amino acid sequence of the region is H Sequence and V L These are sequences that, while related to one another, may not naturally occur in the human antibody germline repertoire in vivo.

[0126] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming molecules of approximately 75-80 kDa consisting of covalently linked light and heavy chains (half antibodies). These forms have proven extremely difficult to separate, even after affinity purification.

[0127] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure provides a method for determining the frequency of occurrence of the second form in the hinge, C H 2nd Area or C H Antibodies with one or more mutations in three regions are included, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0128] Terms such as "specifically binds" or "binds specifically to" mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6 M or less (e.g., a smaller K D indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to FXI have been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, as used herein, multispecific antibodies that bind to FXI protein and one or more additional antigens, or bispecific antibodies that bind to two different regions of FXI, are nevertheless considered to be "specifically binding" antibodies.

[0129] The antibody of the present disclosure may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been separated and / or recovered from the identified antibody and at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally occurs or is naturally produced, is an "isolated antibody" for the purposes of this disclosure. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0130] The anti-FXI antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein with sequences available, for example, from public antibody sequence databases. Once obtained, antibodies and antigen-binding fragments containing one or more mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present disclosure.

[0131] The present disclosure also includes anti-FXI antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-FXI antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or less, 8 or less, 6 or less, or 4 or less conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences set forth in Table 1 herein.

[0132] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes can include portions of sugars, phosphoryl groups, or sulfonyl groups on an antigen.

[0133] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is at least about 95%, more preferably about 96%, 97%, about 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0134] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using predefined gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions are not expected to substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0135] Sequence similarity for polypeptides, also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of best overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0136] Antibody Features The present disclosure provides a method for the preparation of a medicament having a K of less than about 500 pM as measured by surface plasmon resonance at 25° C. or 37° C. DAccording to certain embodiments, the present disclosure includes anti-FXI antibodies that bind to the A2 domain of human FXI at a K of less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 80 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM, or less than about 1 pM for human FXI. D The present invention includes an anti-FXI antibody that binds at

[0137] The present disclosure provides a method for the preparation of a medicament having a K of less than about 1,000 pM as measured by surface plasmon resonance at 25° C. or 37° C. D According to certain embodiments, the present disclosure includes anti-FXI antibodies that bind to activated human FXI (FXIa) at a K of less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 250 pM, less than about 100 pM, less than about 80 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 3 pM, or less than about 1 pM for human FXI. D The present invention includes an anti-FXI antibody that binds at

[0138] The present disclosure includes anti-FXI antibodies that bind to human FXI with a dissociation half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25° C. or 37° C. According to certain embodiments, the present disclosure includes anti-FXI antibodies that bind to human FXI with a t1 / 2 of greater than about 20 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 minutes, greater than about 150 minutes, greater than about 300 minutes, greater than about 350 minutes, greater than about 400 minutes, greater than about 450 minutes, greater than about 500 minutes, greater than about 550 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, greater than about 1100 minutes, or greater than about 1200 minutes.

[0139] The present disclosure includes anti-FXI antibodies that bind to human FXIa with a dissociation half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25° C. or 37° C. According to certain embodiments, the present disclosure includes anti-FXI antibodies that bind to human FXI with a t1 / 2 of greater than about 20 minutes, greater than about 50 minutes, greater than about 100 minutes, greater than about 120 minutes, greater than about 150 minutes, greater than about 300 minutes, greater than about 350 minutes, greater than about 400 minutes, greater than about 450 minutes, greater than about 500 minutes, greater than about 550 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, greater than about 1100 minutes, or greater than about 1200 minutes.

[0140] The present disclosure includes anti-FXI antibodies that may or may not bind to non-human FXI. As used herein, when the antibody is tested in an antigen binding assay, such as surface plasmon resonance, it has a K of greater than about 1000 nM in such an assay. D or does not show any antigen binding, the antibody "does not bind" to a particular antigen (e.g., monkey, mouse, or rat FXI). Another assay format that can be used to determine whether an antibody binds or does not bind to a particular antigen according to this aspect of the disclosure is an ELISA.

[0141] It is generally known in the art that activated FXI (FXIa) activates factor IX by selectively cleaving the arg-ala and arg-val peptide bonds. Factor IXa then forms a complex with factor VIIIa (FIXa-FVIIIa) to activate factor X. The present disclosure provides a method for the preparation of factor X with an IC of less than about 100 pM. 50 The present invention relates to an anti-FXI antibody that inhibits FXI-mediated activation of human FX in plasma by at least about 85% at a concentration of 0.01% or more. 50Values ​​can be calculated as the concentration of antibody required to activate FXI-mediated signaling to half-maximal observed signal. Thus, according to certain embodiments, IC50 values ​​are less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, or less than about 5 pM when measured using the assay format described in Example 4 herein, or a substantially similar assay format. 50 and an anti-FXI antibody that mediates at least about 85% of human FXI-mediated activation of human FX in plasma.

[0142] The present disclosure also provides an IC of less than about 50 pM. 50 The present invention also includes anti-FXI antibodies that inhibit FXIa-mediated activation of human FX in plasma by at least about 25% at IC 50 Values ​​can be calculated as the concentration of antibody required to activate FXIa-mediated signaling to half-maximal signal observed. Thus, according to certain embodiments, IC50 values ​​are less than about 200 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM, less than about 3 pM, less than about 2 pM, or less than about 1 pM when measured using the assay format described in Example 4 herein, or a substantially similar assay format. 50 and comprises an anti-FXI antibody that mediates human FXIa-mediated activation of human FX in plasma by at least about 25%.

[0143] The present disclosure includes anti-FXI antibodies that preferentially bind to the apple 2 domain (A2) as demonstrated by direct binding to an A2 domain construct or by competition with one or more specific A2 binding antibodies, as shown in Examples 5 and 6, respectively. In one embodiment, the antibodies or antigen-binding fragments thereof disclosed herein do not bind to the catalytic domain of FXI.

[0144] The present disclosure includes anti-FXI antibodies that prolong activated partial thromboplastin time (aPTT), a measure of intrinsic pathway thrombosis, in human plasma while having no measurable effect on prothrombin time (PT), a measure of extrinsic pathway thrombosis. In one embodiment, aPTT is measured in pooled human plasma treated with ellagic acid, and PT is measured in pooled human plasma treated with tissue factor using a hemostasis analyzer, as exemplified in Example 7. It is generally known in the art that ellagic acid stimulates the intrinsic pathway of thrombosis in vitro, and tissue factor stimulates the extrinsic pathway of thrombosis. Here, the anti-FXI antibody is less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, 1 nM to 100 nM, 1 nM to 100 nM, 1 nM to 50 nM, 100 pM to 50 nM, 5 nM to 50 nM, 5 nM to 40 nM, 5 nM to 15 nM, 10 nM to 20 nM, 15 nM to 25 nM, 20 nM to 30 nM, 2 At a concentration of 5 nM to 35 nM, 30 nM to 40 nM, 35 nM to 45 nM, 40 nM to 50 nM, 45 nM to 55 nM, 50 nM to 60 nM, 55 nM to 65 nM, 60 nM to 100 nM, 65 nM to 75 nM, 70 nM to 80 nM, 75 nM to 85 nM, 80 nM to 90 nM, 85 nM to 95 nM, 90 nM to 100 nM, or 95 nM to 105 nM, the aPTT is extended by about 2-fold without doubling the PT.

[0145] The present disclosure includes anti-FXI antibodies that inhibit the generation of thrombin via the intrinsic pathway (endogenous thrombin) in human plasma in vitro, with little or no effect on the generation of thrombin via the extrinsic pathway (exogenous thrombin). In one embodiment, pathway-specific thrombin generation is determined in vitro by a thrombin generation assay using a calibrated automated thrombogram, as exemplified in Example 7, where thrombin generation profiles are generated and peak thrombin concentrations are determined in ellagic acid-treated plasma and tissue factor-treated plasma with and without anti-FXI antibodies. Thus, in one embodiment, the anti-FXI antibody is at a concentration of 0.1 nM to 100 nM, 1 nM to 100 nM, 5 nM to 500 nM, 5 nM to 100 nM, 10 nM to 100 nM, 10 nM to 50 nM, 5 nM to 15 nM, 10 nM to 20 nM, 25 nM to 35 nM, 30 nM to 40 nM, 35 nM to 45 nM, 40 nM to 50 The anti-FXI antibody inhibits endogenous thrombin production at any concentration up to 500 nM, 45 nM-55 nM, 50 nM-60 nM, 55 nM-65 nM, 60 nM-65 nM, 20 nM or more, 25 nM or more, 30 nM or more, 35 nM or more, 40 nM or more, 45 nM or more, 50 nM or more, 55 nM or more, 5 nM or more, 10 nM or more, or 15 nM or more, wherein the anti-FXI antibody does not affect exogenous thrombin production at any concentration up to 500 nM.

[0146] The present disclosure includes an anti-FXI antibody that increases activated partial thromboplastin time (aPTT) in a primate in vivo by at least 2-fold without measurably affecting prothrombin time (PT), wherein an anti-FXI antibody is administered to a primate, plasma is obtained from the primate, the plasma is contacted with ellagic acid or tissue factor, and the aPTT or PT, respectively, is determined in an assay as exemplified in Example 8. In one embodiment, the primate is a human. In one embodiment, the primate is a monkey.

[0147] In one embodiment, the anti-FXI antibody is parenterally administered at a dose of 0.01 mg / kg to 20 mg / kg, 0.1 mg / kg to 10 mg / kg, 1 mg / kg to 10 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, or about 15 mg / kg.

[0148] In one embodiment, the aPTT is prolonged by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, or at least 6-fold with anti-FXI treatment compared to without anti-FXI treatment.

[0149] In one embodiment, the anti-FXI mediated aPTT prolonging effect persists in a subject after receiving a dose of anti-FXI antibody for at least 1 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 3 months, at least 4 months, at least 5 months, or at least 6 months.

[0150] The present disclosure includes an anti-FXI antibody that inhibits intrinsic pathway peak thrombin activity in a primate in vivo without measurably affecting extrinsic pathway peak thrombin activity, where an anti-FXI antibody is administered to the primate, plasma is obtained from the primate, the plasma is contacted with ellagic acid or tissue factor, and then endogenous thrombin or exogenous thrombin generation, respectively, is determined in a thrombin generation assay as exemplified in Example 8. In one embodiment, the primate is a human. In one embodiment, the primate is a monkey.

[0151] In one embodiment, the anti-FXI antibody is parenterally administered at a dose of 0.01 mg / kg to 20 mg / kg, 0.1 mg / kg to 10 mg / kg, 1 mg / kg to 10 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, or about 15 mg / kg.

