Factor XI catalytic domain binding antibodies and methods of use thereof

Abstract

The present disclosure provides antibodies that bind to catalytic domains of Factor XI (FXI) and methods of use thereof. According to certain embodiments, the antibody is an antagonist antibody that inhibits blood clot formation achieved through an intrinsic pathway without affecting hemostasis, as shown by the effect of the antagonist antibody on extension of aPTT without affecting PT. As such, these antagonist antibodies can be used in therapeutic regimens for the treatment of blood clotting diseases or disorders or with clot formation as risk factors, such as, but not limited to, atrial fibrillation. In certain embodiments, the present disclosure comprises antibodies that bind to FXI and mediate clot formation or thrombosis. The antibodies of the present disclosure may be fully human, non-naturally occurring antibodies.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 423,272, filed on November 7, 2022. The entire content of the foregoing application is hereby expressly incorporated by reference. Technical Field

[0003] The present disclosure relates to antibodies that bind to the catalytic domain (CAT) of factor XI (FXI), compositions comprising such antibodies, and methods of using the same. Background Art

[0004] The formation of blood clots (i.e., thrombi) is initiated either by (a) the contact pathway or by (b) the extrinsic pathway. The two pathways converge via (c) the common pathway to activate thrombin, which acts as a serine protease to convert soluble fibrinogen into insoluble fibrin strands. Cross-linked fibrin is the major component of blood clots, as well as aggregated platelets and red blood cells.

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

[0006] The contact pathway differs from the extrinsic pathway in that the contact pathway is less involved in hemostasis but still affects clot formation. Here, coagulation is initiated by intrinsic events such as the release of polyphosphates from platelets or the extrusion of histone- and DNA-loaded neutrophil extracellular traps (NETs) from neutrophils, which activate 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 polyphosphates produced by platelets also activate FXI in a feed-forward manner to amplify clot formation.

[0007] FXI is the zymogen of the plasma protease FXIa, which maintains thrombin generation through activation by FIX. FXI is a 160 kDa disulfide-linked homodimer, where each subunit consists of apple domains A1-A4 and a catalytic domain (referred to herein as "CAT" or "CD") from the N-terminus to the C-terminus. The disulfide bond is located between the A4 domains of each subunit. The FXI subunit is activated by cleavage of one or both of the Arg-Ile bonds located between the A4 and CAT domains to form FXIa. It is generally believed that the cleavage of the Arg-Ile bond is catalyzed by FXIIa and / or thrombin. SUMMARY OF THE INVENTION

[0008] Provided herein are an isolated monoclonal antibody 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 antibody or antigen-binding fragment thereof may specifically bind to the CAT domain of FXI. The isolated antibodies and antigen-binding fragments of the present disclosure can be used to treat diseases and disorders associated with FXI activity or expression.

[0009] In its broadest aspects, the present disclosure provides anti-FXI antibodies that block FXI activity or activate and reduce blood clot formation. These antibodies can be used to prevent, treat, or reduce the incidence of blood clot formation in the bloodstream or tissues of patients in need or to reduce the negative effects of such blood clot formation. Preferably, the anti-FXI antibodies attenuate thrombus formation without disturbing hemostasis.

[0010] In certain embodiments, the anti-FXI antibodies can be used to treat various coagulation disorders or diseases, where treating the disease involves the use of anticoagulant therapy and where there is a risk of patient bleeding due to the use of anticoagulant therapy. Those indications, disorders, or diseases include high-risk atrial fibrillation, primary venous thromboembolism (VTE) prophylaxis, extended VTE treatment, prevention of recurrent ischemia after acute coronary syndrome, end-stage renal disease, medical devices (e.g., mechanical heart valves, ventricular assist devices, small-bore grafts, central venous catheters, etc.), extracorporeal circuits, and the like.

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

[0012] Exemplary anti-FXI antibodies of the present disclosure are listed in Tables 1A-1C herein. Tables 1A-1C list the amino acid sequences of the exemplary heavy chain regions (HC) and light chain regions (LC) of exemplary anti-FXI antibodies. In one embodiment, the HC includes a heavy chain variable region (HCVR), and the light chain includes a light chain variable region (LCVR).

[0013] The present disclosure provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain (HC), and the HC comprises an amino acid sequence selected from any of 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.

[0014] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or antigen-binding fragment thereof comprises a light chain (LC), and the LC comprises an amino acid sequence selected from any of 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.

[0015] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or antigen-binding fragment thereof comprises a pair of heavy chain variable region (HCVR) and light chain variable region (LCVR) amino acid sequences (HCVR / LCVR), which comprises any HCVR sequence of the HCVR sequences of the HC listed in Tables 1A - 1C paired with any LCVR amino acid sequence of the LCVR amino acid sequences of the LC listed in Tables 1A - 1C. According to certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof that comprises the HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-FXI antibodies listed in Tables 1A - 1C.

[0016] Thus, in a first aspect, the present disclosure provides an isolated antibody or an 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) contained within the heavy chain variable region (HCVR), the HCVR is contained within the heavy chain region (HC), and the HCVR comprises an amino acid sequence as listed 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) contained within the light chain variable region (LCVR), the LCVR is contained within the light chain region (LC), and the LCVR comprises an amino acid sequence as listed in Tables 1A - 1C or a substantially similar sequence having at least 90% sequence identity thereto.

