Factor XI catalytic domain-binding antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies that bind to the catalytic domain of factor XI (FXI) and methods of use thereof. According to certain embodiments, the antibodies are antagonistic antibodies that inhibit clot formation via the intrinsic pathway without affecting hemostasis, as demonstrated by their ability to prolong aPTT without affecting PT. Thus, these antagonistic antibodies can be used in the treatment or therapeutic regimen of blood clotting diseases or disorders 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 priority to U.S. Provisional Application No. 63 / 423,272, filed November 7, 2022. The entire contents of the foregoing application are expressly incorporated herein by reference.

[0002] The present disclosure relates to antibodies that bind to the catalytic domain (CAT) of factor XI (FXI), compositions comprising these antibodies, and methods of their use. [Background technology]

[0003] 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 a blood clot.

[0004] The extrinsic pathway regulates hemostasis in 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.

[0005] 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 feedforward manner, amplifying clot formation.

[0006] FXI is the zymogen of the plasma protease FXIa, which sustains thrombin generation through FXI 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 (referred to herein as "CAT" or "CD"). A disulfide bond is present between the A4 domains of each subunit. FXI subunits are activated by cleavage of one or both Arg-Ile bonds located between the A4 and CAT domains to form FXIa. Cleavage of the Arg-Ile bond is generally believed to be catalyzed by FXIIa and / or thrombin. Summary of the Invention [Means for solving the problem]

[0007] Provided herein are isolated monoclonal antibodies and antigen-binding fragments thereof that bind, e.g., specifically bind, to the CAT domain of factor XI (FXI). In any of the embodiments disclosed herein, the antibodies or antigen-binding fragments thereof can specifically bind to the CAT 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.

[0008] In its broadest aspect, the present 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 of, 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.

[0009] In certain embodiments, anti-FXI antibodies may be useful in treating a variety of blood clotting disorders or diseases where treatment 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, prevention of primary venous thromboembolism (VTE), treatment of extensive VTE, prevention of recurrent ischemia after 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.

[0010] 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, for example, to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0011] Exemplary anti-FXI antibodies of the present disclosure are listed in Tables 1A-1C herein. Tables 1A-1C show the amino acid sequences of exemplary heavy chain regions (HC) and light chain regions (LC) of exemplary anti-FXI antibodies. In one embodiment, the HC comprises a heavy chain variable region (HCVR) and the light chain comprises a light chain variable region (LCVR).

[0012] The present disclosure provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising an HC comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to FXI, comprising an LC comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Tables 1A-1C, 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 antibodies or antigen-binding fragments thereof that bind to FXI, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR) in which any of the HCVR sequences of a HC listed in Tables 1A-1C are paired with any of the LCVR amino acid sequences of a LC listed in Tables 1A-1C. According to certain embodiments, the present disclosure provides antibodies or antigen-binding fragments thereof that comprise an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0015] Accordingly, in a first aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to serum coagulation factor XI (FXI), wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprised within a heavy chain region (HC) comprising an amino acid sequence set forth in Tables 1A-1C, or a substantially similar sequence having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprised within a light chain region (LC) comprising an amino acid sequence set forth in Tables 1A-1C, or a substantially similar sequence having at least 90% sequence identity thereto.

[0016] 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 5 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 300 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 95 minutes as measured by surface plasmon resonance at 25°C or 37°C; (f) inhibiting the activation of factor Xa (FXa) by FXI in normal dilution plasma by at least about 85% to about 87% with an IC50 of less than about 39 pM to less than about 190 pM; (g) inhibiting the activation of factor Xa (FXa) by FXIa in normal dilution plasma by at least about 25% to about 35% with an IC50 of at least about 10 nM; (h) preferentially binds to the CAT domain (i.e., catalytic domain) compared to either full-length FXI or an FXI domain such as apple domain 2 (A2), PKA1, PKA3, or PKA4, as determined by label-free biolayer interferometry; (i) competing for binding to FXI with an antibody that specifically binds to an epitope within the FXI CAT domain and an epitope that overlaps with the FXI CAT domain; (j) increasing the activated partial thromboplastin time (aPTT), a measure of intrinsic clotting time, in primate samples in vitro by at least 2, 2.5, 3, 3.5, 3.8, or 4-fold without measurably affecting the prothrombin time (PT), a measure of extrinsic clotting time; (k) inhibiting intrinsic pathway peak thrombin activity by 1% to 6% in primate samples in vitro; (l) prolonging 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 20 nM or greater, but does not affect extrinsic pathway thrombin generation at doses up to about 500 nM.

[0017] In one embodiment, the present 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 heavy chain variable region (HCVR) complementarity-determining region (CDR) comprising an amino acid sequence within the heavy chain region (HC) amino acid sequence set forth in Tables 1A-1C; and (b) a light chain variable region (LCVR) CDR comprising an amino acid sequence within the light chain region (LC) amino acid sequence set forth in Tables 1A-1C.

[0018] In one embodiment, the antibody or antigen-binding fragment thereof that binds to FXI comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the HC sequence of SEQ ID NO: 18 or a substantially similar sequence having at least 90% sequence identity thereto, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the LC sequence of SEQ ID NO: 20 or a substantially similar sequence having at least 90% sequence identity thereto.

[0019] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises an HCVR having an amino acid sequence within the HC amino acid sequences provided in Tables 1A-1C below.

[0020] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI further comprises an LCVR having an amino acid sequence within the LC amino acid sequences provided in Tables 1A-1C below.

[0021] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises an HCVR having an amino acid sequence within the HC amino acid sequence provided in Tables 1A-1C below, and an LCVR having an amino acid sequence within the LC amino acid sequence provided in Tables 1A-1C below.

[0022] 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 provided within the HC / LC amino acid sequences of Tables 1A-1C below.

[0023] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence included in the HC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence included in the HC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence included in the HC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0026] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence contained within the LC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0027] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence contained within the LC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0028] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence contained within the LC amino acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0029] 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) in which any of the HCDR3 amino acid sequences listed in Tables 1A-1C is paired with any of the LCDR3 amino acid sequences listed in Tables 1A-1C. 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 Tables 1A-1C. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is SEQ ID NO: 8 / 16.

[0030] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to FXI, comprising a set of six CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3) contained in any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0031] 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 CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., 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 for identifying CDR sequences within antibodies.

[0032] In one embodiment, the present disclosure provides: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 8; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 14, 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: 16.

[0033] In one embodiment, the isolated antibody or antigen-binding fragment thereof comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of SEQ ID NOs: 4-6-8-12-14-16.

[0034] 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 for binding to FXI with a reference antibody, wherein the reference antibody preferentially binds to the catalytic domain of FXI.

[0035] 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 the catalytic domain of FXI.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 10 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 5 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 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a t½ of greater than about 10 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 15 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 16 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 t½ of greater than about 1,000 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 300 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 t 1 / 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 t 1 / 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 t 1 / 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 binds to human FXIa with a t1 / 2 of greater than about 50 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0064] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a t1 / 2 of greater than about 75 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0065] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXIa with a t1 / 2 of greater than about 95 minutes as measured by surface plasmon resonance at 25°C or 37°C.

[0066] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI preferentially binds to the CAT domain (i.e., the catalytic domain) compared to full-length FXI, PKA1, PKA2, PKA3, and / or PKA4, as measured by label-free biolayer interferometry.

[0067] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI competes for binding to FXI with an antibody that specifically binds to an epitope within and overlapping with the FXI CAT domain.

[0068] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI increases the activated partial thromboplastin time (aPTT), a measure of intrinsic clotting time, by at least 2.5-fold in vitro in primates without measurably affecting the prothrombin time (PT), a measure of extrinsic clotting time.

[0069] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds FXI inhibits in vitro in a primate intrinsic pathway peak thrombin activity by about 5% to 15%, about 1% to 20%, about 0.5% to 25%, about 3% to 5%, about 4% to 6%, about 5% to 7%, about 6% to 8%, about 7% to 9%, about 8% to 10%, about 9% to 11%, about 10% to 12%, about 11% to 13%, about 12% to 14%, about 13% to 15%, about 14% to 16%, about 15% to 17%, about 16% to 18%, about 17% to 19%, or about 18% to 20%.

[0070] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI prolongs the aPTT in human plasma by about 2-fold in vitro 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, ≦55 nM, ≦50 nM, ≦45 nM, ≦40 nM, ≦39 nM, ≦38 nM, ≦37 nM, ≦36 nM, ≦35 nM, ≦34 nM, ≦33 nM, ≦32 nM, ≦31 nM, ≦30 nM, ≦25 nM, or ≦20 nM, wherein the anti-FXI prolongs the aPTT by about 2-fold without doubling the PT.

[0071] In one embodiment, the 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 to 100 nM, about 15 nM to 500 nM, about 20 nM to 60 nM, about 25 nM to 50 nM, ≥ 15 nM, ≥ 16 nM, ≥ 17 nM, ≥ 18 nM, ≥ 19 nM, ≥ 20 nM, ≥ 21 nM, ≥ 22 nM, ≥ 23 nM, ≥ 24 nM, ≥ 25 nM, ≥ 26 nM, ≥ 27 nM, ≥ 28 nM, ≥ 29 nM, ≥ 30 nM, ≥ 31 nM, ≥ 32 nM, ≥ 33 nM, ≥ 34 nM, ≥ 35 nM, ≥ 36 nM, ≥ 37 nM, ≥ 38 nM, ≥ 39 nM, or ≥ 40 nM. Here, anti-FXI inhibits intrinsic pathway thrombin generation without any effect on extrinsic pathway thrombin generation, even at doses up to about 500 nM.