[0152] In one embodiment, the peak endogenous thrombin (i.e., thrombin generated via ellagic acid) activity with anti-FXI treatment compared to no anti-FXI treatment is between 1% and 100%, 5% and 95%, 10% and 90%, 20% and 80%, 1% and 10%, 5% and 20%, 10% and 30%, 15% and 40%, 20% and 50%, 25% and 60%, 5% and 15%, 10% and 25% and 30% and 40% and 5 ... % to 20%, 15% to 25%, 20% to 30%, 25% to 35%, 30% to 40%, 35% to 45%, 40% to 50%, 45% to 55%, 50% to 60%, 55% to 65%, 60% to 70%, 65% to 75%, 70% to 80%, 75% to 85%, 80% to 90%, 85% to 95%, 90% to 100%, 95% to 105%, or greater than 100% inhibited.

[0153] In one embodiment, anti-FXI mediated inhibition of peak endogenous thrombin activity persists in a subject after receiving a dose of anti-FXI antibody for at least 1 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 3 months, at least 4 months, at least 5 months, or at least 6 months.

[0154] When a binding characteristic of an antibody of the present disclosure (e.g., any of the binding characteristics mentioned herein above) is disclosed in terms of "measured by surface plasmon resonance", it means that the relevant binding characteristics of the interaction between the antibody and the antigen are measured using a surface plasmon resonance instrument (e.g., a Biacore® instrument, GE Healthcare) using standard Biacore assay conditions as illustrated in Example 3 herein, or a substantially similar assay format. In certain embodiments, the binding parameters are measured at 25° C., while in other embodiments, the binding parameters are measured at 37° C.

[0155] The present disclosure includes antibodies or antigen-binding fragments thereof that specifically bind to FXI, comprising an HCVR and / or LCVR, the HCVR and / or LCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences and / or LCVR amino acid sequences listed in Table 1.

[0156] The antibodies of the present disclosure may have one or more of the biological characteristics described above, or any combination thereof. The above list of biological characteristics of the antibodies of the present disclosure is not intended to be comprehensive. Other biological characteristics of the antibodies of the present disclosure will be apparent to those of skill in the art from a review of the present disclosure, including the Examples herein.

[0157] Epitope mapping and related techniques The epitope to which an antibody of the present disclosure binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the FXI protein. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of FXI. In some embodiments, the epitope is located at or near the surface of FXI, e.g., within a domain that interacts with any one of its ligands, e.g., FXIIa, thrombin, and FIX. In other embodiments, the epitope is located at or near a surface of FXI that does not interact with a FXI ligand, e.g., at a location on the surface of FXI that does not interfere with the interaction between FXI and its ligand when an antibody binds to such an epitope.

[0158] 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 polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, as described, for example, in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide that an antibody interacts with is hydrogen / deuterium exchange, detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal.Chem.73 / 256A-265A.

[0159] The present disclosure includes anti-FXI antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein). Similarly, the present disclosure also includes anti-FXI antibodies that compete with any of the specific exemplary antibodies described herein for binding to FXI (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein).

[0160] Whether an antibody binds to the same epitope as a reference anti-FXI antibody or competes with a reference anti-FXI antibody for binding can be easily determined by using routine methods known in the art and exemplified herein. For example, to determine whether a test antibody binds to the same epitope as a reference anti-FXI antibody of the present disclosure, the reference antibody is bound to FXI protein. The ability of the test antibody to bind to FXI molecules is then evaluated. If the test antibody can bind to FXI after saturation binding with the reference anti-FXI antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-FXI antibody. On the other hand, if the test antibody cannot bind to FXI molecules after saturation binding with the reference anti-FXI antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-FXI antibody of the present disclosure. Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed loss of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed loss of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antibodies bind to the same (or overlapping) epitope if a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a portion of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0161] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-FXI antibody, the binding methodology described above is performed in two directions. In the first direction, the reference antibody is allowed to bind to FXI protein under saturating conditions, and then the binding of the test antibody to the FXI molecule is evaluated. In the second direction, the test antibody is allowed to bind to FXI protein under saturating conditions, and then the binding of the reference antibody to the FXI molecule is evaluated. If in both directions, only the first (saturating) antibody can bind to the FXI molecule, it is concluded that the test antibody and the reference antibody compete for binding to FXI (see, for example, the assay format described in the Examples herein, where FXI protein is captured on a sensor tip and the FXI-coated sensor tip is treated with a reference antibody [mAb-1] and a test anti-FXI antibody [mAb-2] sequentially and in both binding orders). As will be appreciated by those of skill in the art, an antibody that competes with a reference antibody for binding does not necessarily have to bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0162] Preparation of human antibodies The anti-FXI antibody of the present disclosure may be a fully human, but non-naturally occurring antibody. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known method may be used in the context of the present disclosure to generate human antibodies that specifically bind to human FXI.

[0163] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody against an allergen is first isolated, having a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the generation of a transgenic mouse whose genome is operably linked to an endogenous mouse constant region locus, comprising a human heavy chain variable region and a human light chain variable region, such that the mouse produces an antibody comprising the human variable region and the mouse constant region in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy chain constant region and the human light chain constant region. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0164] In general, VELOCIMMUNE® mice are challenged with an antigen of interest and lymphocytes (such as B cells) are harvested from the mice that express antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy 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 the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0165] As described in the experimental section below, isolated high affinity chimeric antibodies having human variable regions and mouse constant regions are characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies of the present disclosure, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the specific application, the high affinity antigen binding and target specificity characteristics reside in the variable regions.

[0166] In certain embodiments, it may be desirable to test anti-human FXI antibodies in mice or rats that have been engineered to express the human FXI receptor. These mice or rats may be beneficial in situations where the anti-FXI antibodies may only bind to human FXI but do not cross-react with mouse or rat FXI. Any method known to those skilled in the art may be used to generate such FXI-humanized mice and rats.

[0167] In general, the antibodies of the present disclosure have very high affinities, as measured by binding to antigen immobilized in either a solid or solution phase, typically on the order of about 10 -12 ~about 10 -9 K of M D has.

[0168] biological equivalent The anti-FXI antibodies and antibody fragments of the present disclosure encompass proteins having amino acid sequences that differ from those of the described antibodies but that retain the ability to bind to human FXI. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, DNA sequences encoding the anti-FXI antibodies of the present disclosure contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode anti-FXI antibodies or antibody fragments that are essentially biologically equivalent to the anti-FXI antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid sequences and DNA sequences are discussed above.

[0169] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes whose absorption rates and extents do not differ significantly when administered at the same molar dose in either single or multiple doses under similar experimental conditions. Some antibodies are to be considered equivalents or pharmaceutical substitutes if their extents of absorption are equivalent but their absorption rates are not, and such differences in absorption rates can be considered bioequivalent because they are intentional and are reflected in the labeling, e.g., are not essential to achieving effective body drug concentrations for long-term use, and are not medically significant for the particular formulation studied.

[0170] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and efficacy.

[0171] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched one or more times compared to therapy sustained without switching between the reference product and the biological product without an expected increase in risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy.

[0172] In one embodiment, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mechanism of action for the condition(s) of use, to the extent that such mechanism is known.

[0173] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals where the concentration of the antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict human bioavailability data, (c) in vivo studies in humans or other mammals where the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0174] Biologically equivalent variants of the anti-FXI antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antibodies can include variants of anti-FXI antibodies that contain amino acid changes that modify the glycosylation characteristic of the antibody, for example, mutations that eliminate or remove glycosylation.

[0175] Species selectivity and species cross-reactivity The present disclosure provides, according to certain embodiments, anti-FXI antibodies that bind to human FXI but not to FXI from other species. The present disclosure also includes anti-FXI antibodies that bind to human FXI and FXI from one or more non-human species. For example, an anti-FXI antibody of the present disclosure may bind to human FXI but may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee FXI, depending on the case. According to certain exemplary embodiments of the present disclosure, an anti-FXI antibody is provided that specifically binds to human FXI but does not bind, or only weakly binds, to mouse or rat FXI.

[0176] multispecific antibody The antibodies of the present disclosure can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains specific for two or more target polypeptides. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-FXI antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.

[0177] The present disclosure includes bispecific antibodies in which one immunoglobulin arm binds to human FXI and the other immunoglobulin arm is specific for a second antigen. The FXI binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences set forth in Table 1 herein.

[0178] An exemplary bispecific antibody format that can be used in the context of the present disclosure is H 3 domain and second IgC H The first and second IgC H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds protein A and is a second IgC H The third domain contains a mutation that reduces or abolishes Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). H 3 may further comprise a Y96F modification (according to IMGT and Y436F in EU). H Further modifications that may be found within 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I according to IMGT, EU), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I according to IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to IMGT, EU). Variations on the bispecific antibody formats described above are contemplated as being within the scope of this disclosure.

[0179] Other exemplary bispecific formats that may be used in the context of the present disclosure include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobodies, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 These include, but are not limited to, bispecific formats (for a review of the above formats, see, e.g., Klein et al. 2012, mAbs 4:6,1-11, and references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid linkages, e.g., using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

[0180] Therapeutic Formulations and Administration The present disclosure provides pharmaceutical compositions comprising the anti-FXI antibody or antigen-binding fragment thereof of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide improved mobility, delivery, tolerability, and the like. Many suitable formulations can be found in a formulary known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0181] The dose of the antibody administered to the patient may vary depending on the age and size of the patient, the target disease, the condition, the route of administration, and the like. The preferred dose is typically calculated by body weight or body surface area. In adult patients, it may be advantageous to administer the antibody of the present disclosure intravenously, usually at a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. Effective dosages and schedules for administering anti-FXI antibodies may be empirically determined, e.g., the progress of the patient may be monitored by periodic evaluation, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages may be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0182] Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions of the present disclosure (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intravitreal, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (such as, for example, oral mucosa, rectal and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0183] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device has easy application in delivering the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0184] Numerous reusable pen and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure, including, for example, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN™, to name a few. Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR pen (sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA pen (Abbott Labs, Abbott Park IL), to name a few.

[0185] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used, see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, controlled release systems can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0186] The injectable preparations may include dosage forms for intravenous injection, intravitreal injection, intraocular injection, subcutaneous injection, intradermal injection, and intramuscular injection, infusion, and the like. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium that is conventionally used for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As oily media, for example, sesame oil, soybean oil, and the like are used, which may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, and the like. The injection solution thus prepared is preferably filled into a suitable ampoule.

[0187] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into a suitable unit dose dosage form to suit the dosage of the active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per unit dose dosage form, and particularly in the form of injection, it is preferable to contain about 5 to about 100 mg of the antibody, and for other dosage forms, it is preferable to contain about 10 to about 250 mg.

[0188] Diagnostic Uses of Antibodies The disclosure includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-FXI antibody (e.g., an anti-FXI antibody comprising any of the HCVR / LCVR sequences or CDR sequences set forth in Table 1 herein). The therapeutic composition may comprise any one or more of the anti-FXI antibodies or antigen-binding fragments thereof disclosed herein and a pharma- ceutically acceptable carrier or diluent.

[0189] The antibodies of the present disclosure are useful, inter alia, for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by FXI expression or activity. The FXI antagonist antibodies of the present disclosure may be used to treat or prevent thrombosis, particularly thrombosis of the intrinsic pathway, while minimizing adverse effects on hemostasis and clot formation via the extrinsic pathway.