[0017] In one embodiment, the anti-FXI antibody or antigen-binding fragment thereof exhibits one or more properties selected from the group consisting of:

[0018] (a) is an antagonist antibody;

[0019] (b) with a K of less than about 5 pM D Binding to human FXI as measured by surface plasmon resonance at 25°C or 37°C;

[0020] (c) with a K of less than about 300 pM D Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C;

[0021] (d) with a dissociation half-life (t) greater than about 1,000 minutes 1 / 2 ) binds to human FXI as measured by surface plasmon resonance at 25°C or 37°C;

[0022] (e) with a dissociation half-life (t 1 / 2 ) binds to human FXIa as measured by surface plasmon resonance at 25°C or 37°C;

[0023] (f) inhibits activation of factor Xa (FXa) by FXI in normal dilute plasma by at least about 85% to about 87% with an IC50 of less than about 39 pM to less than about 190 pM;

[0024] (g) inhibits activation of factor Xa (FXa) by FXIa in normal diluted plasma by at least about 25% to about 35% with an IC50 of at least about 10 nM;

[0025] (h) preferentially binds to the CAT domain (i.e., the catalytic domain) relative to full-length FXI or any of the FXI domains in the apple domain 2 (A2), PKA1, PKA3, or PKA4, as determined by label-free biolayer interferometry;

[0026] (i) competes for binding to FXI with an antibody that specifically binds to an epitope within and overlapping the CAT domain of FXI;

[0027] (j) increases the activated partial thromboplastin time (aPTT), which is a measure of the intrinsic clotting time, in vitro in a primate sample by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 3.8-fold or 4-fold, without measurably affecting the prothrombin time (PT), which is a measure of the extrinsic clotting time;

[0028] (k) inhibits 1%-6% of intrinsic pathway peak thrombin activity in primate samples in vitro;

[0029] (l) prolongs the aPTT in human plasma by about two-fold in vitro at a concentration of about ≤ 33 nM without doubling the PT; and / or

[0030] (m) inhibits in vitro the generation of intrinsic pathway thrombin in human plasma at a concentration of about ≥20 nM, and has no effect on the generation of extrinsic pathway thrombin at a dose of up to about 500 nM.

[0031] In one embodiment, the present disclosure provides an antibody or an antigen-binding fragment thereof that binds to factor XI (FXI), wherein the antibody or the antigen-binding fragment thereof comprises: (a) complementarity-determining regions (CDRs) of a heavy-chain variable region (HCVR), the HCVR comprising amino acid sequences within the amino acid sequences of the heavy-chain region (HC) listed in Tables 1A-1C; and (b) CDRs of a light-chain variable region (LCVR), the LCVR comprising amino acid sequences within the amino acid sequences of the light-chain region (LC) listed in Tables 1A-1C.

[0032] In one embodiment, the antibody or the antigen-binding fragment thereof that binds to FXI comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained in 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 in the LC sequence of SEQ ID NO:20 or a substantially similar sequence having at least 90% sequence identity thereto.

[0033] In one embodiment, the isolated antibody or the antigen-binding fragment thereof that binds to FXI comprises an HCVR having an amino acid sequence within the HC amino acid sequence provided in Table 1A-1C below.

[0034] In one embodiment, the isolated antibody or the antigen-binding fragment thereof that binds to FXI further comprises an LCVR having an amino acid sequence within the LC amino acid sequence provided in Table 1A-1C below.

[0035] In one embodiment, the isolated antibody or the antigen-binding fragment thereof that binds to FXI comprises an HCVR having an amino acid sequence within the HC amino acid sequence provided in Table 1A-1C below; and an LCVR having an amino acid sequence within the LC amino acid sequence provided in Table 1A-1C below.

[0036] In one embodiment, the isolated antibody or the antigen-binding fragment thereof that binds to FXI comprises CDRs of an HCVR / LCVR amino acid sequence pair provided within the HC / LC amino acid sequences in Table 1A-1C below.

[0037] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1 (HCDR1), and the HCDR1 comprises an amino acid sequence included within 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.

[0038] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR2 (HCDR2), and the HCDR2 comprises an amino acid sequence included within 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.

[0039] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR3 (HCDR3), and the HCDR3 comprises an amino acid sequence included within 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.

[0040] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a light chain CDR1 (LCDR1), and the LCDR1 comprises an amino acid sequence included 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.

[0041] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a light chain CDR2 (LCDR2), and the LCDR2 comprises an amino acid sequence included 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.

[0042] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, wherein the antibody or the antigen-binding fragment thereof comprises a light chain CDR3 (LCDR3), and the LCDR3 comprises an amino acid sequence included 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.

[0043] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, the antibody or antigen-binding fragment thereof comprising a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3 / LCDR3), which comprises any HCDR3 amino acid sequence listed in Tables 1A-1C paired with any LCDR3 amino acid sequence listed in Tables 1A-1C. According to certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof that comprises a pair of HCDR3 / LCDR3 amino acid sequences contained 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.