[0072] 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 Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Tables 1A-1C, or a substantially similar sequence 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 LCVR amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Tables 1A-1C, 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 HCDR1 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Tables 1A-1C, 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 HCDR2 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Tables 1A-1C, 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 HCDR3 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Tables 1A-1C, 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 LCDR1 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Tables 1A-1C, 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 LCDR2 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Tables 1A-1C, 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 nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Tables 1A-1C, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Tables 1A-1C, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0080] The present disclosure also provides nucleic acid molecules encoding HCVRs, wherein the HCVRs comprise a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), and the HCDR1, HCDR2, HCDR3 amino acid sequence sets are as defined by any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0081] The present disclosure also provides nucleic acid molecules encoding LCVRs, wherein the LCVRs comprise a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), and the LCDR1, LCDR2, LCDR3 amino acid sequence sets are as defined by any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0082] The present disclosure also provides nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Tables 1A-1C, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Tables 1A-1C. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Tables 1A-1C, 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 Tables 1A-1C, 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 an HCVR and an LCVR, wherein the HCVR and LCVR are both derived from the same anti-FXI antibody listed in Tables 1A-1C.

[0083] In a third aspect, the present disclosure provides recombinant expression vectors 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 present disclosure includes recombinant expression vectors comprising any of the nucleic acid molecules described above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences set forth in Tables 1A-1C. Also included within the scope of the present disclosure are host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that permit the production of the antibodies or antibody fragments, and recovering the antibodies and antibody fragments so produced.

[0084] The present disclosure includes anti-FXI antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesired glycosylation sites may be useful, or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to enhance 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).

[0085] 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 pharmaceutically acceptable carrier.

[0086] 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 neurodegenerative disease or disorder.

[0087] 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 Tables 1A-1C, or contacting FXI with a pharmaceutical composition comprising a biologically effective amount of an antagonist anti-FXI antibody of Tables 1A-1C.

[0088] In certain embodiments, the biological activity is blood coagulation or blood coagulation as a result of the intrinsic coagulation pathway, but not blood coagulation as a result of the extrinsic (i.e., tissue factor-induced) pathway, and when FXI or FXIa is contacted with an antagonist anti-FXI antibody, blood coagulation as a result of the intrinsic coagulation pathway, but not blood coagulation or blood coagulation as a result of the extrinsic pathway, is inhibited or otherwise reduced.

[0089] 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 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, alleviated, inhibited, or prevented by targeting FXI and / or inactivating FXI-mediated blood coagulation.

[0090] In one embodiment, the anti-FXI antibodies of the present disclosure may provide a method for treating pathological intrinsic coagulation without adversely affecting hemostasis. In one embodiment, 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 of natural proteins that prevent clotting (such as antithrombin, protein C, and protein S), elevated homocysteine ​​levels, elevated fibrinogen or dysfunctional fibrinogen levels (dysfibrinogenemia), abnormal fibrinolytic systems including elevated levels of Factor VIII, Factor IX, and / or XI, plasminogen deficiency, 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, oral contraceptives (birth control pills), and the like. the use of estrogen in combination with other drugs may provide a method of treating any one of the following diseases, disorders, coagulation side effects, and indirect coagulation effects: supplemental use of estrogen, including estrogen-releasing hormone (estrogen-releasing hormone) and estrogen-releasing hormone (estrogen-releasing hormone) in combination with other drugs ...

[0091] A seventh aspect of the present disclosure provides a method for preventing thrombosis in a subject without adversely affecting hemostasis, the method comprising administering to the subject a therapeutically effective amount of an FXI antagonist antibody of Tables 1A-1C, or a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof.

[0092] In one embodiment, the above method can be achieved by administering to a subject in need thereof an antagonistic anti-FXI antibody or an antigen-binding fragment thereof, wherein the antagonistic anti-FXI antibody comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence shown in Tables 1A to 1C, 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 shown in Tables 1A to 1C, or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0093] In one embodiment, the method of the present disclosure can be achieved by administering an antagonist FXI antibody of the present disclosure, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the HC sequence of SEQ ID NO: 18 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 LC sequence of SEQ ID NO: 20 or a substantially similar sequence thereof having at least 90% sequence identity thereto.

[0094] In one embodiment, the antibody or antigen-binding fragment thereof comprises an HC having the amino acid sequence of SEQ ID NO: 18, or an HCVR having the amino acid sequence of an HCVR contained within SEQ ID NO: 18. In one embodiment, the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody or antigen-binding fragment thereof comprises an HCVR having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2.

[0095] In one embodiment, the antibody or antigen-binding fragment thereof comprises an LC having the amino acid sequence of SEQ ID NO: 20, or an LCVR having the amino acid sequence of an LCVR contained within SEQ ID NO: 20. In one embodiment, the antibody or antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody or antigen-binding fragment thereof comprises an LCVR having an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 10.

[0096] In one embodiment, the antibody or antigen-binding fragment thereof comprises an HC having the amino acid sequence of SEQ ID NO:18 and an LC having the amino acid sequence of SEQ ID NO:20.

[0097] In one embodiment, the antibody or antigen-binding fragment thereof comprises the CDRs of the HC / LC amino acid sequence pair of SEQ ID NOs: 18 / 20.

[0098] In one embodiment, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10.

[0099] In one embodiment, the antibody or antigen-binding fragment thereof comprises: (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO: 4; (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO: 6; (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO: 7; (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO: 12; (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO: 14, and (f) comprises an LCDR3 domain having the amino acid sequence of SEQ ID NO: 16.

[0100] 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: 4-6-8-12-14-16.

[0101] In one aspect, disclosed herein is a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 4-6-8-12-14-16. In one embodiment, the nucleic acid encodes an antibody or antigen-binding fragment thereof having a HCVR comprising SEQ ID NO: 2 and / or a LCVR comprising SEQ ID NO: 10. In one embodiment, the nucleic acid encodes an antibody or antigen-binding fragment thereof having a HC comprising SEQ ID NO: 18 and / or a LC comprising SEQ ID NO: 20.

[0102] In one aspect, disclosed herein is a nucleic acid encoding an antibody or antigen-binding fragment thereof comprising a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the nucleic acid sequences of SEQ ID NOs: 3-5-7-11-13-15. In one embodiment, the nucleic acid encodes an antibody or antigen-binding fragment thereof having a HCVR comprising SEQ ID NO: 1 and / or a LCVR comprising SEQ ID NO: 9. In one embodiment, the nucleic acid encodes an antibody or antigen-binding fragment thereof having a HC comprising SEQ ID NO: 17 and / or a LC comprising SEQ ID NO: 19.

[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 contemplated to be suitable for treatment with the antibodies of the present disclosure, which may include any disease or condition in which harmful clot formation is a risk, particularly, but not limited to, conditions in which intrinsic coagulation and hemostasis are a risk to the patient.

[0105] Other embodiments will become apparent upon consideration of the following detailed description. [Brief explanation of the drawings]

[0106] [Figure 1]1 shows a size analysis of the complexes formed between human coagulation factor XI and REGN7528 and REGN7531. [Figure 2] 1 shows a size analysis of the complexes formed between human coagulation factor XI and REGN7503, REGN7505, and REGN7508. [Figure 3] 1 shows a size analysis of the complex formed between human coagulation factor XI and REGN7508. [Figure 4] 1 shows a size analysis of the complex formed between human coagulation factor XI and REGN9932. [Figure 5] 1 shows a comparison of the size analysis of the complex formed between human coagulation factor XI and REGN7508 and the size analysis of the complex formed between human coagulation factor XI and REGN9932. [Figure 6A] FIG. 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in plasma of female cynomolgus monkeys using a thrombin generation assay (TGA). [Figure 6B] FIG. 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in plasma of female cynomolgus monkeys using a thrombin generation assay (TGA). [Figure 6C] FIG. 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in plasma of female cynomolgus monkeys using a thrombin generation assay (TGA). [Figure 7A] 1 shows a comparison of the effects of anti-FXI mAbs on the extrinsic coagulation pathway in plasma of female cynomolgus monkeys using TGA. [Figure 7B] 1 shows a comparison of the effects of anti-FXI mAbs on the extrinsic coagulation pathway in plasma of female cynomolgus monkeys using TGA. [Figure 7C] 1 shows a comparison of the effects of anti-FXI mAbs on the extrinsic coagulation pathway in plasma of female cynomolgus monkeys using TGA. [Figure 8A] 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 8B]1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 8C] 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 9A] 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 9B] 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 9C] 1 shows a comparison of the effects of anti-FXI mAbs on the intrinsic coagulation pathway in pooled female plasma using TGA. [Figure 10A] Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 10B] Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 10C] Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 11A] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 11B] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 11C] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 12A] Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 12B] Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 12C]Shows the effect of REGN9932 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 13A] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 13B] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 13C] Shows the effect of REGN7508 on the intrinsic coagulation pathway in six single human donors using TGA. [Figure 14] 1 is an overview of the coagulation pathway. [Figure 15] 1 is an example of a thrombogram. [Figure 16A] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using a clotting assay. [Figure 16B] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using a clotting assay. [Figure 16C] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using a clotting assay. [Figure 17A] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using a clotting assay. [Figure 17B] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using a clotting assay. [Figure 17C] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using a clotting assay. [Figure 18A] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using TGA. [Figure 18B]1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using TGA. [Figure 18C] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using TGA. [Figure 18D] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma using TGA. [Figure 19] 1 shows the effect of REGN7508 at a second concentration set on the intrinsic coagulation pathway in pooled human donor plasma using TGA. [Figure 20A] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using TGA. [Figure 20B] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using TGA. [Figure 20C] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using TGA. [Figure 20D] 1 shows the effect of REGN7508 on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus donor plasma using TGA. [Figure 21] 1 shows the effect of REGN7508 at a second concentration set on the intrinsic coagulation pathway in pooled cynomolgus donor plasma using TGA. DETAILED DESCRIPTION OF THE INVENTION