[0190] The present disclosure includes methods of treating or preventing thrombosis by administering an anti-FXI antibody, or an antigen-binding fragment thereof, to a patient in need of such treatment, as disclosed elsewhere herein.

[0191] In one embodiment, the anti-FXI antibodies of the present disclosure are useful in treating conditions such as Factor V Leiden, prothrombin gene mutations, deficiencies in natural proteins that block clotting (e.g., antithrombin, protein C, and protein S), elevated homocysteine ​​levels, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), elevated levels of Factor VIII, Factor IX, and / or XI, plasminogen deficiencies, plasminogen dysregulation, and elevated levels of plasminogen activator inhibitor (PAI-1), atrial fibrillation, cancer, side effects of some drugs used to treat cancer, such as tamoxifen, bevacizumab, thalidomide, and lenalidomide, recent trauma or surgery. , placement of a central venous catheter, obesity, pregnancy, supplemental use of estrogen including oral contraceptives (birth control pills), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other diseases leading to reduced activity, heparin-induced thrombocytopenia (reduction of platelets in the blood due to heparin or low molecular weight heparin preparations), prolonged airplane travel, antiphospholipid syndrome, deep vein thrombosis or pulmonary embolism, myeloproliferative disorders such as polycythemia vera or essential thrombocytosis, paroxysmal nocturnal hemoglobinuria, inflammatory bowel syndrome, HIV / AIDS, nephrotic syndrome, COVID-19 infection or spike protein immunization.

[0192] In the context of the methods of treatment described herein, the anti-FXI antibodies can be administered as monotherapy (ie, as the sole therapeutic agent) or in combination with one or more additional therapeutic agents.

[0193] Combination Therapies and Formulations The present disclosure includes compositions and therapeutic formulations comprising any of the anti-FXI antibodies described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0194] The anti-FXI antibodies of the present disclosure may be used to treat a variety of conditions, including Factor V Leiden, prothrombin gene mutations, deficiencies in natural proteins that block clotting (e.g., antithrombin, protein C, and protein S), elevated homocysteine ​​levels, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), elevated levels of Factor VIII, Factor IX, and / or XI, plasminogen deficiencies, plasminogen dysregulation, and elevated levels of plasminogen activator inhibitor (PAI-1), atrial fibrillation, cancer, side effects of some of the drugs used to treat cancer, such as tamoxifen, bevacizumab, thalidomide, and lenalidomide, recent trauma or surgery, It may be co-formulated with one or more drugs used to treat conditions such as placement of a central venous catheter, obesity, pregnancy, supplemental use of estrogen including oral contraceptives (birth control pills), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other diseases leading to reduced activity, heparin-induced thrombocytopenia (a decrease in platelets in the blood due to heparin or low molecular weight heparin preparations), long distance air travel, antiphospholipid syndrome, deep vein thrombosis or pulmonary embolism, myeloproliferative disorders such as polycythemia vera or essential thrombocytosis, paroxysmal nocturnal hemoglobinuria, inflammatory bowel syndrome, HIV / AIDS, nephrotic syndrome, COVID-19 infection or spike protein immune effects.

[0195] The anti-FXI antibodies of the present disclosure may also be administered in combination and / or co-formulated with antivirals, antibiotics, analgesics, antioxidants, COX inhibitors, and / or NSAIDs. Anti-FXI antibodies may also be used in combination with other types of therapy, including stem cell therapy, glaucoma filtration surgery, laser surgery, or gene therapy.

[0196] The additional therapeutically active ingredient(s), e.g., any of the agents listed above or derivatives thereof, may be administered immediately prior to, simultaneously with, or immediately following administration of the anti-FXI antibody of the present disclosure (for purposes of this disclosure, such administration regimes will be considered administration of the anti-FXI antibody "in combination with" the additional therapeutically active ingredient). The present disclosure includes pharmaceutical compositions in which the anti-FXI antibody of the present disclosure is co-formulated with one or more of the additional therapeutically active ingredient(s) described elsewhere herein.

[0197] Dosing regimen According to certain embodiments of the present disclosure, multiple doses of an anti-FXI antibody (or a pharmaceutical composition comprising a combination of an anti-FXI antibody and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a period of time. The method according to this aspect of the present disclosure includes sequentially administering multiple doses of an anti-FXI antibody of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of an anti-FXI antibody is administered to a subject at different times, e.g., different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods that include sequentially administering to a patient a single initial dose of an anti-FXI antibody, followed by one or more secondary doses of an anti-FXI antibody, and then optionally one or more tertiary doses of an anti-FXI antibody.

[0198] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the anti-FXI antibody of the present disclosure. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose may all contain the same amount of anti-FXI antibody, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of anti-FXI antibody contained in the initial dose, secondary dose, and / or tertiary dose differs from each other during the course of treatment (e.g., increased or decreased as needed). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0199] In certain exemplary embodiments of the present disclosure, each secondary dose and / or tertiary dose is administered within 1 to 26 (e.g., 1, 11 / 2, 2, 21 / 2, 3, 31 / 2, 4, 41 / 2, 5, 51 / 2, 6, 61 / 2, 7, 71 / 2, 8, 81 / 2, 9, 91 / 2, 10, 101 / 2, 11, 111 / 2, 12, 121 / 2, The phrase "immediately preceding dose" as used herein means a dose of anti-FXI antibody administered to a patient prior to administration of the next dose in a sequence with no intervening doses.

[0200] The method according to this aspect of the disclosure may include administering any number of secondary and / or tertiary doses of an anti-FXI antibody to a patient. For example, in certain embodiments, only a single secondary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to a patient. Similarly, in certain embodiments, only a single tertiary dose is administered to a patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to a patient. The administration regimen may be carried out for the life of a particular subject or indefinitely until such treatment is no longer therapeutically necessary or advantageous.

[0201] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the present disclosure, the frequency at which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical testing.

[0202] The present disclosure includes dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once per week, once per two weeks, once per three weeks, once per month, once per two months, etc.), followed by two or more maintenance doses administered to the patient less frequently. For example, according to this aspect of the disclosure, if a loading dose is administered at a monthly frequency, maintenance doses may be administered to the patient once per six weeks, once per two months, once per three months, etc.

[0203] Diagnostic Uses of Antibodies The anti-FXI antibodies of the present disclosure may be used to detect and / or measure FXI or FXI-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-FXI antibody or a fragment thereof may be used to diagnose a condition or disease characterized by aberrant expression of FXI (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for FXI may include, for example, contacting a sample obtained from a patient with an anti-FXI antibody of the present disclosure, where the anti-FXI antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-FXI antibody may be used for diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S, or 125 The FXI may be a radioisotope such as I, or a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure FXI in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0204] Samples that can be used in the FXI diagnostic assay according to the present disclosure include any tissue or body fluid sample that can be obtained from a patient that contains a detectable amount of FXI protein or a fragment thereof under normal or pathological conditions. Generally, the level of FXI in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal FXI levels or activity) is measured to first establish a baseline or standard level of FXI. This baseline level of FXI can then be compared to the level of FXI measured in a sample obtained from an individual suspected of having a disease or condition associated with FXI. EXAMPLES

[0205] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. 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 at or near atmospheric pressure.

[0206] Example 1 Generation of human antibodies against the A2 domain of FXI Human antibodies against the A2 domain of FXI were generated in mice containing DNA encoding human immunoglobulin heavy chain and kappa light chain variable regions. In one embodiment, human antibodies were generated in VELOCIMMUNE® mice. In one embodiment, VelocImmune® (VI) mice were immunized with human FXI. Antibody immune responses were monitored by FXI-specific immunoassays. For example, serum was assayed for specific antibody titers against purified full-length FXI. Antibody-producing clones were isolated using both B-cell sorting technology (BST) and hybridoma techniques. For example, when a desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing FXI-specific antibodies.

[0207] Anti-FXI antibodies have also been isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Patent No. 7,582,298, specifically incorporated herein by reference in its entirety. Using this method, several fully human anti-FXI antibodies (i.e., antibodies with human variable and human constant domains) have been obtained.

[0208] The biological properties of exemplary antibodies produced by the methods of this example, controls, and comparators are described in detail in the Examples set forth below.

[0209] Example 2 Heavy chain variable region sequence and light chain variable region sequence Columns 2 and 4 of Table 1 list the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of exemplary anti-FXI antibodies of the disclosure. The corresponding nucleic acid sequence identifiers for exemplary anti-FXI antibodies of the disclosure are listed in columns 3 and 5 of Table 1. [Table 1]

[0210] SEQ ID NO:17 comprises the following one letter amino acid sequence: AAS.

[0211] SEQ ID NO:18 comprises the following nucleotide sequence: GCTGCATCC.

[0212] An exemplary full length anti-FXI antibody comprises a fully human Fc gamma 4 heavy chain (i.e., IgG4 Fc) and a fully human light chain sequence. However, as will be appreciated by those skilled in the art, an antibody having a particular Fc isotype may be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc may be converted to an antibody having a human IgG4, etc.), but in any event, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0213] Control constructs used in the following examples For comparison purposes, a control construct designated COMP3448 (an anti-FXI A2 domain antibody) was included in the experiments disclosed herein: a "monoclonal antibody specific for fXI" having the VH / VL sequence of antibody "14E11" described in U.S. Patent No. 8,388,959, the entire contents of which are expressly incorporated herein by reference in their entirety.

[0214] Example 3 Biacore binding kinetics of anti-FXI monoclonal antibodies binding to different FXI reagents measured at 25°C and 37°C The equilibrium dissociation constants (K D ) was determined using a real-time surface plasmon resonance-based Biacore 8K biosensor. All binding studies were performed at 25°C and 37°C in a running buffer of 10 mM HEPES, 300 mM NaCl, and 0.05% v / v detergent Tween®-20, pH 7.4 (HBS-P). The surface of a Biacore CM5 sensor chip was first derivatized by amine coupling with a mouse anti-human Fc-specific monoclonal antibody (Regeneron, Tarrytown) to capture anti-FXI monoclonal antibodies. Binding studies were performed with human FXI (zymogen; factor XI protein, human plasma, Enzyme Research Laboratories, South Bend, IN, catHFXI 1111) and FXIa (activated; factor XIa protein, human plasma, Enzyme Research Laboratories, South Bend, IN, cat.HFXIa 1111a). Different concentrations of hFXI and hFXIa (25 nM to 0.39 nM, 4-fold serial dilutions) were first prepared in HBS-P running buffer and injected over the surface of anti-human Fc-captured anti-FXI monoclonal antibody at a flow rate of 30 μL / min for 3 min, during which dissociation of monoclonal antibody-bound FXI reagent was monitored for 10 min in HBS-P running buffer. The association rate (k a ) and dissociation rate (k d The binding dissociation equilibrium constant (K) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

number

[0215] The binding kinetic parameters for hFXI or hFXIa binding to the anti-FXI monoclonal antibodies of the presently disclosed subject matter and isotype controls at 25° C. and 37° C. are shown in Tables 2 and 3.