[0044] The present disclosure also provides an antibody or an antigen-binding fragment thereof that binds to FXI, the antibody or antigen-binding fragment thereof 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.

[0045] 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 the CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, 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 can also be used to identify CDR sequences within antibodies.

[0046] In one embodiment, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to FXI, the isolated antibody or antigen-binding fragment thereof comprising:

[0047] (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:4;

[0048] (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO:6;

[0049] (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO:8;

[0050] (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO:12;

[0051] (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO:14; and

[0052] (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO:16.

[0053] 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 NO:4-6-8-12-14-16.

[0054] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI comprises an antibody or antigen-binding fragment thereof that competes with a reference antibody for binding to FXI, wherein the reference antibody preferentially binds to the catalytic domain of FXI.

[0055] 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.

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

[0057] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to human FXI with a K DBinds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0058] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 250 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0059] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 100 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0060] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 50 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0061] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 25 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0062] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 10 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0063] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 5 pM D Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0064] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a dissociation half-life (t 1 / 2 ) greater than about 10 minutes and binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0065] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t of greater than about 20 minutes 1 / 2 and binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0066] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t of greater than about 60 minutes 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0067] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 2 hours 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0068] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 5 hours 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0069] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 10 hours 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0070] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 15 hours 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0071] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 16 hours 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0072] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 1,000 minutes 1 / 2 Binds to human FXI, as measured by surface plasmon resonance at 25°C or 37°C.

[0073] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K less than about 100 nM D Binds to human FXIa, as measured by surface plasmon resonance at 25°C or 37°C.

[0074] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K less than about 10 nM D Binds to human FXIa, as measured by surface plasmon resonance at 25°C or 37°C.

[0075] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 1,000 pM. D Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0076] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 500 pM. D Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0077] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a K of less than about 300 pM. D Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0078] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a dissociation half-life (t 1 / 2 ) binds to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0079] In one embodiment, the isolated antibody or antigen-binding fragment thereof is isolated at a t greater than about 10 minutes. 1 / 2 Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0080] In one embodiment, the isolated antibody or antigen-binding fragment thereof is isolated at a t greater than about 15 minutes. 1 / 2 Binding to human FXI as measured by surface plasmon resonance at 25°C or 37°C.

[0081] In one embodiment, the isolated antibody or antigen-binding fragment thereof is isolated at a t greater than about 20 minutes. 1 / 2 Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0082] In one embodiment, the isolated antibody or antigen-binding fragment thereof is isolated at a t greater than about 25 minutes. 1 / 2 Binding to human FXIa as measured by surface plasmon resonance at 25°C or 37°C.

[0083] In one embodiment, the isolated antibody or antigen-binding fragment thereof is isolated at a t greater than about 50 minutes.1 / 2 Binds to human FXIa, as measured by surface plasmon resonance at 25°C or 37°C.

[0084] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 75 minutes 1 / 2 Binds to human FXIa, as measured by surface plasmon resonance at 25°C or 37°C.

[0085] In one embodiment, the isolated antibody or antigen-binding fragment thereof has a t greater than about 95 minutes 1 / 2 Binds to human FXIa, as measured by surface plasmon resonance at 25°C or 37°C.

[0086] 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) relative to full-length FXI, PKA1, PKA2, PKA3, and / or PKA4, as determined by label-free biolayer interferometry.

[0087] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI competes with an antibody that specifically binds to and overlaps an epitope within the FXI CAT domain for binding to FXI.

[0088] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI increases the activated partial thromboplastin time (aPTT) (which is a measure of the intrinsic clotting time) in primates by at least 2.5-fold in vitro, without measurably affecting the prothrombin time (PT) (which is a measure of the extrinsic clotting time).

[0089] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI inhibits the intrinsic pathway peak thrombin activity in primates by about 5%-15%, about 1%-20%, about 0.5%-25%, about 3%-5%, about 4%-6%, about 5%-7%, about 6%-8%, about 7%-9%, about 8%-10%, about 9%-11%, about 10%-12%, about 11%-13%, about 12%-14%, about 13%-15%, about 14%-16%, about 15%-17%, about 16%-18%, about 17%-19%, or about 18%-20% in vitro.

[0090] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI doubles the aPTT in human plasma in vitro at the following concentrations: about 100 pM - 100 nM, about 1 nM - 50 nM, about 5 nM - 40 nM, about 10 nM - 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. Here, without doubling the PT, the anti-FXI doubles the aPTT.

[0091] In one embodiment, the isolated antibody or antigen-binding fragment thereof that binds to FXI inhibits the generation of thrombin in the intrinsic pathway in human plasma in vitro at the following concentrations: about 10 nM - 100 nM, about 15 nM - 500 nM, about 20 nM–60 nM, about 25 nM–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, at a dose of up to about 500 nM, the anti-FXI inhibits the generation of thrombin in the intrinsic pathway without affecting the generation of thrombin in the extrinsic pathway.

[0092] In a second aspect, the present disclosure provides nucleic acid molecules encoding anti-FXI antibodies or portions thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR 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.

[0093] The present disclosure also provides nucleic acid molecules encoding 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 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.