[0107] Before the present disclosure is described, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which 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 one of ordinary skill in the art to which this disclosure belongs. As used herein, the term "about," when used in connection with a specific recited numerical value, means that the value may vary by 1% or less from the recited value. 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 as being 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: 41). Human FXI containing a myc-myc-hexahistidine tag is set forth as SEQ ID NO: 42 (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: 43 (construct hFXI_PKA1) is a chimera comprising the apple 1 domain of human kallikrein B1 (PKA1) (amino acids G20 to C104 of human kallikrein B1 [SEQ ID NO: 48]) at amino acids 1-85, amino acids H105 to V625 of human FXI (hFXI) at amino acids 86-60, and a myc-myc-hexagistidine tag at amino acids 607-634.

[0112] For example, SEQ ID NO: 44 (construct hFXI_PKA2) is a chimera containing amino acids E19 to S108 of hFXI at amino acids 1 to 90, the apple 2 domain (PKA2, also referred to as "A2") of hKLKB1 (amino acids C111 to C193, SEQ ID NO: 48) at amino acids 91 to 174, amino acids A195 to V625 of hFXI at amino acids 175 to 605, and a myc-myc-hexagistidine tag at amino acids 606 to 633.

[0113] For example, SEQ ID NO: 45 (construct hFXI_PKA3) is a chimera containing amino acids E19 to L198 of hFXI at amino acids 1 to 180, the apple 3 domain of hKLKB1 (PKA3) (amino acids C201 to C284, SEQ ID NO: 48) at amino acids 181 to 264, amino acids H285 to V625 of hFXI at amino acids 265 to 605, and a myc-myc-hexagistidine tag at amino acids 606 to 633.

[0114] For example, SEQ ID NO: 46 (construct hFXI_PKA4) is a chimera containing amino acids E19 to V289 of hFXI at amino acids 1 to 271, the Apple 4 domain of hKLKB1 (PKA4) (amino acids C292 to C375, SEQ ID NO: 48) at amino acids 272 to 355, amino acids M376 to V625 of hFXI at amino acids 356 to 605, and a myc-myc-hexagistidine tag at amino acids 606 to 633.

[0115] For example, SEQ ID NO: 47 (construct hKLKB1.mmh) is a chimera containing amino acids G20 to A638 of hKLKB1 at amino acids 1 to 619, and a myc-myc-hexagistidine tag at amino acids 620 to 647.

[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 Tables 1A-1C herein. The term "anti-FXI antibody" also includes antibody-drug conjugates (ADCs) comprising an anti-FXI antibody or 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 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 catalytic domain of FXI or an epitope within the catalytic domain of FXI. The term "antibody" includes immunoglobulin molecules comprising 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 VL). H The heavy chain constant region comprises a C H 1. C H 2 and C H Each light chain comprises three domains: a light chain variable region (herein LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V LThe regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of an anti-FXI antibody (or antigen-binding portion thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0118] As used herein, the term "antibody" also includes antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains, and optionally 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. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[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 associated with the domain H For antigen-binding fragments containing domains, V H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region is a dimer, with 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 dimer. H or V L It may include a domain.

[0121] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H-C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any arrangement of variable and constant domains, including any of the exemplary arrangements listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A 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 LIt may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain arrangements listed above in non-covalent association with the domains (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 comprise 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 clot formation. However, the antibodies of the present disclosure function as antagonist antibodies, functioning as inhibitors of FXI or FXIa activity and simultaneously as inhibitors of intrinsic pathway thrombosis / 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 a patient's bloodstream.

[0124] As used herein, the term "human antibody" is intended to include non-naturally occurring human antibodies. The term includes antibodies 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] 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 that are 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, including splicing of human immunoglobulin gene sequences into other DNA sequences. In certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) to thereby modify the V of the recombinant antibody. H Area and V L The amino acid sequence of the region is human germline V 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 dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming approximately 75-80 kDa molecules 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 antibody hinge region isotype. 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 various intact IgG isotypes. H Area 2 or C H Antibodies with one or more mutations in the three regions are included, which may be desirable, for example, to improve the yield of the desired antibody form in production.

[0128] The terms "specifically binds" or "binds specifically to" and the like 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 defined as binding to at least about 1 x 10 -6 can be characterized by an equilibrium dissociation constant less than or equal to M (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 used herein, an antibody that specifically binds to FXI has been identified by surface plasmon resonance, e.g., BIACORE™. Furthermore, as used herein, a multispecific antibody that binds to the FXI protein and one or more additional antigens, or a bispecific antibody that binds to two different regions of FXI, is nevertheless considered to be an antibody that "specifically binds."

[0129] An antibody of the present disclosure may be an isolated antibody. As used herein, an "isolated antibody" refers to an antibody that has been identified, separated, and / or recovered from 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 it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present 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 can 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 identified by comparing the amino acid sequences disclosed herein to 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 antagonist 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 comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes anti-FXI antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Tables 1A-1C 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 the antigen and have different biological effects. Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include carbohydrate, phosphoryl, or sulfonyl moieties on the antigen.

[0133] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) using 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 for 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 cases, encode a polypeptide having the same or substantially the same amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0134] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT, using predetermined gap weights, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. 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 do not 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 upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids 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 replacement is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al. (1992) Science 256:1443-1445, incorporated herein by reference. A "moderately conservative" replacement 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 measures of similarity 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 alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to databases containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, 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 characteristics The present disclosure provides a soluble soluble cellulose derivative having a K of less than about 500 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 the catalytic domain of human FXI with 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. Dand anti-FXI antibodies that bind to human FXI.

[0137] The present disclosure provides a soluble cellulose derivative 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. D and anti-FXI antibodies that bind to human FXI.

[0138] The present disclosure includes anti-FXI antibodies that bind to human FXI with a dissociation half-life (t) 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 t 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 (t) 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 FXIa with a t 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, an antibody that, when tested in an antigen binding assay such as surface plasmon resonance, 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). According to this aspect of the disclosure, another assay format that can be used to determine whether an antibody binds or does not bind to a particular antigen 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 IC using the assay format described in Example 4, or a substantially similar assay format. 50 The value can be calculated as the concentration of antibody required to activate FXI-mediated signaling to half-maximal observed signal. Thus, according to certain embodiments, the present disclosure provides an IC50 of 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 comprises an anti-FXI antibody that mediates human FXI-mediated activation of human FX in plasma by at least about 85%.

[0142] The present disclosure also provides a method for producing a compound having an IC of less than about 50 pM. 50The present invention also includes anti-FXI antibodies that inhibit FXIa-mediated activation of human FX in plasma by at least about 25% at IC using the assay format described in Example 4, or a substantially similar assay format. 50 The value can be calculated as the concentration of antibody required to activate FXIa-mediated signaling to half the observed maximal signal. Thus, according to certain embodiments, the present disclosure provides an IC50 of 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 catalytic domain (CAT), as demonstrated by direct binding to a CAT domain construct or by competition with one or more specific CAT-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 apple domain (e.g., the A2 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, as exemplified in Examples 6 and 9, 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. 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 may have a concentration of 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, 25 nM At concentrations of 30nM to 35nM, 30nM to 40nM, 35nM to 45nM, 40nM to 50nM, 45nM to 55nM, 50nM to 60nM, 55nM to 65nM, 60nM to 100nM, 65nM to 75nM, 70nM to 80nM, 75nM to 85nM, 80nM to 90nM, 85nM to 95nM, 90nM to 100nM, or 95nM to 105nM, the aPTT is prolonged by approximately 2-fold without doubling the PT.

[0145] The present disclosure includes anti-FXI antibodies that inhibit thrombin generation via the intrinsic pathway (endogenous thrombin) in human plasma in vitro, with little or no effect on thrombin generation 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 Examples 6 and 9. Here, thrombin generation profiles are generated and peak thrombin concentrations are determined in ellagic acid-treated plasma and tissue factor-treated plasma, with or without anti-FXI antibodies. Thus, in one embodiment, the anti-FXI antibody has 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 concentrations of 45 nM to 55 nM, 50 nM to 60 nM, 55 nM to 65 nM, 60 nM to 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, where the anti-FXI antibody does not affect exogenous thrombin production at any concentration up to 500 nM.