[0216] As shown in Table 2, anti-FXI monoclonal antibodies bound to hFXI with KD values ​​ranging from 35.1 pM to 7.53 pM at 25° C. As shown in Table 2, anti-hFXI monoclonal antibodies bound to hFXI with KD values ​​ranging from 13.7 pM to 48.3 pM at 37° C.

[0217] As shown in Table 3, the anti-FXI monoclonal antibodies bound to hFXIa with KD values ​​ranging from 257 pM to 269 pM at 25° C. As shown in Table 3, the anti-FXI monoclonal antibodies bound to hFXIa with KD values ​​ranging from 763 pM to 893 pM at 37° C. [Table 2-1] [Table 2-2] [Table 3]

[0218] Example 4 Activated partial thromboplastin time bioassay Testing Procedure: The BIOPHEN Factor XIa kit (HYPHEN BioMed, Neuville-sur-Oise, FR, cat. #220412) was used to assess the ability of anti-FXI antibodies of the present disclosure to inhibit the activity of zymogen factor FXI (FXI) or pre-activated FXIa, which leads to the generation of active factor Xa (FXa). Inhibition by antibodies of the present disclosure was determined by measuring the reduction in the amount of chromogenic substrate converted by FXa (BIOPHEN kit component R3). All reagents in the BIOPHEN kit were used in the assay, except for Reagent 1B (human factor IX) and the FXIa calibrator (Cal).

[0219] To test dose-dependent activity of FXI or FXIa, normal human plasma was first diluted to 0.65% plasma in the provided Tris-BSA buffer (used as dilution buffer for the assay) and then serially diluted to 0.021% plasma with a no plasma control. Normal human plasma was also diluted to either 0.13% or 0.15% plasma. Antibodies (anti-FXI, control, and comparator) were serially diluted from a starting concentration of either 500 nM or 300 nM to a concentration of 5.1 pM with buffer only samples. For inhibition of zymogen FXI, anti-FXI antibodies were pre-incubated with diluted plasma for 30 min at 25° C., followed by an additional 30 min incubation with 0.32 μM aPTT-XL ellagic acid at 25° C. For inhibition of active FXIa, diluted plasma was preactivated with 0.32 μM aPTT-XL ellagic acid for 30 min at 25° C. and subsequently incubated with anti-FXI antibody for 30 min at 25° C.

[0220] After the plasma was incubated with ellagic acid and antibodies, Reagent 1A (Human FX, FVIII:C, fibrin polymerization inhibitor) was added and incubated for 5 min at 37°C. Reagent 2 (thrombin, phospholipids, and calcium) was then added and incubated for 5 min at 37°C. Finally, Reagent 3 (SXa-11 FXa substrate) was added and incubated for 30 min at 37°C. Absorbance was measured on a FLEXSTATION 3 Plate Reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 405 nm. Results were analyzed using nonlinear regression (4-parameter logistic) with PRISM® 6 software (GraphPad, La Jolla, CA) to determine EC 50 and IC 50 The percent inhibition was calculated based on the following equation 2:

number

[0221] In this equation, the absorbance 希釈血漿 " refers to the absorbance measurement at 405 nm of diluted plasma (either 0.13% or 0.15% plasma) activated with 0.32 μM aPTT-XL ellagic acid to cleave FXI to FXIa without the addition of any antibody. 阻害 " refers to the minimum absorbance measurement at 405 nm from the dose response of a particular antibody with diluted plasma activated with 0.32 μM ellagic acid. 無血漿対照 " refers to the absorbance measurement at 405 nm of Tris-BSA buffer alone in the absence of any plasma. [Table 4]

[0222] As shown in Table 4, anti-FXI / FXIa antibodies have IC values ​​ranging from 39 to 220 pM (39 pM in one example). 50The anti-FXI / FXIa antibodies of the present disclosure also exhibited inhibition of FXI in diluted normal plasma with IC values ​​ranging from 680 pM to greater than 10 nM (in one example, 10 nM), with maximal inhibition ranging from 85-87% (in one example, 85%). 50 The control mAbs inhibited FXIa in diluted plasma with IC values ​​ranging from 22% to 25%, and in one embodiment, 25%. 50 The comparative mAbs also showed inhibition of FXI with IC values ​​ranging from >10 nM to >5 nM, with maximum inhibition ranging from 86% to 106%. 50 The IC values ​​showed inhibition of FXIa, with maximum inhibition ranging from 35% to 38%. The isotype control mAb showed no inhibition of FXIa, but inhibition of FXI at high antibody concentrations, with IC 50 Values ​​ranged from >100 nM to 120 nM with maximum inhibition ranging from 58 to 102% inhibition.

[0223] Example 5 Anti-FXI binding specificity to FXI subunits The binding specificity of various anti-FXI monoclonal antibodies to different truncated apple domains of human FXI was determined using a real-time label-free biolayer interferometry assay on an OCTETHTX biosensor (Pall ForteBio Corp., Port Washington, NY). The experiments were performed in a buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant TWEEN®-20, 1 mg / mL bovine serum albumin (BSA), pH 7.4 (HBS-EBT) at 25° C. with the plate shaking at a speed of 1000 rpm. Binding of anti-FXI monoclonal antibodies to their respective epitopes on recombinant human FXI protein was tested using human FXI expressed with a C-terminal myc-myc-hexahistidine tag (hFXI-mmh; SEQ ID NO: 22), human FXI PKA1 domain expressed with a C-terminal myc-myc-hexahistidine tag (hFXI-PKA1-mmh; SEQ ID NO: 23), human FXI PKA2 domain expressed with a C-terminal myc-myc-hexahistidine tag (hFXI-PKA2-mmh; SEQ ID NO: 24), human FXI PKA3 domain expressed with a C-terminal myc-myc-hexahistidine tag (hFXI-PKA3-mmh; SEQ ID NO: 25), and human FXI PKA4 domain expressed with a C-terminal myc-myc-hexahistidine tag (hFXI-PKA4-mmh; SEQ ID NO: 26) (collectively FXI-mmh reagents). PK (plasma kallikrein) marked FXI-mmh reagents replace the identified apple domain with the corresponding PK apple domain, meaning that the binding specificity is determined by the non-binding of the antibody to its respective epitope on the recombinant human FXI protein described herein. For example, because hFXI-PKA2-mmh expresses plasma kallikrein apple domain 2, the mAb of interest is hypothesized to bind only to hFXI-PKA1-mmh, hFXI-PKA3-mmh, and hFXI-PKA4-mmh, but not to hFXI-PKA2-mmh.

[0224] FXI-mmh reagents were first individually captured on anti-Penta-His antibody-coated OCTET biosensor tips (Pall Fortebio Inc, cat.#18-5122) by immersing the biosensor tips in wells each containing a 100 nM solution of human FXI-mmh reagent for 2 minutes. The resulting antigen-captured biosensor tips were then individually saturated with each anti-FXI monoclonal antibody by immersing them in wells containing a 133 nM solution of anti-FXI monoclonal antibody for 180 seconds. The biosensor tips were then washed with HBS-ETB buffer between each step of the experiment. Real-time binding responses were monitored throughout the course of the experiment, and binding responses were recorded at the end of each step. The binding specificity responses of anti-FXI monoclonal antibodies were compared with different truncated FXI-mmh reagents, as shown in Table 5. [Table 5]

[0225] Example 6 Competition assay 6.1: Competitive binding of Apple 2 domains Binding competition between anti-FXI monoclonal antibodies was determined using a real-time label-free biolayer interferometry assay on an OCTETHTX biosensor (Pall ForteBio Corp., Port Washington, NY). All experiments were performed in 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant Tween®-20, 1 mg / mL BSA (Octet HBS-P buffer) at 25° C. with plates shaking at a speed of 1000 rpm. To assess whether any two antibodies could compete with each other for binding to their respective epitopes on human FXI (ERL) (Factor XI protein from human plasma, Ensyme Research Laboratories, South Bend, IN, cat. #HFXI 1111), approximately 1.5-2.8 nM of anti-human FXI monoclonal antibody was first captured on an anti-hFc antibody-coated (AHC) OCTET biosensor tip (Pall ForteBio Corp., #18-5060, Port Washington, NY) by immersing the tip in a well containing 50 μg / mL of anti-human FXI monoclonal antibody (hereafter referred to as first mAb) solution for 5 min. The antibody-captured biosensor tip was then saturated with a blocking H4H isotype (human IgG4) control monoclonal antibody (hereafter referred to as blocking mAb) by immersing the tip in a well containing 50 μg / mL of blocking mAb solution for 4 min. The biosensor tip was then immersed in a well containing a co-complexed solution of 25 nM hFXI (ERL, #HFXI 1111) and 1 μM of a second anti-human FXI monoclonal antibody (hereafter referred to as second mAb) that had been pre-incubated for 2 h. The biosensor tip was washed with octet HBS-P buffer between each step of the experiment.

[0226] Real-time binding responses were monitored during the course of the experiment and binding responses were recorded at the end of each step. The responses of a second mAb pre-complexed with human FXI binding to the first mAb were corrected for background binding and compared to determine the competitive / non-competitive behavior of the different anti-FXI monoclonal antibodies.

[0227] Table 6 explicitly defines the relationship of competing antibodies in both directions, regardless of the order of binding, where anti-FXI mAb REGN9933 competes for binding to plasma-derived hFXI with H4H29801P2, which is known to bind to the A2 (apple 2) domain of FXI. [Table 6]

[0228] 6.2:FXI conflict binding A second binding competition between an anti-FXI monoclonal antibody and a negative isotype control was performed using the following materials and methods.

[0229] material Equipment used: Octet HTX 384 RED Temperature: 25℃ Running buffer: HBS-P + 1mg / ml BSA, pH 7.4 Sensor type: ForteBIO Anti-hFc Capture flow rate / time: 1000 rpm, 300 sec (mAb-1), 300 sec association (Ag mAb-2 premix) 180 sec dissociation mAb-2

[0230] method Approximately 1.5-2.8 nm of α-human FXI mAb was captured by immersing an α-hFc coated Octet biosensor in a well containing 50 μg / mL of α-human FXI mAb for 5 min. H4H hFc (isotype control) was used as a negative control. Unoccupied α-hFc Octet sensors were saturated by immersing in a well containing blocking mAb solution (50 μg / mL of H4H human Fc (isotype control)1) for 4 min. 25 nM hFXI(ERL) was pre-incubated with 1 uM of α-FXI mAb in buffer containing 50 μg / mL of H4H hFc for at least 1 h. The blocking mAb saturated Octet biosensor was then immersed in a well containing a premix of αC5 mAb and hC5 for 5 min. At the end of each cycle, the α-hFc Octet sensor was regenerated in 10 mM HCl. During analysis, the self-self background signal (mAb binding to the capture surface) was subtracted from the entire column. The binding response of mAb-1 was recorded and competing / non-competing mAbs were binned based on their respective mAb-2 binding responses.

[0231] Results and Discussion [Table 7] [Table 8]

[0232] The subject mAbs REGN9933 and H4H29801P2 cross-competed in both directions.