[0094] The present disclosure also provides a nucleic acid molecule encoding any HCDR1 amino acid sequence among the HCDR1 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR1 nucleic acid sequence among 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.

[0095] The present disclosure also provides a nucleic acid molecule encoding any HCDR2 amino acid sequence among the HCDR2 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR2 nucleic acid sequence among 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.

[0096] The present disclosure also provides a nucleic acid molecule encoding any HCDR3 amino acid sequence among the HCDR3 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCDR3 nucleic acid sequence among 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.

[0097] The present disclosure also provides a nucleic acid molecule encoding any LCDR1 amino acid sequence among the LCDR1 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR1 nucleic acid sequence among 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.

[0098] The present disclosure also provides a nucleic acid molecule encoding any LCDR2 amino acid sequence among the LCDR2 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR2 nucleic acid sequence among 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.

[0099] The present disclosure also provides a nucleic acid molecule encoding any LCDR3 amino acid sequence among the LCDR3 amino acid sequences listed in Tables 1A-1C; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any LCDR3 nucleic acid sequence among 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.

[0100] The present disclosure also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1, HCDR2, HCDR3), and wherein the set of HCDR1, HCDR2, HCDR3 amino acid sequences is defined as in any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0101] The present disclosure also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1, LCDR2, LCDR3), and wherein the set of LCDR1, LCDR2, LCDR3 amino acid sequences is defined as in any of the exemplary anti-FXI antibodies listed in Tables 1A-1C.

[0102] The present disclosure also provides a nucleic acid molecule encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any HCVR amino acid sequence among the HCVR amino acid sequences listed in Tables 1A-1C, and wherein the LCVR comprises the amino acid sequence of any LCVR amino acid sequence among the LCVR amino acid sequences listed in Tables 1A-1C. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any HCVR nucleic acid sequence among 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 LCVR nucleic acid sequence among 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 of this aspect according to the present disclosure, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-FXI antibody listed in Tables 1A-1C.

[0103] In a third aspect, the present disclosure provides recombinant expression vectors capable of expressing polypeptides comprising the heavy or light chain variable regions of an anti-FXI antibody. For example, the present disclosure encompasses recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and / or CDR sequences listed in Tables 1A - 1C. Also within the scope of the present disclosure are host cells into which such vectors have been introduced, and methods of producing an antibody or a portion thereof by culturing the host cells under conditions permitting the production of the antibody or antibody fragment and recovering the antibody and antibody fragment so produced.

[0104] The present disclosure encompasses anti-FXI antibodies having a modified glycosylation pattern. In some embodiments, for example, modifications can be used to remove undesired glycosylation sites, or antibodies lacking a fucose moiety present on the oligosaccharide chain to increase antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al., (2002) Journal of Biological Chemistry (JBC) 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0105] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising at least one antibody or antigen-binding fragment of the present disclosure that specifically binds to FXI and a pharmaceutically acceptable carrier.

[0106] In a related aspect, the present 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 the anti-FXI antibody. The second therapeutic agent can be used to alleviate at least one symptom of a neurodegenerative disease or disorder.

[0107] In a fifth aspect, the present disclosure provides a method for enhancing FXI-mediated biological activity, 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.

[0108] In certain embodiments, the biological activity is coagulation or coagulation due to the intrinsic coagulation pathway and not due to the extrinsic (i.e., e.g., tissue factor-induced) pathway; and when FXI or FXIa is contacted with the antagonist anti-FXI antibody, coagulation or coagulation due to the intrinsic coagulation pathway and not due to the extrinsic pathway is inhibited or otherwise reduced.

[0109] In a sixth aspect, the present disclosure provides a method of 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 an antigen-binding portion of an antibody of the present disclosure. The method of treatment according to this aspect of the present disclosure comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an antibody or an antigen-binding fragment of an antibody of the present disclosure. The disorder being treated is any disease or condition that is ameliorated, alleviated, inhibited, or prevented by targeting FXI and / or by inactivating FXI-mediated blood clotting.

[0110] In one embodiment, the anti-FXI antibody of the present disclosure can provide a method of treating pathologic intrinsic blood clotting without adversely affecting hemostasis. In one embodiment, the anti-FXI antibody of the present disclosure can provide a method of treating a disease, disorder, a blood clotting side effect of any of the following, an indirect blood clotting effect: Factor V Leiden; prothrombin gene mutation; deficiencies in natural proteins that prevent blood clotting (such as antithrombin, protein C, and protein S); elevated levels of homocysteine; elevated levels of fibrinogen or dysfibrinogen (dysfibrinogenemia); elevated levels of factor VIII, factor IX, and / or factor XI; abnormal fibrinolytic system, including hypoplasminogenemia, dysplasminogenemia, 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; central venous catheter placement; obesity; pregnancy; use of supplemental estrogen, including oral contraceptives); hormone replacement therapy; prolonged bed rest or immobility; heart attack; congestive heart failure; stroke and other diseases resulting in reduced activity; heparin-induced thrombocytopenia (reduction in platelets in the blood due to heparin or low molecular weight heparin preparations); long-haul air travel; antiphospholipid antibody syndrome; deep vein thrombosis or pulmonary embolism; myeloproliferative disorders, such as polycythemia vera or essential thrombocythemia; paroxysmal nocturnal hemoglobinuria; inflammatory bowel syndrome; HIV / AIDS; nephrotic syndrome; COVID-19 infection or spike protein immune effects, etc.