[0146] The present disclosure includes anti-FXI antibodies that increase activated partial thromboplastin time (aPTT) in primates in vivo by at least two-fold without measurably affecting prothrombin time (PT), wherein the primate is administered an anti-FXI antibody, plasma is obtained from the primate, the plasma is contacted with ellagic acid or tissue factor, and then the aPTT or PT, respectively, is determined in an assay as exemplified in Example 7. 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 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 prolongation effect persists in the 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 anti-FXI antibodies that inhibit intrinsic pathway peak thrombin activity in a primate in vivo without measurably affecting extrinsic pathway peak thrombin activity, wherein the primate is administered an anti-FXI antibody, 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 7. 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 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 is increased by 1% to 100%, 5% to 95%, 10% to 90%, 20% to 80%, 1% to 10%, 5% to 20%, 10% to 30%, 15% to 40%, 20% to 50%, 25% to 60%, 5% to 15%, 10% Inhibited by up 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 more than 100%.

[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 property of an antibody of the present disclosure (e.g., any of the binding properties referred to herein above) is disclosed in terms of "measured by surface plasmon resonance," it means that the relevant binding property for the interaction between the antibody and antigen is measured using a surface plasmon resonance instrument (e.g., a Biacore® instrument, GE Healthcare) using standard Biacore assay conditions exemplified in Example 3 herein, or a substantially similar assay format. In certain embodiments, the binding parameter is measured at 25°C; in other embodiments, the binding parameter is 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 comprising an amino acid sequence selected from any of the HCVR and / or LCVR amino acid sequences listed in Tables 1A-1C.

[0156] The antibodies of the present disclosure may have one or more of the aforementioned biological properties, or any combination thereof. The foregoing list of biological properties of the antibodies of the present disclosure is not intended to be comprehensive. Other biological properties 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 the 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 the 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 routine cross-blocking assays, as described, for example, in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. Additionally, 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 amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange, as detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing 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, revealing deuterium-labeled residues corresponding 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 Tables 1A-1C 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 Tables 1A-1C herein).

[0160] Whether an antibody binds to the same epitope as a reference anti-FXI antibody or competes for binding with a reference anti-FXI antibody can be readily 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 an FXI protein. The ability of the test antibody to bind to an FXI molecule 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 from the reference anti-FXI antibody. On the other hand, if the test antibody cannot bind to an FXI molecule after saturation binding with the reference anti-FXI antibody, the test antibody may bind to the same epitope as 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. These types of experiments 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-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured, for example, 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 also reduce or eliminate binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also 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 above-described binding method is performed in two orientations. In the first orientation, the reference antibody is allowed to bind to FXI protein under saturating conditions, followed by assessment of the binding of the test antibody to the FXI molecule. In the second orientation, the test antibody is allowed to bind to FXI protein under saturating conditions, followed by assessment of the binding of the reference antibody to the FXI molecule. In both orientations, if only the first (saturating) antibody can bind to the FXI molecule, it is concluded that the test and reference antibodies compete for binding to FXI. (See, for example, the assay format described in the Examples herein, in which FXI protein is captured on a sensor chip and the FXI-coated sensor chip is treated with the reference and test anti-FXI antibodies sequentially and in both binding orders.) As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily need to bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0162] Preparation of human antibodies The anti-FXI antibodies of the present disclosure may be fully human, but non-naturally occurring antibodies. 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., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against an allergen having a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves generating 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 antibodies 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 and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0164] Generally, VELOCIMMUNE® mice are administered an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to the desired heavy and light chain isotype constant regions. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding antigen-specific chimeric antibodies or the light and heavy chain variable domains 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 murine constant regions are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The murine 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 properties 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 antibody can bind only to human FXI but 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 on either a solid or solution phase, typically on the order of 10 -12 ~about 10 -9 K of M D It has.

[0168] bioequivalence 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 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 anti-FXI antibodies of the present disclosure contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequence, 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 bioequivalents if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that exhibit no significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under similar experimental conditions. Some antibodies are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their rate is not; however, they may be considered bioequivalent because such differences in absorption rate are intentional, reflected in the labeling, and are not considered essential, for example, to achieving effective body drug concentrations during chronic use and are not considered medically significant for the particular pharmaceutical product 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 between the reference product and the biological product one or more times without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or reduced efficacy, compared to continued treatment without switching.

[0172] In one embodiment, two antigen binding proteins are bioequivalent if they both operate 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. Measures of bioequivalence include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolites as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro tests that correlate with and are reasonably predictive of in vivo bioavailability data in humans, (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) 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 by 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 unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other contexts, biologically equivalent antibodies can include variants of anti-FXI antibodies that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0175] Species selectivity and species cross-reactivity According to certain embodiments, the present disclosure provides 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, in some cases, 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. According to certain exemplary embodiments of the present disclosure, anti-FXI antibodies are provided that specifically bind to human FXI but do not bind, or only weakly bind, to mouse or rat FXI.

[0176] multispecific antibodies Antibodies of the present disclosure may be monospecific or multispecific (e.g., bispecific). Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. Anti-FXI antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be operatively linked (e.g., by chemical bonding, genetic fusion, noncovalent bonding, or other means) 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 can comprise any of the HCVR / LCVR or CDR amino acid sequences shown in Tables 1A-1C herein.

[0178] An exemplary bispecific antibody format that can be used in the context of the present disclosure is H 3 domain and second Ig C H The first and second Ig C domains involve the use of 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 to protein A and the second Ig C H The 3 domain contains a mutation that reduces or eliminates binding to Protein A, such as the H95R modification (according to IMGT exon numbering, H435R according to EU numbering). H 3 may further comprise a Y96F modification (Y436F by EU, by IMGT). H Additional 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 by EU by IMGT), N44S, K52N and V82I for IgG2 antibodies (N384S, K392N and V422I by IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU by IMGT). Variations on the above bispecific antibody formats are contemplated as being within the scope of the present disclosure.

[0179] Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats are included (for a review of the aforementioned 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: December 4, 2012]).

[0180] Therapeutic Formulations and Administration The present disclosure provides pharmaceutical compositions comprising the anti-FXI antibodies or antigen-binding fragments thereof of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide for improved transport, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion 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 an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated based on body weight or body surface area. For adult patients, it may be advantageous to administer the antibody of the present disclosure intravenously, typically 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. The frequency and duration of treatment may be adjusted depending on the severity of the condition. Effective dosages and schedules for administering anti-FXI antibodies may be determined empirically, for example, by monitoring the patient's progress through periodic evaluations and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0182] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, 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, or by absorption through epithelial or mucocutaneous linings (e.g., 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 is easily used to deliver the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0184] Numerous reusable pen and autoinjector delivery devices have utility in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX75 / 25™ pen, the HUMALOG™ pen, the HUMALIN70 / 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 IV ... Examples of disposable pen delivery devices that have utility for subcutaneous delivery of the 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 auto-injector (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, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in 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] Injectable preparations may include dosage forms for intravenous injection, intravitreal injection, intraocular injection, subcutaneous injection, intradermal injection, and intramuscular injection, infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injections include, for example, saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections prepared in this manner are preferably filled into appropriate ampoules.

[0187] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into a suitable unit dosage form to accommodate the dose of the active ingredient. Such unit 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 dosage form. In particular, injections contain about 5 to about 100 mg of the antibody, and other dosage forms preferably contain about 10 to about 250 mg.

[0188] Diagnostic Uses of Antibodies The present 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 Tables 1A-1C herein). The therapeutic composition can comprise any one or more of the anti-FXI antibodies or antigen-binding fragments thereof disclosed herein and a pharmaceutically 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 can be used to treat or prevent thrombosis, particularly intrinsic pathway thrombosis, 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 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 prevent clotting (e.g., antithrombin, protein C, and protein S), elevated homocysteine ​​levels, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), abnormal fibrinolytic systems including elevated levels of Factor VIII, Factor IX, and / or XI, plasminogen deficiency, 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, oral contraceptives (birth control pills), and the like. the use of estrogen supplements, including estrogen-releasing hormone (estrogen-releasing hormone), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other diseases leading to decreased activity, heparin-induced thrombocytopenia (a decrease in 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 immunization.

[0192] In the context of the methods of treatment described herein, 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 conditions such as Factor V Leiden, prothrombin gene mutations, deficiencies of natural proteins that prevent clotting (e.g., antithrombin, protein C, and protein S), elevated homocysteine ​​levels, elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia), abnormal fibrinolytic systems including elevated levels of Factor VIII, Factor IX, and / or XI, plasminogen deficiency, 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, oral contraceptives (birth control pills), and the like. It may be co-formulated with one or more drugs used to treat conditions such as supplemental use of estrogen, including estrogen-releasing hormone (estrogen-releasing hormone), hormone replacement therapy, prolonged bed rest or immobility, heart attack, congestive heart failure, stroke and other conditions leading to decreased activity, heparin-induced thrombocytopenia (a decrease in 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 effects.