[0233] Example 7 Functional plasma assays for clotting time and thrombin generation (TGA) Coagulation factors, together with platelets, are blood components involved in the process of hemostasis during vascular injury. It is well established that these components can be the driving force of thrombosis if an imbalance occurs in their regulation (i.e., production and / or activity). Thrombotic diseases were thought to arise mainly from abnormal activation of the extrinsic pathway (via tissue factor), but recently, clinical studies using antisense oligonucleotides for pre-F11 knee arthroplasty were found to prevent postoperative venous thrombosis (Buller HR, Bethune C, Bhanot S, Gailani D, Monia BP, Raskob GE, Segers A, Verhamme P, Weitz JI. Factor XI antisense oligonucleotide for prevention of venous thrombosis. N Engl J Med. 2015 Jan 15; 372(3): 232-40). Therefore, inhibition of FXI activity is important in reducing thrombotic coagulation (Weitz JI. Factor XI and factor XII as targets for new anticoagulants. Thromb Res. 2016 May;141 Suppl 2:S40-5).

[0234] This example demonstrates that anti-FXI antibodies bind to and inhibit the activity of human FXI in four functional plasma assays: 1) activated partial thromboplastin time (aPTT), 2) prothrombin time (PT), 3) thrombin generation assay (TGA) induced by ellagic acid, and 4) TGA induced by tissue factor. The aPTT test evaluates all clotting factors in the intrinsic and common pathways of the clotting cascade by measuring the time it takes for a clot to form after the addition of calcium and ellagic acid, whereas the PT test evaluates all clotting factors in the extrinsic and common pathways of the clotting cascade after the addition of calcium and tissue factor. The ellagic acid induced TGA measures the rate and amount of thrombin generated via the intrinsic and common pathways, whereas the tissue factor induced TGA measures the rate and amount of thrombin generated via the extrinsic and common pathways.

[0235] Testing Procedure: aPTT was determined on a Diagnostica STAGO START4 Hemostasis Analyzer (Diagnostica Stago, Parsippany, NJ) in the following manner: A total of 50 μl of pooled normal human plasma was added to the cuvette at 37° C. After 1 min, 5 μl of 2-fold serially diluted test substances (antibodies or small molecule inhibitors) in PBS were added to the cuvette and incubated for 5 min. Then, 50 μl of APPT-XL Ellagic Acid (Thermo Scientific, Waltham, MA) was added and incubated for 300 s, after which 50 μl of 20 mM calcium chloride (Thermo Scientific, Waltham, MA) was added to start the reaction. The measured clotting times of the test substance concentrations were normalized to baseline (drug-free) plasma clotting times and plotted against the log molar concentration of the test substance. The results were analyzed using nonlinear regression (4-parameter logistics) with PRISM5 software (GraphPad, La Jolla, CA) to obtain doubling time concentrations.

[0236] PT was determined on a Diagnostica Stago START4 Hemostasis Analyzer (Diagnostica Stago, Parsippany, NJ) in the following manner: A total of 50 μl of pooled normal human plasma was added to the cuvette at 37° C. After 1 min, 5 μl of 2-fold serially diluted test substances (antibodies or small molecule inhibitors) in PBS were added to the cuvette and incubated for 5 min. Then, 100 ul of tissue factor (TriniCLOT PT Excel, Diagnostica Stago, Parsippany, NJ, cat. #T1106) was added to initiate the reaction. The measured clotting times of the test substance concentrations were normalized to baseline (drug-free) plasma clotting times and plotted against the log molar concentration of the test substance. The results were analyzed using nonlinear regression (4-parameter logistics) with PRISM5 software (GraphPad, La Jolla, CA) to obtain doubling time concentrations.

[0237] The thrombin generation profile under the intrinsic coagulation pathway was determined on a Diagnostica Stago Calibrated Automated Thrombogram (Stago, Parsippany, NJ) in the following manner: 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 two-fold serially diluted test substances (antibodies or small molecule inhibitors) in PBS were added to the wells of the microplate and incubated for 30 minutes. 15 ul of APPT-XL Ellagic Acid (Thermo Scientific, Waltham, MA, cat. #95059-804) was diluted in Microparticle (MP) Reagent (Diagnostica Stago, cat. #86222) and then added to the wells and incubated for 45 minutes. Then, 15 μl of Fluo Flu Cal Substrate (Diagnostica Stago, cat. #86197) was added immediately before the continuous 90-minute reading of the microplate. The measured real-time thrombin concentration values ​​were plotted against time to obtain a thrombogram for each concentration of test substance used.

[0238] The thrombin generation profile under the extrinsic coagulation pathway was determined on a Diagnostica Stago Calibrated Automated Thrombogram (Diagnostica Stago, Parsippany, NJ) in the following manner: 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 two-fold serially diluted test substances (antibodies or small molecule inhibitors) in PBS were added to the wells of the microplate and incubated for 30 minutes. 15 μl of tissue factor PPP reagent (Diagnostica Stago, cat. #86194) was added to the wells and incubated for 45 minutes. Then, 15 μl of Fluo Flu Cal substrate (Diagnostica Stago, cat. #86197) was added immediately before the continuous 90-minute reading of the microplate. The measured real-time thrombin concentration values ​​were plotted against time to obtain a thrombogram for each concentration of test substance used.

[0239] Summary of results and conclusions: Dose-response curves were generated to determine the effect of each drug on plasma aPTT and PT. An IgG4 isotype control mAb (control mAb) had no effect on aPTT (Figure 1) or PT. REGN9933 prolonged the aPTT (Figure 1) without increasing the PT (not shown). The FXI-A2 binding comp mAb (comp mAb) COMP3448 also prolonged the aPTT without increasing the PT.

[0240] The efficiency of a drug to inhibit clotting activity is indexed by an arbitrary "doubling time" concentration or C2xt (the concentration of drug required to extend clotting time by 2-fold over baseline values). These extrapolated C2xt values ​​(i.e., curves that cross the "doubling time line") for drugs and controls are discussed below. REGN9933 reached C2xt at 33 nM or less for aPTT (intrinsic pathway). At the maximum tested of 4 μM, REGN9933 was not found to double PT clotting time. comp mAb COMP3448 required 62 nM to reach aPTT C2xt but did not reach PT CC2xt at 4 μM. These data show the superiority of REGN9933 in achieving C2xt at significantly lower antibody concentrations compared to COMP3448.

[0241] The effects of these mAbs were assessed for their ability to inhibit thrombin generation when plasma was challenged with ellagic acid or tissue factor (i.e., extended time to detection of thrombin = lag time, reduction in thrombin peak, and reduction in total amount of thrombin generated = endogenous thrombin generation potential). The control mAb had no effect on thrombin generation (Figure 2). The subject anti-FXI / FXIa mAb showed a slight reduction in thrombin generation at 16 nM, but significantly or completely inhibited thrombin production at concentrations of 31 nM and above (Figure 3), indicating that the subject anti-FXI mAb suppresses activation of the intrinsic pathway which in turn prevents thrombin generation. The comp mAb showed a similar response to the subject anti-FXI / FXIa mAb with respect to thrombin generation, with near complete inhibition at 31 nM and above (Figure 4).

[0242] Tissue factor-induced thrombin generation was not affected by the control isotype mAb (Figure 5). The subject anti-FXI / FXIa mAb REGN9933 had a slight to mild inhibitory effect on tissue factor-induced thrombin generation (Figure 6). Comp mAb COMP3448 had the least effect on tissue factor-induced thrombin generation (Figure 7).

[0243] The concentrations of test substances required to increase the lag time by 2-fold and halve the peak thrombin and total thrombin generation when coagulation was activated by ellagic acid or tissue factor are summarized in Table 9. [Table 9]

[0244] Example 8 Pharmacokinetic study of the subject anti-FXI antibody drug substance in cynomolgus monkeys The objective of this study was to determine intravenous single-dose pharmacodynamic / pharmacokinetic (PK / PD) parameters of an anti-FXI monoclonal antibody (mAb) in cynomolgus monkeys over an 8-week period.

[0245] Female cynomolgus monkeys (n=39) aged 2-4 years weighing 2-4 kg were acclimated to laboratory housing for at least 2 weeks prior to the start of mAb dosing. Animals were assigned to treatment groups based on established social groups, where a stratified randomization scheme was used to incorporate body weights from one animal from each social unit to assign animals to study groups (Table 8). Animals were housed under standard conditions (temperature 18°C-29°C, relative humidity 30%-70%, respectively) and maintained on a light (12 h) / dark (12 h) cycle. Food (PMI LABDIET FIBER-PLUS Monkey Diet 5049 biscuits, LabDiet, St. Louis, MO) was provided twice daily and water was provided ad libitum. [Table 10]

[0246] The control group (Group 1) was administered vehicle (10 mM histidine, pH 6.0) intravenously at a volume of 2 mL / kg. Groups 2-7 were administered the appropriate mAb (control and comparator) intravenously at 1, 3, or 10 mg / kg. The dose volume of 2 mL / kg for each animal was based on most recent body weight measurement. Blood samples were collected (venous blood draws) multiple times prior to and throughout the 8-week study period. Blood was processed to measure drug levels in serum and both target levels and clotting activity in plasma.

[0247] Drug levels in serum were measured using an enzyme-linked immunosorbent assay (ELISA) with mouse anti-human IgG Fc as the capture mAb and biotinylated anti-human Ig kappa light chain specific as the detection mAb. NeutrAvidin conjugated with horseradish peroxidase (NeutrAvidin-HRP) was used to convert a luminol-based substrate into a signal proportional to the concentration of total FXI mAb concentration.

[0248] Plasma target (FXI) levels were measured using the Affinity Biologicals Factor XI ELISA kit (FXI-AG) (Ancaster, ON). The manufacturer's protocol was followed to determine monkey FXI concentrations.

[0249] Plasma coagulation activity was determined in functional assays measuring clotting time and thrombin generation. Clotting time was determined using a Diagnostica Stago START 4 ​​Hemostasis Analyzer (Diagnostica Stago, Parsippany, NJ) to measure activated partial thromboplastin time (aPTT), which measures clotting activated by intrinsic pathway activator (ellagic acid), and prothrombin time (PT), which measures clotting activated by extrinsic pathway activator (tissue factor). Time to clotting was reported as fold change over baseline. Thrombin generation was measured with a Stago Diagnostica Calibrated Automated Thrombogram (CAT) (Diagnostica Stago, Parsippany, NJ). Thrombin generation assays (TGA) were performed using the intrinsic pathway activator ellagic acid (EA) or the extrinsic pathway activator tissue factor (TF). TGA data are reported as percentage change from baseline for the following parameters: lag time, peak thrombin concentration, and total thrombin concentration.

[0250] Summary of results and conclusions: Pharmacokinetic (PK) parameters were estimated using noncompartmental and population compartmental analyses. The mAb concentration-time profile after an intravenous bolus was characterized by an initial short distribution phase, followed by a linear beta elimination phase, and a terminal target-mediated elimination phase (Tables 11 and 12). 最大 A dose-proportional increase in was observed with intravenous administration (Table 13), and the dose-normalized C 最大 (C 最大 / dose) was found to be comparable across the mAb treatment groups. Dose (AUC inf Drug exposure (AUC inf) were found to increase at higher doses, indicating a greater than dose-proportional increase in exposure across groups, which would be consistent with nonlinear kinetics of the mAb. These observations are consistent with parallel linear and nonlinear target-mediated clearance (TMC), where the observed decrease in clearance is a function of increasing dose. Target (FXI) in plasma increased approximately 2-3 fold from baseline in all animals except those receiving comp mAb COMP3448 (A2 binder) (Tables 14 and 15). Higher doses of mAb did not show dose-dependent retention of target FXI concentrations.