[0111] A seventh aspect of the present disclosure provides a method of preventing thrombosis in a subject without adversely affecting hemostasis, the method comprising administering to the subject a therapeutically effective amount of an FXI antagonist antibody of Tables 1A - 1C or a pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof.

[0112] In one embodiment, the above method can be achieved by administering an antagonist anti-FXI antibody or an antigen-binding fragment thereof to a subject in need, wherein the antagonist anti-FXI antibody comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR), and the HCVR comprises the amino acid sequence listed 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) contained within the light-chain variable region (LCVR), and the LCVR comprises the amino acid sequence listed in Tables 1A-1C or a substantially similar sequence having at least 90% sequence identity thereto.

[0113] 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 an 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 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.

[0114] In one embodiment, the antibody or an 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 the HCVR contained within SEQ ID NO:18. In one embodiment, the antibody or an antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO:2. In one embodiment, the antibody or an 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.

[0115] In one embodiment, the antibody or an 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 the LCVR contained within SEQ ID NO:20. In one embodiment, the antibody or an antigen-binding fragment thereof comprises an LCVR having the amino acid sequence of SEQ ID NO:10. In one embodiment, the antibody or an antigen-binding fragment thereof comprises an LCVR having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:10.

[0116] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO:18; and a light chain (LC) having the amino acid sequence of SEQ ID NO:20.

[0117] In one embodiment, the antibody or antigen-binding fragment thereof comprises the CDRs of the HC / LC amino acid sequence pair of SEQ ID NO:18 / 20.

[0118] In one embodiment, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10.

[0119] In one embodiment, the antibody or antigen-binding fragment thereof comprises:

[0120] (a) an HCDR1 domain having the amino acid sequence of SEQ ID NO:4;

[0121] (b) an HCDR2 domain having the amino acid sequence of SEQ ID NO:6;

[0122] (c) an HCDR3 domain having the amino acid sequence of SEQ ID NO:7;

[0123] (d) an LCDR1 domain having the amino acid sequence of SEQ ID NO:12;

[0124] (e) an LCDR2 domain having the amino acid sequence of SEQ ID NO:14; and

[0125] (f) an LCDR3 domain having the amino acid sequence of SEQ ID NO:16.

[0126] 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 sequence of SEQ ID NO:4-6-8-12-14-16.

[0127] On the one hand, the present disclosure provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NO: 4-6-8-12-14-16. In one embodiment, the nucleic acid encodes an antibody or an antigen-binding fragment thereof having an HCVR comprising SEQ ID NO: 2 and / or an LCVR comprising SEQ ID NO: 10. In one embodiment, the nucleic acid encodes an antibody or an antigen-binding fragment thereof having an HC comprising SEQ ID NO: 18 and / or an LC comprising SEQ ID NO: 20.

[0128] On the one hand, the present disclosure provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a set of six CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) having the nucleic acid sequences of SEQ ID NO: 3-5-7-11-13-15. In one embodiment, the nucleic acid encodes an antibody or an antigen-binding fragment thereof having an HCVR comprising SEQ ID NO: 1 and / or an LCVR comprising SEQ ID NO: 9. In one embodiment, the nucleic acid encodes an antibody or an antigen-binding fragment thereof having an HC comprising SEQ ID NO: 17 and / or an LC comprising SEQ ID NO: 19.

[0129] In one embodiment, the disease or disorder to be treated with the anti-FXI antibodies of the present disclosure is thrombosis and any complications caused by thrombosis.

[0130] It is contemplated that any disease or disorder associated with FXI activity or expression is suitable for treatment with the antibodies of the present disclosure. Such disorders can include any disease or condition where there is a risk of harmful clot formation, particularly but not limited to those conditions where intrinsic coagulation and hemostasis are a risk to the patient.

[0131] Other embodiments will become apparent by reading the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 Size analysis of the complex formed between human coagulation factor XI and REGN7528 and REGN7531 is depicted.

[0133] Figure 2 Size analysis of the complex formed between human coagulation factor XI and REGN7503, REGN7505, and REGN7508 is depicted.

[0134] Figure 3Depicts size analysis of the complex formed between human coagulation factor XI and REGN7508.

[0135] Figure 4 Depicts size analysis of the complex formed between human coagulation factor XI and REGN9932.

[0136] Figure 5 Depicts comparison of size analysis of the complex formed between human coagulation factor XI and REGN7508 and the complex formed between human coagulation factor XI and REGN9932.

[0137] Figures 6A - 6C Shows the effect of anti-FXI mAb on the intrinsic coagulation pathway in female cynomolgus monkey plasma using thrombin generation assay (TGA).

[0138] Figures 7A - 7C Shows the effect of anti-FXI mAb on the extrinsic coagulation pathway in female cynomolgus monkey plasma using TGA.