[0195] The anti-FXI antibodies of the present disclosure may also be administered in combination with 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), for example, any of the above-listed agents or derivatives thereof, may be administered before, concurrently with, or after administration of the anti-FXI antibody of the present disclosure (for purposes of this disclosure, such administration regimens will be considered to be administration of the anti-FXI antibody "in combination" with the additional therapeutically active ingredient(s). 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 ingredients mentioned herein) may be administered to a subject over a predetermined period of time. The method according to this aspect of the present disclosure comprises 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, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure includes methods comprising 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 an 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 second dose. The initial, secondary, and tertiary doses may all contain the same amount of anti-FXI antibody but generally may differ from one another with respect to administration frequency. However, in certain embodiments, the amount of anti-FXI antibody contained in the initial, secondary, and / or tertiary doses differ from one another during the course of treatment (e.g., adjusted up or down as needed). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" 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 of the secondary and / or tertiary doses is administered within 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 The term "immediately preceding dose" as used herein refers to a dose of anti-FXI antibody administered to a patient prior to administration of a subsequent dose, with no intervening doses, in a multiple dose series.

[0200] Methods according to this aspect of the disclosure can include administering any number of secondary and / or tertiary doses of 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 can be carried out for the lifetime 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 with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the individual patient's needs 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 less frequently to the patient. For example, according to this aspect of the disclosure, if the loading dose is administered monthly, the 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 can also be used to detect and / or measure FXI or FXI-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-FXI antibody or a fragment thereof can 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 can 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 can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S or 125 The FXI may be a radioisotope such as I, 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 assays according to the present disclosure include any tissue or body fluid sample obtainable from a patient that contains a detectable amount of FXI protein or a fragment thereof under normal or pathological conditions. Generally, FXI levels are measured 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) 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. [Example]

[0205] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present disclosure, 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 specified, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

[0206] Example 1: Generation of human antibodies against the CAT domain of FXI Human antibodies against the CAT 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. The antibody immune response was monitored by an FXI-specific immunoassay. 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. The hybridoma cell lines were screened and selected to identify cell lines producing FXI-specific antibodies.

[0207] Anti-FXI antibodies were isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Patent No. 7,582,298, which is 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) were obtained.

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

[0209] Example 2: Heavy and light chain region sequences Table 1A shows the amino acid sequence identifiers for the heavy and light chain regions of exemplary anti-FXI antibodies of the disclosure. Tables 1B and 1C show the nucleic acid (DNA) and amino acid (PEP) sequence identifiers for the heavy and light chain regions of antibodies of the disclosure. [Table 1A] [Table 1B] [Table 1C]

[0210] 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 understood by those skilled in the art, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a murine IgG1 Fc can be converted to an antibody with a human IgG4 Fc, etc.), but in either case, the variable domains (including the CDRs) will remain the same and the antigen binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0211] Example 3: Biacore binding kinetics of anti-FXI monoclonal antibodies binding to different FXI reagents measured at 25°C and 37°C The purpose of this experiment was to determine the kinetics and specificity of hFXI(ERL), hFXIa(ERL), hFXI.mmh (REGN3848), and mfFXI.mmh (REGN3883) binding to anti-FXI mAb and a comparative mAb at 25° C. and 37° C. The comparative mAb (REGN6166 or COMP6166) is described, for example, in U.S. Patent No. 10,465,011.

[0212] material Equipment used: Biacore 8k and T200-RED Temperature: 25℃ and 37℃ Running buffer: HBS-P and 300 mM NaCl, pH 7.4 Sensor type: Anti-human Fc mAb (REGN2567) Flow rate / time: 30 μl / min hFXI binding - 180 seconds hFXI dissociation 600 seconds

[0213] method Approximately 27.1–41.9 RU of anti-FXI mAb was captured by anti-hFc mAb (REGN2567) on a CM5 sensor surface. Next, 30 nM stock solutions of hFXI(ERL), hFXIa(ERL), hFXI.mmh (REGN3848), and mfFXI.mmh (REGN3883) were prepared and serially diluted 3-fold to create 10 nM, 3.3 nM, and 1.1 nM solutions. All FXI solutions were then injected into a Biacore 8K at 30 μL / min for 180 s, and dissociation was monitored for 10 min.

[0214] result [Table 2]

[0215] At 25°C and 37°C, anti-FXI mAbs have K values ​​of 4.54 pM to 119 pM and 3.09 pM to 36.5 pM, respectively. D It bound to human FXI(ERL) at 1000kJ / mL. [Table 3]

[0216] At 25°C and 37°C, the anti-FXI mAb had K of 31.1 pM and 25.6 pM, respectively. D It bound to human FXIa (ERL) at 1000kJ / mL. [Table 4]

[0217] At 25°C and 37°C, anti-FXI mAbs have K values ​​of 5.34 pM to 116 pM and 32.6 pM to 387 pM, respectively. D It bound to human FXI.mmh (REGN3848) at 1000kJ / mL. [Table 5]

[0218] At 25°C and 37°C, anti-FXI mAbs have K values ​​of 4.81 pM-64.4 pM and 43.5 pM-268 pM, respectively. D The values ​​were measured by binding to monkey FXI.mmh (REGN3883).

[0219] Example 4: Activated Partial Thromboplastin Time Bioassay Testing Procedure: The BIOPHEN Factor XIa kit (HYPHEN BioMed, Neuville-sur-Oise, FR, catalog number 220412) was used to assess the ability of the disclosed anti-FXI antibodies to inhibit the activity of zymogen factor FXI (FXI) or pre-activated FXIa, leading to the generation of active factor Xa (FXa). Inhibition by the disclosed antibodies was determined by measuring the reduction in the amount of chromogenic substrate converted by FXa (BIOPHEN kit component R3). All reagents from the BIOPHEN kit were used in the assay, except for Reagent 1B (human factor IX) and the FXIa calibrator (Cal).

[0220] To test the 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 the 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 with buffer-only samples from a starting concentration of either 500 nM or 300 nM to a concentration of 5.1 pM. For inhibition of zymogen FXI, anti-FXI antibodies were preincubated with diluted plasma for 30 minutes at 25°C, followed by an additional 30-minute 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, followed by incubation with anti-FXI antibody for 30 min at 25°C.

[0221] After incubating the plasma with ellagic acid and antibody, Reagent 1A (human FXa, FVIII:C, fibrin polymerization inhibitor) was added and incubated at 37°C for 5 minutes. Reagent 2 (thrombin, phospholipids, and calcium) was then added and incubated at 37°C for 5 minutes. Finally, Reagent 3 (SXa-11 FXa substrate) was added and incubated at 37°C for 30 minutes. 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 (four-parameter logistic) with Prism® 6 software (GraphPad, La Jolla, CA) to determine EC 50 and IC 50 The values ​​were obtained. The percent inhibition was calculated based on the following equation 2:

number

[0222] Tabular data overview: [Table 6]

[0223] Summary of results and conclusions: As shown in Table 6, anti-FXI / FXIa antibodies inhibited FXI in diluted normal plasma with an IC50 value of 190 pM and a maximum inhibition range of 87%. Anti-FXI / FXIa antibodies of the present disclosure also inhibited FXIa in diluted plasma with an IC50 value of greater than 10 nM and a maximum inhibition range of 35%. Comparative mAbs inhibited FXI with an IC50 value of 38 pM and a maximum inhibition of 108%. Comparative mAbs also inhibited FXIa with an IC50 value of 480 pM and a maximum inhibition of 95%. The isotype control mAb did not inhibit FXIa but did inhibit FXI at high antibody concentrations, with IC50 values ​​ranging from greater than 100 nM to 120 nM and maximum inhibition ranging from 58 to 102%.

[0224] Example 5: Complex formation 5.1: Size analysis of the complex formed between human coagulation factor XI and the target mAb [REGN7508] Experimental conditions Sample preparation A solution of anti-hFXI mAb and hFXI was prepared in an equimolar ratio and incubated at room temperature for 2 hours. After incubation, the complex solution was injected onto the column.

[0225] SEC-MALS conditions Samples were fractionated on a tandem Waters ACQUITY UPLC BEH® 200 SEC column (1.7 μm, 4.6 mm × 150 mm) pre-equilibrated with 10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0, at a flow rate of 0.3 mL / min.

[0226] Protein species eluting from the column were monitored by three in-line detectors: an absorbance detector (280 nm), a multi-angle light scattering (MALS) detector, and a refractive index detector.

[0227] Data analysis The molar masses of the free ligand and free drug samples were determined by protein complexation analysis. For complexed samples, the corrected dn / dc and UV values ​​determined from the protein complexation analysis of the free sample were used.

[0228] Data analysis was performed using Astra™ software version 7.3.1.9.

[0229] result Overall, as can be seen in Figure 1, REGN7528 and REGN7531 showed the clearest evidence of widespread "paper dolling."

[0230] In comparison, as seen in Figure 2, REGN7503 and REGN7505 appeared to form smaller, less heterogeneous complexes, and only the control mAb [REGN7508] showed the presence of a 1:1 complex with FXI. [Table 7]

[0231] Consideration SEC-MALS was used to rapidly assess the relative size distribution of complexes formed between human coagulation factor XI (hFXI) and a panel of anti-hFXI mAbs to eliminate mAbs exhibiting extensive “paper doll ring” and provide additional characterization to support time-constrained decisions.