[0251] The inhibitory activity of anti-FXI mAbs (subject and comparator) was assessed by functional assays measuring plasma clotting time or thrombin generation. At each time point, an aliquot of plasma was used to determine clotting time as assessed by activated partial thromboplastin time (aPTT) and prothrombin time (PT) assays, which measure clotting activity of intrinsic and extrinsic clotting activity, respectively. Data are presented as clotting time relative to baseline clotting time for aPTT (Tables 16 and 17) and PT (Tables 18 and 19). The comp mAb and subject anti-FXI mAb showed a similar 2-fold prolongation (i.e., inhibitory effect) on aPTT clotting time. Changes from baseline in PT clotting time were not affected by any of the mAbs tested or by increasing doses of any of the mAbs.

[0252] Coagulation activity was also assessed by measuring the profile of thrombin generation when activated by an intrinsic pathway activator (ellagic acid) or an extrinsic pathway activator (tissue factor). One measured parameter from the thrombin generation curve was the lag time, which evaluates the time required to generate thrombin after the activator is added to the plasma. Data represented fold change from baseline lag time values. The 1 mg / kg dose of mAb extended the lag time by about 2-fold for about 7 days in ellagic acid-activated plasma (Tables 20 and 21). The lag time was extended by about 3-4-fold above baseline times for up to about 3 weeks for the 3 and 10 mg / kg doses of mAb when activated with ellagic acid. The lag time was not extended when tissue factor was used as the activator of coagulation (Tables 22 and 23).

[0253] The second parameter measured in the thrombin generation assay was peak thrombin, which evaluates the concentration at which thrombin generation peaked. Data were expressed as a percentage of baseline thrombin peak. The mAb was able to significantly inhibit thrombin peak concentrations (when induced by ellagic acid) to approximately 5-15% of baseline at the three concentrations of mAb used, but the effect was found to be short-lived (approximately 5 days) at the 1 mg / kg dose (Tables 24 and 25). 3 mg / kg reduced for up to 2 weeks, and the 10 mg / kg mAb dose reduced for approximately 4 weeks. The effect of the mAbs on peak thrombin when tissue factor was used as an activator was variable, with peak thrombin concentrations increasing and decreasing by 50% (Tables 26 and 27).

[0254] The third parameter measured in the thrombin generation assay was endogenous thrombin generation potential, which evaluates the total concentration of thrombin generated during activation. Data was expressed as a percentage of baseline total thrombin generation. mAbs showed similar effects on inhibition of total thrombin generation as they did on peak thrombin inhibition when ellagic acid was used to activate coagulation (Tables 28 and 29). Similarly, results were variable when tissue factor was used as the activator (Tables 30 and 31).

Table 11

Table 12-1

Table 12-2

Table 13

Table 14-1

Table 14-2

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21-1

Table 21-2

Table 22

Table 23-1

Table 23-2

[0255] Example 9 First-order pharmacokinetics 9.1: In vitro pharmacology A series of studies were conducted to evaluate the efficacy and safety of REGN9933 using in vitro assays. The objectives of the in vitro studies included: (a) determining REGN9933 binding affinity and specificity for FXI from human and non-human species, and human FXIa, (b) characterizing REGN9933-mediated blockade of the intrinsic and extrinsic coagulation pathways in plasma from human and cynomolgus monkey donors, (c) evaluating the subject mAb REGN9933-FXI and subject mAb REGN9933-FXIa immune complexes for binding to C1q, and (d) evaluating the potential risk of IgG4P antibodies such as REGN9933 to induce peripheral blood mononuclear cell (PBMC) proliferation and cytokine release.

[0256] The binding interactions between REGN9933 and human, cynomolgus monkey, rabbit, and mouse FXI or human FXIa were evaluated using surface plasmon resonance (SPR)-based assays. In these assays, REGN9933 bound to human FXI and FXIa, and cynomolgus monkey FXI with subnanomolar affinity. REGN9933 also bound weakly to rabbit FXI, but not to mouse FXI (Example 9.1.1).

[0257] The ability of REGN9933 to block the coagulation pathway in pooled plasma from either human or cynomolgus monkey donors was evaluated in vitro using coagulation assays and thrombin generation assays (TGA). In these assays, REGN9933 exerted a concentration-dependent effect on the intrinsic coagulation pathway in pooled human plasma and had a minor effect on the extrinsic pathway. REGN9933 also exerted a concentration-dependent effect on the intrinsic coagulation pathway in pooled cynomolgus monkey plasma, but not on the extrinsic coagulation pathway (Example 9.1.2.1).

[0258] REGN9933 contains a hinge-stabilized fragment-crystallizable (Fc) constant domain from IgG4 (designated IgG4P), which is unlikely to form immune complexes capable of binding C1q because IgG4 does not bind C1q in the same way as IgG1 (Patel, 2015). Nevertheless, to assess the potential binding of REGN9933-FXI and REGN9933-FXIa complexes to C1q, an enzyme-linked immunosorbent assay (EIA) was performed. In this assay, REGN9933-FXI and REGN9933-FXIa complexes did not demonstrate positive binding to C1q (Example 9.1.2.2).

[0259] Results from cell-based in vitro experiments demonstrate that IgG4P antibodies that do not specifically target immune cell surface molecules, such as REGN9933, are unlikely to induce cytokine release or proliferation of PBMCs (Example 9.1.3).

[0260] In vitro functional characterization of the subject mAbs 9.1.1: Determination of Kinetic and Equilibrium Binding Parameters of the Subject mAb Interactions with FXI from Human, Cynomolgus Monkey, Rabbit, and Mouse, and FXIa from Human In this study, SPR technology was used to determine the binding affinity of REGN9933 to plasma-derived human FXI and FXIa proteins, as well as recombinant human, cynomolgus, rabbit, and mouse FXI proteins produced with a C-terminal myc-myc-hexahistidine (mmH)-tag. Human FXI (E19-V625) shares 96%, 86%, and 79% amino acid sequence identity with cynomolgus, rabbit, and mouse FXI, respectively. Various concentrations of soluble FXI or FXIa proteins were injected over surface-captured REGN9933 at 25°C and pH 7.4, followed by a dissociation phase. Kinetic binding parameters for human, cynomolgus, and mouse proteins were determined using a 1:1 binding model with mass transport limitations, and binding affinity for the rabbit protein was determined using a 1:1 steady-state binding model.

[0261] In studies using plasma-derived human proteins, REGN9933 bound to human FXI (hFXI) and FXIa (hFXIa) with equilibrium dissociation constants (KD) of 14.4 and 141 pM, respectively (data not shown).

[0262] In studies using purified proteins produced with mmH-tags, the subject anti-FXI mAb REGN9933 bound to recombinant human (hFXI.mmH) and cynomolgus monkey (Macaca fascicularis) (MfFXI.mmH) FXI with K values ​​of 144 and 104 pM, respectively, and showed weak but detectable binding to recombinant rabbit FXI (rbFXI.mmH) with a K value of 171 nM. The subject anti-FXI mAb REGN9933 showed no detectable binding to recombinant mouse FXI (mFXI.mmH) up to the highest concentration tested (50 nM) (data not shown).

[0263] 9.1.2.1: In Vitro Evaluation of the Ability of the Subject Anti-FXI Antibody Drug Substances to Block Coagulation Pathways in Pooled Human or Cynomolgus Monkey Plasma In this study, the ability of REGN9933 to block coagulation pathways in pooled plasma from either human or cynomolgus monkey donors was evaluated in vitro using clotting assays and TGAs, in which coagulation was induced in plasma with either EA or TF, respectively, to measure the effect of REGN9933 on the intrinsic or extrinsic coagulation pathways.

[0264] A summary of the results of the coagulation assays and TGA using human and cynomolgus plasma is shown in Table 32. [Table 32]

[0265] In clotting assays, the subject anti-FXI mAb REGN9933 increased aPTT in a concentration-dependent manner relative to baseline (no antibody), with up to 2.7-fold and 2.1-fold increases observed in human (FIG. 8A) and cynomolgus monkey (data not shown) plasma, respectively, over the range of antibody concentrations tested (19 nM to 1.2 μM). A doubling of aPTT relative to baseline was estimated to occur at 25 nM and 1.1 μM in human and cynomolgus monkey plasma, respectively. No change in PT relative to baseline was observed in either human or cynomolgus monkey plasma at the highest antibody concentration tested (1.2 μM) (FIG. 8B).

[0266] In TGA in which thrombin generation was induced by EA via the intrinsic pathway in human plasma, the subject anti-FXI mAb REGN9933 increased the lag time of thrombin generation by up to 4.9-fold over baseline (no antibody), reduced peak thrombin levels to 8% of baseline, and reduced endogenous thrombin capacity to 28% of baseline (Figures 9A and 9B). The subject anti-FXI mAb REGN9933 exerted these effects in a concentration-dependent manner, achieving a maximal effect at concentrations of 250 nM or higher (Figure 9A). When thrombin generation was induced by TF via the extrinsic pathway, the subject anti-FXI mAb REGN9933 slightly reduced peak thrombin levels to 86% of baseline and reduced endogenous thrombin capacity to 89% of baseline (Figures 9C and 9D). The subject anti-FXI mAb REGN9933 exerted these effects in a concentration-dependent manner, achieving maximal effects at concentrations of 125 nM or greater (FIG. 9C). Up to the highest antibody concentration tested (500 nM), no concentration-dependent increase in the lag time of thrombin generation was observed with the subject anti-FXI mAb REGN9933 (FIG. 9C).

[0267] In TGA in which thrombin generation was induced by EA via the intrinsic pathway in cynomolgus monkey plasma, the subject anti-FXI mAb REGN9933 increased the lag time of thrombin generation by up to 2.2-fold over baseline (no antibody), reduced peak thrombin levels to 43% of baseline, and reduced endogenous thrombin capacity to 66% of baseline (data not shown). The subject anti-FXI mAb REGN9933 exerted these effects in a concentration-dependent manner, achieving a maximal effect at concentrations of 62 nM or higher. When thrombin generation was induced by TF via the extrinsic pathway, no concentration-dependent increase in the lag time of thrombin generation or reduction in peak thrombin or endogenous thrombin capacity was observed with the subject anti-FXI mAb REGN9933 up to the maximum antibody concentration tested (500 nM) (data not shown).