[0139] Figures 8A - 8C Shows the effect of anti-FXI mAb on the intrinsic coagulation pathway in pooled female human plasma using TGA.

[0140] Figures 9A - 9C Shows the effect of anti-FXI mAb on the intrinsic coagulation pathway in pooled female human plasma using TGA.

[0141] Figures 10A - 10C Shows the effect of REGN9932 on the intrinsic coagulation pathway in six individual human donors obtained using TGA.

[0142] Figures 11A - 11C Shows the effect of REGN7508 on the intrinsic coagulation pathway in six individual human donors obtained using TGA.

[0143] Figures 12A - 12C Shows the effect of REGN9932 on the extrinsic coagulation pathway in six individual human donors obtained using TGA.

[0144] Figures 13A - 13C Shows the effect of REGN7508 on the extrinsic coagulation pathway in six individual human donors obtained using TGA.

[0145] Figure 14 Is an overview of the coagulation pathway.

[0146] Figure 15 Is an example of a thromboagram.

[0147] Figures 16A - 16CShows the effects of REGN7508 obtained using a coagulation assay on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma.

[0148] Figures 17A - 17C Shows the effects of REGN7508 obtained using a coagulation assay on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus monkey donor plasma.

[0149] Figures 18A - 18D Shows the effects of REGN7508 obtained using TGA on the intrinsic and extrinsic coagulation pathways in pooled human donor plasma.

[0150] Figure 19 Shows the effects of a second set of concentrations of REGN7508 obtained using TGA on the intrinsic coagulation pathway in pooled human donor plasma.

[0151] Figures 20A - 20D Shows the effects of REGN7508 obtained using TGA on the intrinsic and extrinsic coagulation pathways in pooled cynomolgus monkey donor plasma.

[0152] Figure 21 Shows the effects of a second set of concentrations of REGN7508 obtained using TGA on the intrinsic coagulation pathway in pooled cynomolgus monkey donor plasma. Detailed Description

[0153] Before describing the present disclosure, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present disclosure will be defined only by the appended claims.

[0154] 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 pertains. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value may vary from the recited value by no more than 1%. For example, as used herein, the expression "about 100" encompasses 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0155] Although any methods and materials similar or equivalent to those described herein may 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 in this specification are incorporated herein by reference in their entirety.

[0156] Definitions

[0157] The expression "FXI", etc. (also known as "coagulation factor XI" or "factor XI") refers to a human plasma serine protease (unless specified as from another species), which includes the amino acid sequence (SEQ ID NO: 41) listed in amino acid residues 19 to 625 of accession number NP_000119.1. Human FXI containing a myc-myc-hexahistidine tag is shown as SEQ ID NO: 42 (where amino acid residues 1-607 are human FXI and amino acid residues 608-635 are the myc-myc-hexahistidine tag).

[0158] In some cases, cell lines are prepared that express: FXI protein; subunits of FXI protein; and chimeric proteins containing one or more FXI subunits, tag sequences, and plasma kallikrein protein sequences. For example, SEQ ID NO: 43 (construct hFXI_PKA1) is a chimera that contains the apple 1 domain (PKA1) of human kallikrein B1 (amino acids G20-C104 of human kallikrein B1 [SEQ ID NO: 48]) at amino acids 1-85, amino acids H105-V625 of human FXI (hFXI) at amino acids 86-60, and a myc-myc-hexahistidine tag at amino acids 607-634.

[0159] For example, SEQ ID NO: 44 (construct hFXI_PKA2) is a chimera that contains amino acids E19-S108 of hFXI at amino acids 1-90, the apple 2 domain (PKA2, also known as "A2") of hKLKB1 (amino acids C111-C193 SEQ ID NO: 48) at amino acids 91-174, amino acids A195-V625 of hFXI at amino acids 175-605, and a myc-myc-hexahistidine tag at amino acids 606-633.

[0160] For example, SEQ ID NO: 45 (construct hFXI_PKA3) is a chimera that contains amino acids E19-L198 of hFXI at amino acids 1-180, the apple 3 domain (PKA3) of hKLKB1 (amino acids C201-C284 SEQ ID NO: 48) at amino acids 181-264, amino acids H285-V625 of hFXI at amino acids 265-605, and a myc-myc-hexahistidine tag at amino acids 606-633.

[0161] For example, SEQ ID NO:46 (construct hFXI_PKA4) is a chimera that contains amino acids E19-V289 of hFXI at amino acids 1-271, the apple 4 domain of hKLKB1 (PKA4) (amino acids C292-C375 SEQ ID NO:48) at amino acids 272-355, amino acids M376-V625 of hFXI at amino acids 356-605, and a myc-myc-hexahistidine tag at amino acids 606-633.

[0162] For example, SEQ ID NO:47 (construct hKLKB1.mmh) is a chimera that contains amino acids G20-A638 of hKLKB1 at amino acids 1-619 and a myc-myc-hexahistidine tag at amino acids 620-647.