[0232] Although SEC-MALS analysis was chosen for convenience, the limited resolution range of the column prohibits detailed interpretation of the stoichiometry, and only qualitative comparisons can be made from this data.

[0233] Overall, among the panel of mAbs, REGN7528 (catalytic domain) and REGN7531 (A3 / A4 domain) showed the clearest evidence of extensive “paper doll ring.”

[0234] In comparison, REGN7503 (A3 / A4 domain) and REGN7505 (A3 / A4 domain) appeared to form a more uniform complex distribution with a smaller overall average molar mass, but the control mAb [REGN7508] (catalytic domain) was the only mAb in the panel that showed a significant 1:1 complex with FXI, as previously observed by A4F-MALS.

[0235] 5.2 Size analysis of the complex formed between human coagulation factor XI and the target mAb [REGN7508] Experimental conditions Sample preparation Samples were prepared in 1× DPBS, pH 7.4, and incubated at room temperature for 2 hours before total protein fractionation by A4F-MALLS (PostNova).

[0236] A4F-MALLS conditions 7 mg of conjugate or 4 mg of mAb or ligand was injected into an A4F short channel equipped with a 350 W spacer and a 10 kDa regenerated cellulose membrane using a mobile phase of 10 mM sodium phosphate, 500 mM NaCl, pH 7.0, and separated using a gradient as follows:

[0237] Flow rate: Channel flow: 1.0 mL / min Focused flow: 1.0 mL / min for 4 minutes. Crossflow: linear gradient from 3.0 mL / min to 0 mL / min over 45 min, followed by 10 min at 0 mL / min.

[0238] Data analysis The molar masses of the free ligand and free antibody samples were determined by protein complex analysis. For the complexed samples, the corrected dn / dc and UV values ​​determined from the protein complex analysis of the free sample were used.

[0239] result [Table 8] 1 Unequal ratios (such as 1:2 and 2:1) are indistinguishable as they have similar MW.

[0240] As seen in Table 8 (above) and Figure 3, the subject mAb [REGN7508] (catalytic domain) preferred lower order complexes with predominant species representing distinct 1:1 and 2:2 complexes with hFXI. [Table 9]

[0241] As seen in Table 9 (above) and Figure 4, [REGN9932] (catalytic domain) formed the highest proportion of low-order complexes with the predominant species representing distinct 1:1 and 2:2 complexes with hFXI.

[0242] As seen in Figure 5, the molar mass and distribution of the complex formed between the subject mAb [REGN7508] (catalytic domain) and hFXI were comparable to those previously observed with [REGN9932] (catalytic domain).

[0243] Consideration Asymmetric flow-field separation combined with multi-angle laser light scattering (A4F-MALLS) was used to assess the relative size distribution of complexes formed between human coagulation factor XI (hFXI, from Enzyme Research Laboratory) and several anti-hFXI mAbs (target mAbs [REGN7508] and [REGN9932]).

[0244] The target mAb [REGN7508] (catalytic domain) preferred lower order complexes with predominant species representing distinct 1:1 and 2:2 complexes with hFXI when mixed at various molar ratios.

[0245] The molar mass and size distribution observed for the complex formed between the subject mAb [REGN7508] (catalytic domain) and hFXI were comparable to those previously observed with [REGN9932] (catalytic domain).

[0246] Example 6: Thrombin Generation Assay (TGA) TGA was performed to measure the following endpoints: Delay time (minutes), clotting time Thrombin peak (nM) ttPeak: Time to peak (min) ·ETP: Endogenous thrombin potential (nM*min) (ETP; area under the curve) Speed: Peak (nM) / ttPeak (min)

[0247] 6.1: TGA profile in pooled human plasma result [Table 10]

[0248] 6.2: TGA profile in cynomolgus monkey plasma (female) A comparison of the effects of anti-FXI mAbs on intrinsic pathway thrombin generation in cynomolgus monkey plasma (female) is shown in Figures 6A, 6B, and 6C.

[0249] A comparison of the effects of anti-FXI mAbs on intrinsic pathway thrombin generation in cynomolgus monkey plasma (female) is shown in Figures 7A, 7B, and 7C.

[0250] 6.3: TGA profile in pooled female plasma A comparison of the effects of anti-FXI mAbs on intrinsic pathway thrombin generation in pooled female plasma is seen in Figures 8A, 8B, and 8C.

[0251] A comparison of the effects of anti-FXI mAbs on intrinsic pathway thrombin generation in pooled female plasma is seen in Figures 9A, 9B, and 9C.

[0252] 6.4: Comparison of anti-FXI / FXIa mAbs for thrombin generation in six single donors A comparison of the effect of anti-FXI mAb [REGN9932] on intrinsic pathway thrombin generation in six single donors is seen in Figures 10A, 10B, and 10C.

[0253] A comparison of the effect of the subject anti-FXI mAb [REGN7508] on intrinsic pathway thrombin generation in six single donors is seen in Figures 11A, 11B, and 11C.

[0254] A comparison of the effect of anti-FXI mAb [REGN9932] on extrinsic pathway thrombin generation in six single donors is seen in Figures 12A, 12B, and 12C.

[0255] A comparison of the effect of the subject anti-FXI mAb [REGN7508] on extrinsic pathway thrombin generation in six single donors is seen in Figures 13A, 13B, and 13C.

[0256] Example 7: Pharmacokinetic study of the subject anti-FXI antibody drug substance in cynomolgus monkeys The objective of this study was to determine the intravenous single-dose pharmacodynamic / pharmacokinetic (PK / PD) parameters of an anti-FXI monoclonal antibody (mAb) in cynomolgus monkeys over an 8-week period.

[0257] Plasma will be collected pre-dose, 5 minutes, 6 hours, and on days 1, 2, 3, 5, 7, 14, 21, 28, 35, 42, 49, and 56.

[0258] The endpoints measured were: 1) Target level (total) 2) hFc level (total) 3) aPTT / PT 4) TGA-EA / TF 5) Modified FXIa Activity Assay (Biophen) 6) CBC (starting at 72 hours) [Table 11]

[0259] Example 8: Determination of kinetic and equilibrium binding parameters for the interaction of the subject anti-FXI antibody drug substance with FXI from human, cynomolgus monkey, rabbit, mouse, and human FXIa 8.1: Experimental design Surface plasmon resonance (SPR) experiments were performed on a Biacore instrument to determine the binding affinity of REGN7508 to plasma-derived human FXI and FXIa proteins, as well as recombinant mmH-tagged forms of human, cynomolgus monkey, rabbit, and mouse FXI proteins. Various concentrations of plasma-derived and recombinant FXI or FXIa proteins were injected over REGN7508 captured on the sensor surface at pH 7.4 and 25°C (all FXI and FXIa proteins) or 37°C (plasma-derived human FXI / FXIa only), followed by a dissociation phase. The change in binding signal was recorded, and specific binding signals were calculated. Kinetic binding parameters were determined by fitting the data to a 1:1 binding model with mass transport limitations.

[0260] Covalent binding of anti-human FcG antibody to the sensor chip surface Mouse anti-human FcG monoclonal antibody (anti-hFcG) was immobilized on the surface of a sensor chip using standard amine coupling chemistry. The coupling procedure was performed at a flow rate of 10 μL / min using filtered and degassed HBS-P (10 mM HEPES, 300 mM NaCl, 0.05% (v / v) polysorbate 20, pH 7.4) as the running buffer. The sensor surface was activated by injecting a 1:1 (volume ratio) mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 M N-hydroxysuccinimide over the chip for 7 minutes. After surface activation, anti-hFcG (20 μg / mL) prepared in 10 mM sodium acetate (pH 5.0) was injected over the activated chip surface for 7 minutes. Remaining active groups on the sensor chip surface were blocked by injecting 1 M ethanolamine for 7 minutes until a final surface density of approximately 1,900 resonance units (RU) was reached. The sensor chip surface was then treated with at least 10 injections of 20 mM phosphoric acid for 12 s each to remove any residual unbound protein and washed with running buffer HBS-P before performing the kinetic binding experiments.

[0261] Kinetic binding interactions of REGN7508 with FXI and FXIa proteins Binding of FXI and FXIa proteins to REGN7508 (Lot No. 9048800001 [all FXI / FXIa proteins] and Lot No. REGN7508-L2 [human FXI / FXIa only]) was measured using HBS-P as the running buffer at pH 7.4 and 25°C or 37°C. REGN7508 was captured by surface-bound anti-hFcG until a signal of 59-101 RU was reached. Plasma-derived and recombinant FXI and FXIa proteins were serially diluted two-fold over the concentration ranges of 0.781 nM to 25.0 nM (hFXI, hFXIa, hFXI.mmH, or MfFXI.mmH), 7.81 nM to 250 nM (rbFXI.mmH), and 1.56 nM to 50 nM (mFXI.mmH) and injected individually over the captured REGN7508 surface at a flow rate of 50 μL / min for 1 min (25°C) or 0.5 min (37°C). After a 20-min dissociation phase, the resulting change in binding signal was recorded. Each concentration was tested in duplicate.