[0268] 9.1.2.2: Evaluation of the subject mAb REGN9933-FXI and -FXIa immune complexes for binding to C1q. Circulating immune complexes (CICs) are formed by the multimerization of antibodies with soluble antigens. The deposition of CICs in tissues and the associated inflammatory response can result in tissue damage at the deposition site. Large immune complexes can also activate the complement component C1q in serum (Rojko, 2014). REGN9933 is a hinge-stabilized IgG4-derived heavy chain Fc constant domain (IgG4 P IgG4 contains IgG1-specific FXIa (referred to as FXIa), and IgG4 does not bind C1q in the same way as IgG1, and is therefore unlikely to form immune complexes capable of binding C1q (Patel, 2015). Nonetheless, EIAs were performed to assess the potential binding of the subject mAb-FXI and subject mAb-FXIa complexes to C1q.

[0269] In this study, REGN9933 was incubated with human FXI or FXIa proteins and added to plate-adsorbed C1q followed by detection using anti-human IgG.

[0270] The subject mAb REGN9933-FXI and subject mAb REGN9933-FXIa conjugates did not demonstrate binding levels considered positive by the assay kit specifications (data not shown). In contrast, C1q binding was detected for the positive control conjugate, heat aggregated human gamma globulin (HAGG).

[0271] 9.1.3: Non-immune cell surface binding IgG4 P In vitro characterization of proliferation and cytokine release following treatment of human peripheral blood mononuclear cells with isotype control antibodies The subject anti-FXI mAb REGN9933 is an IgG4 antibody that does not target immune cell surface molecules. P Such a non-binding IgG4 P The ability of IgG4 to induce cytokine release or proliferation in human PBMCs was examined. P Evaluation was performed using an isotype control antibody [REGN1945].

[0272] IgG4 P The isotype control antibody [REGN1945] did not induce PBMC cytokine release or proliferation in an in-house study (data not shown) performed according to established methods to detect PBMC proliferation and cytokine release induced by a superagonist anti-CD28 antibody (TeGenero TGN1412) (Findlay, 2010) (Vessillier, 2015). In this assay, accumulation of IFN-γ, IL-2, TNF-α, IL-10, IL-6, IL-13, IL-4, and IL-12p70 was assessed after incubation of PBMC with the test antibody. IgG4 with the same primary sequence as TGN1412 (except for the S228P substitution) P Anti-CD28 superagonist antibody [REGN2329] and stimulatory anti-CD3 antibody (OKT3) served as positive controls. IgG4 at concentrations ranging from 0.4 to 10 μg / mL PThe isotype control antibody [REGN1945] had no effect on PBMC proliferation (data not shown) or cytokine release (data not shown) under any of the conditions tested. In contrast, the anti-CD28 positive control antibody mediated significant proliferation of PBMC and significant cytokine release, whereas the anti-CD3 positive control antibody mediated significant cytokine release.

[0273] In summary, IgG4 does not specifically target immune cell surface molecules. P The antibody did not induce cytokine release from or proliferation of PBMCs in standard in vitro assays. This study was performed using IgG4 antibodies that do not specifically target immune cell surface molecules. P The antibodies show a low potential to induce cytokine release syndrome in humans.

[0274] 9.1.4: Discussion The subject m-Ab REGN9933 is a human IgG4-based mAb that binds to FXI / FXIa and selectively inhibits the intrinsic coagulation pathway with minimal effect on the extrinsic coagulation pathway in human plasma. The subject m-Ab REGN9933 is being developed for the prevention and treatment of thromboembolic diseases. A series of non-clinical studies were conducted to evaluate the efficacy and safety of the subject m-Ab REGN9933 using in vitro assays and in vivo models.

[0275] SPR studies showed that the subject m-Ab REGN9933 specifically binds to human FXI and FXIa, as well as to cynomolgus monkey FXI with KD values ​​in the picomolar range. The subject m-Ab REGN9933 also weakly bound to rabbit FXI with a high nanomolar KD value, but no binding to mouse FXI was detected. In vitro clotting time assays and TGA using pooled plasma from human or cynomolgus monkey donors showed that the subject m-Ab REGN9933 blocks intrinsic coagulation pathway activity with a concentration-dependent effect on duration but not magnitude. A slight effect on extrinsic coagulation pathway activity in human plasma and no effect on extrinsic coagulation pathway activity in cynomolgus monkey plasma was observed. Together, these in vitro binding and activity studies demonstrated that the subject m-Ab REGN9933 can bind to FXI and block intrinsic coagulation pathway activity in human and monkey plasma, thereby supporting the use of cynomolgus monkeys as a relevant species for pharmacology and / or toxicology studies with the subject m-Ab REGN9933.

[0276] In an in vitro EIA, the subject m-Ab REGN9933-FXI and subject m-Ab REGN9933-FXIa complexes did not demonstrate positive levels of binding to C1q, suggesting that the subject m-Ab REGN9933 is unlikely to induce an immune complex-mediated inflammatory response.

[0277] 9.2: In vivo pharmacology The subject m-Ab REGN9933-mediated blockade of the coagulation pathway in cynomolgus monkeys was evaluated in a pilot PK / PD study evaluating single IV doses of 1, 3, or 10 mg / kg of the subject m-Ab REGN9933 (Example 9.2.1), and in a study evaluating single doses of 0.5, 5, or 30 mg / kg of the subject m-Ab REGN9933 via the IV route, or a single dose of 30 mg / kg of the subject m-Ab REGN9933 via the SC route (Example 9.2.2).

[0278] REGN9933-mediated blockade of the coagulation pathway was evaluated in coagulation assays and TGAs (Example 9.2.1) using plasma from cynomolgus monkeys receiving a single IV dose of 1, 3, or 10 mg / kg of the subject m-Ab REGN9933, and in TGAs (Example 9.2.2) using plasma from cynomolgus monkeys receiving a single dose of 0.5, 5, or 30 mg / kg of the subject m-Ab REGN9933 via the intravenous (IV) route, or a single dose of 30 mg / kg of the subject m-Ab REGN9933 via the subcutaneous (SC) route. In the coagulation assays performed in each of the aforementioned studies, REGN9933 exerted a PD effect on the intrinsic coagulation pathway (i.e., prolongation of activated partial thromboplastin time [aPTT] relative to baseline) with no effect on the extrinsic coagulation pathway (i.e., no effect on prothrombin time [PT] relative to baseline). In the TGAs performed, the subject m-Ab REGN9933 exerted PD effects on the intrinsic coagulation pathway (i.e., prolonged lag time of thrombin generation, reduced peak thrombin levels, and reduced endogenous thrombin potential induced by ellagic acid [EA] versus baseline) with minimal effects on the extrinsic coagulation pathway (i.e., no effect on lag time of thrombin generation, and slightly reduced peak thrombin levels and endogenous thrombin potential induced by tissue factor [TF] versus baseline). In both studies, the duration of the effects on the intrinsic coagulation pathway (as observed in clotting assays and TGA) was dose-dependent, whereas the slight effects on the extrinsic coagulation pathway (as observed in TGA) were not dose-dependent. The effects on the intrinsic coagulation pathway were likely driven by increasing the subject m-Ab REGN9933 concentration rather than reducing the concentration of FXI.

[0279] 9.2.1: Ex vivo evaluation of single doses of 1, 3, or 10 mg / kg of the subject mAbs IV on coagulation pathway activity in cynomolgus monkeys (R3448 PK 19085) Characterization of the subject m-Ab REGN9933-mediated blockade of the coagulation pathway in cynomolgus monkeys was evaluated as part of a single-dose PK study (data not shown). Female cynomolgus monkeys were administered a single IV slow bolus injection of vehicle (control) or the subject m-Ab REGN9933 at doses of 1, 3, or 10 mg / kg (n=3 per group) and monitored for 8 weeks after dosing. Blood samples were collected from all animals pre-dose and 5 minutes, 6 hours, days 2, 3, 4, 6, 8, 11, 15, 22, 29, 36, 43, 50, and 57 after dosing. Pre-dose measurements served as the baseline for each animal. Concentrations of subject total m-Ab REGN9933 in serum (all drug, regardless of target presence) and total FXI in plasma (subject m-Ab REGN9933 bound and free) were determined by enzyme-linked immunosorbent assay (ELISA). Various PD analyses were performed using plasma samples, including measuring the effect of subject m-Ab REGN9933 on the coagulation pathway using clotting assays and TGA. In these assays, clotting was induced in plasma samples by either EA or TF to measure the effect of subject m-Ab REGN9933 on the intrinsic or extrinsic coagulation pathways, respectively. Total FXI concentrations, as well as measurements from clotting assays and TGA, were normalized to baseline (i.e., pre-dose measurements) and expressed as change versus baseline.

[0280] 9.2.1.1: Assessment of the effect of the subject mAb REGN9933 on total FXI levels in plasma The subject total m-Ab REGN9933 concentration-time profile was characterized by an initial short distribution phase, followed by a short linear beta elimination phase, and a terminal target-mediated elimination phase. Subject peak m-Ab REGN9933 concentrations increased dose-proportionally, but dose-normalized subject m-Ab REGN9933 exposure demonstrated greater than dose-proportional increases in exposure across dose groups (FIG. 10A).

[0281] Increases in total FXI relative to baseline were observed at subject m-Ab REGN9933 doses of 3 mg / kg and above. Statistically significant increases in total FXI relative to baseline were observed only in the 10 mg / kg subject m-Ab REGN9933 group compared to the control group (FIG. 10B). These increases were associated with greater than dose-proportional increases in exposure to subject total m-Ab REGN9933 across dose groups. Subject m-Ab REGN9933 was not associated with a decrease in the concentration of total FXI in plasma, likely due to stabilization of FXI in the circulation by binding to subject m-Ab REGN9933.

[0282] 9.2.1.2: Assessment of the Effects of the Subject m-Ab REGN9933 on aPTT and PT in Plasma The subject m-Ab REGN9933 prolongs activated partial thromboplastin time (aPTT), an indicator of intrinsic pathway activity, relative to baseline at all dose levels tested, with a dose-dependent effect on duration of activity. No effect on prothrombin time (PT), an indicator of extrinsic pathway activity, relative to baseline was observed with the subject m-Ab REGN9933 at any dose level tested (FIG. 11B).

[0283] 9.2.1.3: Evaluation of the effect of REGN9933 on thrombin generation in plasma In EA-induced TGA (TGA-EA), the subject m-Ab REGN9933 prolonged the lag time of thrombin generation (data not shown), reduced peak thrombin levels (data not shown), and reduced endogenous thrombin capacity mediated by the intrinsic pathway (FIG. 12A) at all dose levels tested, with a dose-dependent effect on the duration of activity. In TF-induced TGA (TGA-TF), no effect on the lag time of thrombin generation mediated by the extrinsic pathway was observed at any dose level tested (data not shown). The subject m-Ab REGN9933 reduced peak thrombin levels mediated by the extrinsic pathway (data not shown) and endogenous thrombin capacity (FIG. 12B) at all dose levels tested, relative to baseline. However, the effects of the subject m-Ab REGN9933 on the extrinsic pathway were small in magnitude compared with the effects on the intrinsic pathway, and neither the magnitude nor the duration of the effect on the extrinsic pathway appeared to be dose-dependent.