[0163] As used herein, the expression "anti-FXI antibody" includes both monovalent antibodies having single specificity and bispecific antibodies that include a first arm that binds to FXI and a second arm that binds to a second (target) antigen, wherein the anti-FXI arm includes any HCVR / LCVR or CDR sequence listed in Tables 1A-1C herein. The expression "anti-FXI antibody" also includes antibody-drug conjugates (ADCs) that include an anti-FXI antibody or an antigen-binding portion thereof conjugated to a drug or toxin (i.e., a cytotoxic agent). The expression "anti-FXI antibody" also includes antibody-radionuclide conjugates (ARCs) that include an anti-FXI antibody or an antigen-binding portion thereof conjugated to a radionuclide.

[0164] As used herein, the term "anti-FXI antibody" means any antigen-binding molecule or molecular complex that includes 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 that include four polypeptide chains, two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region includes three domains C H 1, C H 2, and C H 3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region includes one domain (C L 1). V H and V LThe region is further subdivided into hypervariable regions known as complementarity-determining regions (CDRs), which are interspersed with more conserved regions known as framework regions (FRs). Each V H and V L is composed of three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present disclosure, the FRs of the anti-FXI antibody (or its antigen-binding portion) may be identical to the human germline sequence, or may be natural or artificially modified. Amino acid consensus sequences can be defined based on the juxtaposition analysis of two or more CDRs.

[0165] As used herein, the term "antibody" also encompasses antigen-binding fragments of full-length antibody molecules. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. encompass any enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived from, for example, whole antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or readily obtainable from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical methods or using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains in a suitable configuration, or to introduce codons, generate cysteine residues, modify, add or delete amino acids, etc.

[0166] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, trispecific antibodies, tetravalent antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0167] An antigen-binding fragment of an antibody will generally comprise at least one variable domain. The variable domain can have any size or amino acid composition and will generally comprise at least one CDR adjacent to or within a framework having one or more framework sequences. In an antigen-binding fragment having a V L domain-related V H domain, the V H and V L domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0168] In certain embodiments, the antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the present disclosure include: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C L In any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly connected to each other or can be connected by a full or partial hinge or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which results in a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present disclosure can include any of the variable domain configurations and constant domain configurations of the homologous dimers or heterodimers (or other multimers) of the variable domain configurations and constant domain configurations non-covalently associated with each other and / or with one or more monomeric V H or V L domains (e.g., via disulfide bonds).

[0169] Like whole antibody molecules, the antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will generally include at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format that includes the exemplary bispecific antibody formats disclosed herein can be adapted for use in the context of the antigen-binding fragments of the antibodies of the present disclosure using conventional techniques available in the art.

[0170] In certain cases, it may be desirable to antagonize FXI, e.g., for inhibiting the formation of blood clots. However, the antibodies of the present disclosure act as antagonist antibodies that serve as inhibitors of FXI or FXIa activity and simultaneously as inhibitors of intrinsic pathway thrombus / coagulation formation. The antibodies of the present disclosure can act by preventing the interaction between FXI and its upstream activators coagulation factor XII (FXII) and / or coagulation factor II (FII or thrombin). The antibodies of the present disclosure can also act by preventing the interaction between FXI and its downstream target coagulation factor IX (FIX). The antibodies of the present disclosure can also act by sequestering FXI from the bloodstream of a patient.

[0171] As used herein, the term "human antibody" is intended to encompass non-naturally occurring human antibodies. The term encompasses antibodies recombinantly produced in non-human mammals or cells of non-human mammals. The term is not intended to encompass antibodies isolated or produced from human subjects.

[0172] In some embodiments, the antibodies of the present disclosure can be recombinant and / or non-naturally occurring human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (further described below), antibodies isolated from a recombinant combinatorial human antibody library (further described below), antibodies isolated from an animal transgenic for human immunoglobulin genes (e.g., a mouse) (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. In certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when using an animal transgenic for human Ig sequences, to in vivo somatic mutagenesis), and thus the amino acid sequences of the V H and V L regions are related to, but may not naturally exist in the human antibody germline repertoire, although related to the human germline V H and V L sequences.

[0173] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, in which the dimers are held together by inter-chain heavy chain disulfide bonds. In the second form, the dimers are not linked by inter-chain disulfide bonds and form a molecule of approximately 75-80 kDa, which consists of covalently coupled light and heavy chains (half-antibodies). Even after affinity purification, these forms are extremely difficult to separate.

[0174] The frequency of occurrence of the second form in the various intact IgG isotype forms is based on, but not limited to, structural differences related to the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using the human IgG1 hinge. The present disclosure encompasses antibodies having one or more mutations in the hinge region, C H 2 region, or C H 3 region, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0175] The term "specifically binds" or "binds specifically to", etc. means that an antibody or an antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. The characteristics of specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x 10 -6 M or less (e.g., a smaller K D indicating tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies have been identified by surface plasmon resonance (e.g., BIACORE TM ) that specifically bind to FXI. In addition, as used herein, a multispecific antibody that binds to FXI protein and one or more additional antigens, or a bispecific antibody that binds to two different regions of FXI, is still considered an antibody that "specifically binds".

[0176] The antibodies of the present disclosure can be isolated antibodies. As used herein, an "isolated antibody" means an antibody that has been identified and 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 the antibody naturally occurs or is naturally produced is an "isolated antibody" for the purposes of the present disclosure. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, the isolated antibody can be substantially free of other cellular materials and / or chemicals.