[0262] Specific binding signals were obtained by a double-referencing procedure and plotted as SPR sensorgrams. Double-referencing was performed by first subtracting the signal of each injection on the reference surface (anti-hFcG) from the signal on the experimental surface (REGN7508 captured by anti-hFcG), thereby eliminating the contribution from refractive index changes. Additionally, injections of running buffer were performed to allow for subtraction of signal changes resulting from dissociation of captured REGN7508 from the bound anti-hFcG surface. Kinetic parameters were obtained by globally fitting these specific binding signals to a 1:1 binding model with mass transport limitation. The equilibrium dissociation constant (KD) was calculated from the ratio of the dissociation rate constant to the association rate constant (KD = kD / ka). The dissociation half-life (t1 / 2) was calculated by dividing 0.693 (the natural logarithm of 2) by the experimentally determined kD.

[0263] 8.2: Results Binding parameters of the interaction of REGN7508 with FXI and FXIa Kinetic binding parameters for the interaction of REGN7508 with FXI proteins from human, cynomolgus monkey, rabbit, and mouse, as well as human-derived FXIa, were determined using SPR technology. Human FXI (E19-V625) shares 96%, 86%, and 79% amino acid sequence identity with cynomolgus monkey, rabbit, and mouse FXI, respectively. These assays used injections of various concentrations of FXI or FXIa protein over a REGN7508 sensor surface captured at 25°C and pH 7.4. The calculated kinetic binding parameters are summarized in Table 12.

[0264] In SPR experiments performed at 25°C and pH 7.4, REGN7508 bound with picomolar affinity to plasma-derived hFXI and hFXIa, as well as recombinant human FXI (hFXI.mmH).

[0265] REGN7508 also bound to recombinant cynomolgus FXI (MfFXI.mmH) with picomolar affinity. REGN7508 did not bind to recombinant rabbit (rbFXI.mmH) or mouse (mFXI.mmH) proteins up to the highest concentrations tested (250 nM or 50 nM, respectively), demonstrating binding specificity for human and cynomolgus FXI.

[0266] Kinetic binding parameters for the interaction of different lots of REGN7508 with human FXI and FXIa were also determined. REGN7508 (Lot No. 9048800001) used in the toxicology studies described above and REGN7508 (Lot No. REGN7508-L2) used in several nonclinical in vitro and in vivo pharmacology studies exhibited similar binding affinities for human FXI and FXIa. [Table 12]

[0267] The equilibrium dissociation constants (KD) of REGN7508 for plasma-derived human FXI and FXIa were 3.00 and 10.2 pM, respectively. REGN7508 also bound to recombinant human (hFXI.mmH) and cynomolgus monkey (Macaca fascicularis) (MfFXI.mmH) FXI with KD values ​​of 8.07 and 3.73 pM, respectively, but showed no detectable binding to recombinant rabbit (rbFXI.mmH) or mouse (mFXI.mmH) FXI proteins up to the highest concentrations tested (250 nM and 50 nM, respectively).

[0268] Example 9: In vitro functional characterization of REGN7508 9.1: Experimental design The ability of REGN7508 to inhibit coagulation pathways in human and cynomolgus monkey donor plasma was evaluated in vitro using clotting assays and TGA. The effect on the intrinsic coagulation pathway was measured based on aPTT and EA-induced thrombin generation, and the effect on the extrinsic coagulation pathway was measured by PT and TF-induced thrombin generation (Figure 14).

[0269] The aPTT test assesses all clotting factors in the intrinsic and common pathways of the coagulation cascade by measuring the time it takes for a clot to form after the addition of calcium and EA, while the PT test assesses all clotting factors in the extrinsic and common pathways of the coagulation cascade after the addition of calcium and TF.

[0270] EA-induced TGA measures the rate and amount of thrombin generated via the intrinsic and common pathways, while TF-induced TGA measures the rate and amount of thrombin generated via the extrinsic and common pathways.

[0271] aPTT assay (for intrinsic pathway activity) aPTT in the presence of REGN7508 or IgG4P isotype control in either human or cynomolgus monkey donor plasma was measured using a STart4 hemostasis analyzer. Plasma samples (50 μL) were added to a STart® cuvette and incubated with two-fold serial dilutions of either REGN7508 (9 nM to 1.2 μM) or IgG4P isotype control (19 nM to 1.2 μM) for 5 minutes at 37°C. A no-antibody control cuvette containing PBS instead of antibody was also included for baseline measurements. A second set of REGN7508 concentrations was also tested, in 4 nM increments, from 4 nM to 28 nM and 31 nM. 50 μL of aPTT-XL EA was added and incubated for 5 minutes, after which the reaction was initiated by adding 50 μL of 20 mM calcium chloride. The measured clotting time of each test sample was normalized to the plasma clotting time of the no-antibody control. The mean change relative to the no antibody control for each concentration (performed in duplicate) was plotted against antibody concentration.

[0272] The concentration at which the aPTT doubles (doubling time), C2xt, was determined in a second set of experiments in which a range of antibody concentrations (4 nM to 31 nM) was tested in smaller increments of 4 nM. For aPTT clotting times, values ​​in seconds were generated relative to baseline, i.e., the no-antibody control or PBS-only value, corresponding to a 1.0-fold change in aPTT. C2xt was estimated at the intersection of the "doubling time line" and the aPTT curve. The doubling time line was positioned at twice the baseline value in seconds, corresponding to a 2.0-fold change in aPTT for mAb values ​​relative to baseline in GraphPad Prism.

[0273] PT assay (for extrinsic pathway activity) PT in the presence of REGN7508 or IgG4P isotype control in either human or cynomolgus monkey donor plasma was measured using a STart4 hemostasis analyzer. Plasma samples (50 μL) were added to a STart® cuvette and incubated with either REGN7508 or IgG4P isotype control (600 nM and 1.2 μM) for 5 minutes at 37°C. A no-antibody control plasma sample containing PBS instead of antibody was also included for baseline measurements. The reaction was initiated by adding TriniCLOT PT Excel S (TF and calcium, 100 μL). The measured clotting time of each test sample was normalized to the plasma clotting time of the no-antibody control. The mean change relative to the no-antibody control for each concentration (performed in duplicate) was plotted against antibody concentration.

[0274] Thrombin generation assay The thrombin generation profile of REGN7508 or IgG4P isotype control in either human or cynomolgus monkey donor plasma was determined using the Calibrated Automated Thrombogram® platform. Thrombin activity was measured by monitoring the cleavage of a fluorescent substrate and comparing it to a known thrombin activity in a nonclotting sample evaluated in parallel. Plasma samples (55 μL) were added to wells of an Immulon II HB U-bottom microplate and incubated for 30 minutes at 37°C with two-fold serial dilutions of REGN7508 or IgG4P isotype control ranging from 16 nM to 500 nM. Antibody-free control wells containing PBS instead of antibody were also included for baseline measurements. A second set of REGN7508 concentrations was also tested to test intrinsic pathway activity alone, from 4 nM to 31 nM in 4 nM increments, and the IgG4P isotype control was tested at 31 nM.

[0275] Thrombin generation was then induced by adding either 15 μL of aPTT-XL EA (intrinsic pathway activity) or 15 μL of PPP reagent low TF (extrinsic pathway activity) prediluted in MP reagent. After 45 min of incubation at 37°C, 15 μL of Fluo substrate prewarmed in FluCa buffer was added to the wells immediately prior to continuous readings over 90 min in an Immulon II HB U-bottom microplate. The measured real-time thrombin concentration values ​​recorded over the first 60 min were plotted against time, yielding a thrombogram profile for each antibody concentration tested (Figure 15). From each thrombogram, the lag time, peak thrombin, and intrinsic thrombin potential were determined.

[0276] 9.2:Results 9.2.1: Effect of REGN7508 in Coagulation Assays Using Human or Cynomolgus Monkey Donor Plasma Clotting assay using human plasma REGN7508 increased aPTT relative to baseline (no antibody) in a concentration-dependent manner, with a maximum 3.8-fold increase in human plasma at concentrations of 9 nM to 1.2 μM ( FIG. 16A and Table 13 ) and at concentrations of 4 nM to 31 nM tested in a second set of experiments ( FIG. 16B and Table 14 ). In a second set of experiments, a range of antibody concentrations in smaller increments (4 nM to 31 nM) was tested, and a 2-fold increase in aPTT relative to baseline was estimated to occur at 16 nM in human plasma. At the highest antibody concentration tested (1.2 μM), no change in PT relative to baseline was observed in human ( FIG. 16C and Table 13 ) plasma. Up to the highest antibody concentration tested (1.2 μM), no change in either aPTT or PT relative to baseline was observed for the IgG4P isotype control in human plasma. [Table 13] [Table 14]

[0277] Coagulation assay using cynomolgus monkey plasma REGN7508 increased aPTT relative to baseline (no antibody) in a concentration-dependent manner, with a maximum 2.8-fold increase in cynomolgus monkey plasma at concentrations of 9 nM to 1.2 μM ( FIG. 17A and Table 15 ) and at concentrations of 4 nM to 31 nM tested in a second set of experiments ( FIG. 17B and Table 16 ). In the second set of experiments, using an antibody concentration range with smaller increments (4 nM to 31 nM), a 2-fold increase in aPTT relative to baseline was estimated to occur at 14.8 nM in cynomolgus monkey plasma. At the highest antibody concentration tested (1.2 μM), no change in PT relative to baseline was observed in the plasma of cynomolgus monkeys ( FIG. 17C and Table 15 ). Up to the highest antibody concentration tested (1.2 μM), no change in either aPTT or PT relative to baseline was observed for the IgG4P isotype control in cynomolgus monkey plasma. [Table 15] [Table 16]

[0278] 9.2.2 Efficacy of REGN7508 in TGA Using Human or Cynomolgus Monkey Donor Plasma TGA using human plasma When thrombin generation was induced by EA via the intrinsic pathway in human plasma, REGN7508 increased the thrombin generation lag time by up to 6.6-fold relative to baseline (i.e., no antibody), reduced peak thrombin levels to 1% of baseline, and reduced endogenous thrombin capacity to 2% of baseline. REGN7508 exerted these effects in a concentration-dependent manner, with maximal effects achieved at concentrations of ≥125 nM (Figure 18A and Table 17). With the IgG4P isotype control, no increase in thrombin generation lag time was observed up to the highest antibody concentration tested (500 nM), and only slight decreases in peak thrombin levels and endogenous thrombin capacity relative to baseline were observed; these effects were not concentration-dependent (Figure 18B and Table 17).