[0284] Relative to baseline as measured by TGA, statistically significant effects on the intrinsic coagulation pathway were observed up to days 6, 11, and 50 in the subject m-Ab REGN9933 groups at 1, 3, and 10 mg / kg, respectively, compared to the control group (data not shown). All parameters returned to baseline or near baseline levels by the end of the 8-week study.

[0285] 9.2.2: Ex vivo evaluation of a single dose of 0.5, 5, or 30 mg / kg of a subject m-Ab REGN9933 IV, or a single dose of 30 mg / kg of a subject m-Ab REGN9933 SC, on coagulation pathway activity in cynomolgus monkeys A PK / PD study was conducted in female cynomolgus monkeys (data not shown). Ex vivo assays were performed to evaluate the effect of the subject m-Ab REGN9933 on coagulation using blood samples collected over a 10-week monitoring period. The subject m-Ab REGN9933 was given to animals as a single dose at 0.5, 5, or 30 mg / kg (n=5 per group) IV or 30 mg / kg (n=5 per group) SC.

[0286] 9.2.2.1: Assessment of the effect of the subject m-Ab REGN9933 on total FXI levels in plasma The concentration-time profile of subject total m-Ab REGN9933 was characterized by an initial short distribution phase, followed by a short linear beta elimination phase, a terminal target-mediated elimination phase, and a post-target-mediated elimination phase (data not shown). Subject peak m-Ab REGN9933 concentrations increased in a dose-proportional manner (data not shown). Greater than dose-proportional increases in dose-normalized subject m-Ab REGN9933 exposure were observed across dose groups, consistent with nonlinear kinetics that were more pronounced at lower doses and concentrations of subject m-Ab REGN9933 (data not shown). These findings are consistent with the observed decrease in clearance with increasing dose and associated concentrations. Following administration of subject m-Ab REGN9933, concentrations of soluble FXI in plasma increased 1.0-1.9-fold across all dose groups (IV and SC) compared to baseline levels, indicating a drug effect versus baseline. However, there was no correlation with subject m-Ab REGN9933 concentrations in serum (data not shown).

[0287] 9.2.2: Evaluation of the Effects of the Subject m-Ab REGN9933 on aPTT and PT in Plasma Ex vivo clotting time assays were performed using plasma from blood samples collected over time to determine the effect of administration of the subject m-Ab REGN9933 on aPTT, an index of intrinsic pathway activity, and PT, an index of extrinsic pathway activity. Blood sample collections prior to day 3 were performed with the incorrect anticoagulant, ethylenediaminetetraacetic acid (EDTA), whereas sample collections from days 3 through 71 were performed with the preferred anticoagulant, sodium citrate, which interfered less with the performance of the clotting assay. Therefore, only values ​​generated for blood samples from days 3 through 71 were plotted and this and combined values ​​from a previous PK / PD study in cynomolgus monkeys (data not shown) were used to determine historical baselines for aPTT and PT in lieu of study-specific pre-dose measurements in the clotting assays.

[0288] The subject m-Ab REGN9933 prolonged the mean aPTT by 1.5-2.1-fold increases over historical baseline with a dose-dependent effect on duration of activity, but of similar magnitude across all dose groups tested (data not shown). The first occurrence of ≥1.5-fold increases in aPTT over historical baseline was observed on day 3 for all dose levels, but this effect was observed for duration of increase of 1, 19, or 54 days at dose level increments of 0.5 mg / kg (IV), 5 mg / kg (IV), and 30 mg / kg (IV) of subject m-Ab REGN9933, respectively. In addition, ≥1.5-fold increases in aPTT over historical baseline were observed for 47 days following administration of subject m-Ab REGN9933 at 30 mg / kg (SC). Across all dose groups, the maximum mean effect was a 2.1-fold increase in aPTT prolongation.

[0289] No effect on PT was observed versus historical baseline with subject m-Ab REGN9933 at any dose level tested (data not shown).

[0290] 9.2.3: Discussion The subject m-Ab REGN9933-mediated blockade of coagulation pathways was evaluated in blood clotting assays and TGAs using plasma from cynomolgus monkeys receiving a single IV dose of 1, 3, or 10 mg / kg of the subject m-Ab REGN9933, and in TGAs using plasma from cynomolgus monkeys receiving a single dose of 0.5, 5, or 30 mg / kg of the subject m-Ab REGN9933 via IV, or a single dose of 30 mg / kg of the subject m-Ab REGN9933 via SC. In the clotting assays performed in each of the aforementioned studies, the subject m-Ab REGN9933 blocked the intrinsic coagulation pathway with a dose-dependent effect in duration, but not magnitude, and no effect on the extrinsic coagulation pathway was observed. In the TGA performed, the subject m-Ab REGN9933 had a dose-dependent effect on duration but not magnitude that blocked the intrinsic coagulation pathway, with minimal effects observed on the extrinsic coagulation pathway.

[0291] Example 10 Safety Pharmacology Safety pharmacological endpoints were incorporated into a GLP repeat-dose toxicity study conducted in cynomolgus monkeys (data not shown). No test article-related cardiovascular (heart rate, blood pressure, and electrocardiogram), respiratory (breaths / min and pulse oximetry), or central nervous system changes were evident at doses up to 100 mg / kg / week (SC or IV), the highest dose administered.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to the apple 2 (A2) domain of coagulation factor XI (FXI), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) comprising heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and a light chain variable region (LCVR) comprising light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3; (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 5; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 7; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 9; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 15; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19; or (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 33; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 37; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 15; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) An isolated antibody, or an antigen-binding fragment thereof, wherein the LCDR3 comprises the amino acid sequence of SEQ ID NO:

19.

2. the HCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO:3 and the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO:13; or The isolated antibody, or antigen-binding fragment thereof, of claim 1, wherein the HCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 31 and the LCVR comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

13.

3. the HCVR comprises an amino acid sequence comprising SEQ ID NO:3 and the LCVR comprises an amino acid sequence comprising SEQ ID NO:13; or 3. The isolated antibody, or antigen-binding fragment thereof, of claim 2, wherein the HCVR comprises an amino acid sequence comprising SEQ ID NO: 31 and the LCVR comprises an amino acid sequence comprising SEQ ID NO:

13.

4. 1. An isolated antibody, or antigen-binding fragment thereof, that binds to the apple 2 (A2) domain of coagulation factor XI (FXI), wherein the antibody or antigen-binding fragment thereof is a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 90% identity to SEQ ID NO:3, and a light chain variable region (LCVR) comprising an amino acid sequence having at least 90% identity to SEQ ID NO:13; or An isolated antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region (HCVR) comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 31, and a light chain variable region (LCVR) comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

13.

5. the HCVR comprises an amino acid sequence comprising SEQ ID NO:3 and the LCVR comprises an amino acid sequence comprising SEQ ID NO:13; or 5. The isolated antibody, or antigen-binding fragment thereof, of claim 4, wherein the HCVR comprises an amino acid sequence comprising SEQ ID NO: 31 and the LCVR comprises an amino acid sequence comprising SEQ ID NO:

13.

6. (a) HCDR1 comprising the amino acid sequence of SEQ ID NO: 5; (b) HCDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) HCDR3 comprising the amino acid sequence of SEQ ID NO: 9; (d) LCDR1 comprising the amino acid sequence of SEQ ID NO: 15; (e) an LCDR2 comprising the amino acid sequence of SEQ ID NO: 17; and (f) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 19; or (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 33; (b) the HCDR2 comprises the amino acid sequence of SEQ ID NO: 35; (c) the HCDR3 comprises the amino acid sequence of SEQ ID NO: 37; (d) the LCDR1 comprises the amino acid sequence of SEQ ID NO: 15; (e) the LCDR2 comprises the amino acid sequence of SEQ ID NO: 17; and (f) the LCDR3 comprises the amino acid sequence of SEQ ID NO: 19; or an antigen-binding fragment thereof;

7. The antibody or antigen-binding fragment thereof has a K selected from the group consisting of less than about 1,000 pM, less than about 800 pM, less than about 500 pM, less than about 100 pM, and less than about 50 pM, as measured by surface plasmon resonance at 25°C or 37°C. Ｄ 2. The isolated antibody or antigen-binding fragment of claim 1, which binds to human FXI at 200 ng / mL.

8. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to human FXI with a dissociation half-life (t1 / 2) selected from the group consisting of greater than about 10 minutes, greater than about 60 minutes, greater than about 500 minutes, or greater than about 1,000 minutes, as measured by surface plasmon resonance at 25°C or 37°C.

9. The antibody or antigen-binding fragment thereof has an IC ５０ 2. The isolated antibody or antigen-binding fragment of claim 1, which inhibits activation of human coagulation factor X (FX) at 2000-200407.

10. The antibody or antigen-binding fragment thereof has an IC of less than about 40 pM ５０ 2. The isolated antibody or antigen-binding fragment of claim 1, which inhibits activation of human coagulation factor X (FX) at 2000-200407.

11. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment increases activated partial thromboplastin time (aPTT) by at least two-fold.

12. 12. The isolated antibody or antigen-binding fragment thereof of claim 11, wherein the antibody or antigen-binding fragment does not increase prothrombin time (PT).

13. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof inhibits FXIa-mediated thrombin activity by at least 5%, at least 10%, at least 15%, or between 5% and 15%.

14. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment thereof extends the aPTT in human plasma by at least 2-fold at concentrations of 100 nM or less, 75 nM or less, or 50 nM or less.

15. 15. The isolated antibody or antigen-binding fragment of claim 14, wherein the antibody or antigen-binding fragment does not prolong PT.

16. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof inhibits the generation or activation of thrombin via the intrinsic coagulation pathway in human plasma at a concentration of at least 10 nM, at least 25 nM, or at least 50 nM without affecting the generation or activation of thrombin via the extrinsic coagulation pathway.

17. An isolated antibody, or antigen-binding fragment thereof, that competes for binding with the antibody or antigen-binding fragment thereof of claim 1.

18. An isolated antibody, or antigen-binding fragment thereof, that binds to the same epitope as the antibody or antigen-binding fragment thereof described in claim 1.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1 and a pharmaceutically acceptable carrier or diluent.

20. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1.

21. A vector comprising the nucleic acid molecule of claim 20.

22. 22. A cell comprising the vector of claim 21 or the nucleic acid molecule of claim 20.

23. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 18, or the pharmaceutical composition of claim 19, for inhibiting biological activity mediated by FXI.

24. 24. The composition of claim 23, wherein the biological activity is thrombus formation, and thrombus formation is inhibited by contacting FXI with the antibody or antigen-binding fragment thereof.

25. 25. The composition of claim 24, wherein the contact results in a prolongation of the aPTT or a reduction in thrombin activity in plasma.

26. A composition comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 18, or a pharmaceutical composition described in claim 19, for treating or preventing a disease or disorder associated with FXI activity or expression, or ameliorating at least one symptom associated with said disease or disorder associated with FXI activity or expression, in a subject in need of such treatment.

27. 27. The composition of claim 26, wherein the disease or disorder is a disease or disorder of blood clotting or a disease or disorder that confers an increased risk of thrombus formation in the subject.

28. 28. The composition of claim 27, wherein the disease or disorder is atrial fibrillation.

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