[0279] A second set of experiments was performed with REGN7508 concentrations ranging from 4 nM to 31 nM, testing in smaller increments to facilitate greater resolution of the thrombin peak at concentrations below 16 nM, but no gradual dose-response was observed. At REGN7508 concentrations above 16 nM, a sharp change in the PD effect followed by a plateau was observed. Furthermore, a similar concentration-dependent effect on EA-induced thrombin generation was observed under otherwise identical assay conditions (Figure 19 and Table 18).

[0280] When thrombin generation was induced by TF via the extrinsic pathway in human plasma, REGN7508 partially reduced peak thrombin levels and endogenous thrombin capacity to 66% and 68% of baseline, respectively. REGN7508 exerted these effects in a concentration-dependent manner, with maximal effects achieved at concentrations of ≥ 125 nM. With REGN7508, no concentration-dependent increase in the lag time of thrombin generation was observed up to the highest antibody concentration tested (500 nM) (Figure 18C and Table 19). With the IgG4P isotype control, no increase in the lag time of thrombin generation was observed up to the highest antibody concentration tested (500 nM), and no decrease in peak thrombin or endogenous thrombin capacity was observed (Figure 18D and Table 19). [Table 17] [Table 18] [Table 19]

[0281] TGA using cynomolgus monkey plasma When thrombin generation was induced by EA via the intrinsic pathway in cynomolgus monkey plasma, REGN7508 increased the thrombin generation lag time by up to 2.8-fold relative to baseline (i.e., no antibody), reduced peak thrombin levels to 6% of baseline, and reduced endogenous thrombin capacity to 15% of baseline. REGN7508 exerted these effects in a concentration-dependent manner, with maximal effects achieved at concentrations of ≥ 500 nM (Figure 20A and Table 20). With the IgG4P isotype control, no increase in thrombin generation lag time was observed, and no decrease in peak thrombin or endogenous thrombin capacity was observed up to the highest antibody concentration tested (500 nM) (Figure 20B and Table 20).

[0282] A second set of experiments was performed with REGN7508 concentrations ranging from 4 nM to 31 nM, testing in smaller increments to facilitate greater resolution of the thrombin curve at concentrations below 16 nM; however, a gradual dose-response was not observed, as there was a rapid effect once target saturation was achieved. Furthermore, a similar concentration-dependent effect on EA-induced thrombin generation was observed under otherwise identical assay conditions (Figure 21 and Table 21).

[0283] When thrombin generation was induced by TF via the extrinsic pathway in cynomolgus monkey plasma, REGN7508 reduced peak thrombin levels to 55% of baseline and endogenous thrombin capacity to 65% of baseline. REGN7508 exerted these effects in a concentration-dependent manner, with maximal effects achieved at concentrations of ≥125 nM (Figure 20C and Table 22). With REGN7508, no concentration-dependent increase in the lag time of thrombin generation was observed up to the maximum antibody concentration tested (500 nM). With the IgG4P isotype control, no increase in the lag time of thrombin generation was observed, and no decrease in peak thrombin or endogenous thrombin capacity was observed up to the maximum antibody concentration tested (500 nM) (Figure 20D and Table 22). [Table 20] [Table 21] [Table 22]

[0284] conclusion REGN7508 mediated complete blockade of the intrinsic coagulation pathway in human and cynomolgus monkey plasma in a concentration-dependent manner. REGN7508 also mediated partial blockade of the extrinsic coagulation pathway in human and cynomolgus monkey plasma in a concentration-dependent manner, but to a much lesser extent than its effect on the intrinsic pathway.

[0285] Example 10: Evaluation of REGN7508-FXI 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 lead to tissue damage at the site of deposition. Large immune complexes can also activate the complement component C1q in serum (Rojko, 2014).

[0286] REGN7508 contains a hinge-stabilized IgG4-derived heavy chain fragment crystallizable (Fc) constant domain (termed IgG4P), and because IgG4 does not bind C1q as well as IgG1, it is unlikely to form immune complexes capable of binding C1q (Patel, 2015). Nevertheless, enzyme-linked immunosorbent assays (EIA) were performed to assess the potential for C1q binding of REGN7508-FXI and REGN7508-FXIa complexes.

[0287] REGN7508-FXI and REGN7508-FXIa complexes showed no detectable binding to C1q, consistent with minimal effector function activity of IgG4-based antibodies.

[0288] Example 11: Pharmacokinetic and toxicokinetic evaluation of REGN7508 in cynomolgus monkeys Characterization of REGN7508-mediated blockade of the coagulation pathway in cynomolgus monkeys was assessed as part of a single-dose pharmacokinetic (PK) study. The results of these studies demonstrate that REGN7508 mediated inhibition of the intrinsic coagulation pathway with minimal effects on the extrinsic coagulation pathway. The results of these studies further demonstrate that there were no tolerability issues following SC and IV administration of REGN7508. [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 23-5] [Table 23-6] [Table 23-7] [Table 23-8] [Table 23-9]

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that binds to the catalytic (CAT) domain of coagulation factor XI (FXI), wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain (HC) comprising a heavy chain variable region (HCVR) comprising heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 sequences are the HCDR1, HCDR2, and HCDR3 sequences in SEQ ID NO: 18; An isolated antibody or antigen-binding fragment thereof comprising: a light chain (LC) comprising a light chain variable region (LCVR) comprising light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the LCDR1, LCDR2, and LCDR3 sequences in SEQ ID NO:

20.

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

10.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the HC comprises an amino acid sequence comprising SEQ ID NO: 18 and the LC comprises an amino acid sequence comprising SEQ ID NO:

20.

4. An isolated antibody or antigen-binding fragment thereof that binds to the catalytic (CAT) domain of coagulation factor XI (FXI), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain region (HC) comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18, and a light chain region (LC) comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

20.

5. The isolated antibody or antigen-binding fragment thereof of claim 4, wherein the HC comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 18 and the LC comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

20.

6. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, wherein the HC comprises an amino acid sequence comprising SEQ ID NO: 18 and the LC comprises an amino acid sequence comprising SEQ ID NO:

20.

7. The antibody or antigen-binding fragment thereof has a K of less than about 5 pM as measured by surface plasmon resonance at 25° C. or 37° C. Ｄ 7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, which binds to human FXI at

8. The antibody or antigen-binding fragment thereof has a K selected from the group consisting of less than about 800 pM, less than about 500 pM, less than about 100 pM, or less than about 50 pM, as measured by surface plasmon resonance at 25° C. or 37° C. Ｄ 8. The isolated antibody or antigen-binding fragment of claim 7, which binds to human FXI at

9. 9. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 8, 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.

10. The antibody or antigen-binding fragment thereof activates human coagulation factor X (FX) with an IC of less than about 10 nM. ５０ 10. The isolated antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment inhibits

11. The antibody or antigen-binding fragment thereof activates human coagulation factor X (FX) with an IC of less than about 40 pM. ５０ The isolated antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody or antigen-binding fragment inhibits

12. 12. The isolated antibody or antigen-binding fragment of any one of claims 1 to 11, wherein the antibody or antigen-binding fragment thereof increases activated partial thromboplastin time (aPTT) by at least 2.5 fold.

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

14. 14. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 13, 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%.

15. 15. The isolated antibody or antigen-binding fragment of any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof prolongs aPTT in human plasma by at least 2-fold at a concentration of 100 nM or less, 75 nM or less, or 50 nM or less.

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

17. 17. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 16, 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.

18. An isolated antibody or antigen-binding fragment thereof that competes for binding with the antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

19. An isolated antibody or antigen-binding fragment thereof that binds to the same epitope as the antibody or antigen-binding fragment thereof of any one of claims 1 to 18.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier or diluent.

21. An isolated nucleic acid molecule comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 19.

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

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

24. 1. A method for inhibiting a biological activity mediated by FXI, said method comprising: A method comprising contacting FXI or FXIa with a biologically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the pharmaceutical composition of claim 20.

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

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

27. A method 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, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 19, or a pharmaceutical composition described in claim 20, to a subject in need of such treatment.

28. 28. The method of claim 27, 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.

29. 29. The method of claim 28, wherein the disease or disorder is atrial fibrillation.

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