[0177] The anti-FXI antibodies disclosed herein can include 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 determined by comparing the amino acid sequences disclosed herein with sequences available from, for example, public antibody sequence databases. Once obtained, the one or more desired properties of the antibodies and antigen-binding fragments containing one or more mutations, such as improved binding specificity, increased binding affinity, improved or enhanced agonistic or antagonistic biological properties, as appropriate, reduced immunogenicity, etc., can be readily tested. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0178] The present disclosure also encompasses anti-FXI antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure encompasses anti-FXI antibodies having HCVR, LCVR, and / or CDR amino acid sequences that have, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences listed in Tables 1A-1C herein.

[0179] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule called a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope can include a moiety of a sugar, phosphoryl, or sulfonyl group on an antigen.

[0180] When referring to a nucleic acid or a fragment thereof, the terms "substantially identical" or "substantially the same" indicate that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, the nucleotide sequence identity is at least about 95% and more preferably at least about 96%, 97%, 98%, or 99% of nucleobases, as measured by any well-known sequence identity algorithm such as FASTA, BLAST, or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0181] When applied to polypeptides, the terms "substantially similar" or "substantially identical" mean that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned by a program such as GAP or BESTFIT using default gap weights. Preferably, the non-identical residue positions differ due to conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In cases where two or more amino acid sequences differ from each other due to conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. The manner in which this adjustment is made is well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are any changes having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0182] Sequence similarity, also referred to as sequence identity, of polypeptides is usually measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions and other modifications (comprising conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different organism species) or between wild-type proteins and their mutant proteins. See, for example, version 6.1 of GCG. FASTA using default or recommended parameters can also be used to compare polypeptide sequences, which is a program in version 6.1 of GCG. FASTA (e.g., FASTA2 and FASTA3) provides comparison and sequence identity percentage of the best overlap region between the query and search sequence (Pearson (2000), supra). When comparing sequences of the present disclosure with databases containing a large number of sequences from different organisms, another preferred algorithm is a computer program BLAST using default parameters, especially BLASTP or TBLASTN. See, e.g., Altschul et al. (1990) J. Molecular Biology 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0183] Antibody properties

[0184] The present disclosure includes a K of less than about 500 pM. D Anti-FXI antibodies that bind to the catalytic domain of human FXI as measured by surface plasmon resonance at 25°C or 37°C. According to certain embodiments, the present disclosure comprises D Anti-FXI antibodies that bind to human FXI: 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.

[0185] The present disclosure includes a K of less than about 1,000 pM. D Anti-FXI antibodies that bind to activated human FXI (FXIa) as measured by surface plasmon resonance at 25°C or 37°C. According to certain embodiments, the present disclosure comprises antibodies with a K of DAnti-FXI antibodies that bind to human FXI: 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.

[0186] This disclosure encompasses anti-FXI antibodies that bind to human FXI with a dissociation half-life (t 1 / 2 ) greater than about 10 minutes, as measured by surface plasmon resonance at 25 °C or 37 °C. According to certain embodiments, this disclosure encompasses anti-FXI antibodies that bind to human FXI with the following t 1 / 2 Anti-FXI antibodies that bind to human FXI: 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.

[0187] This disclosure encompasses anti-FXI antibodies that bind to human FXIa with a dissociation half-life (t 1 / 2 ) greater than about 10 minutes, as measured by surface plasmon resonance at 25 °C or 37 °C. According to certain embodiments, this disclosure encompasses anti-FXI antibodies that bind to human FXIa with the following t 1 / 2 Anti-FXI antibodies that bind to human FXIa: 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.

[0188] This disclosure encompasses anti-FXI antibodies that may or may not bind to non-human FXI. As used herein, when tested in an antigen-binding assay such as surface plasmon resonance, if the antibody exhibits a K DOr does not exhibit any antigen binding, the antibody "does not bind" to a specific antigen (e.g., monkey, mouse or rat FXI). According to this aspect of the present disclosure, another assay format that can be used to determine whether an antibody binds or does not bind to a specific antigen is ELISA.

[0189] It is generally known in the art that activated FXI (FXIa) activates factor IX by selectively cleaving arg-ala and arg-val peptide bonds. Factor IXa in turn forms a complex with factor VIIIa (FIXa-FVIIIa) and activates factor X. The present disclosure encompasses anti-FXI antibodies that inhibit FXI-mediated activation of human FX in plasma by at least about 85% at an IC 50 Using the assay format described in Example 4 or a substantially similar assay format, the IC 50 value can be calculated as the concentration of antibody required to activate FXI-mediated signal transduction to the observed half-maximal signal. Thus, according to certain embodiments, the present disclosure encompasses anti-FXI antibodies that inhibit FXI-mediated activation of human FX in plasma by at least about 85% at an IC 50 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, as measured using the assay format described in Example 4 herein or a substantially similar assay.

[0190] The present disclosure also encompasses anti-FXI antibodies that inhibit FXIa-mediated activation of human FX in plasma by at least about 25% at an IC 50 Using the assay format described in Example 4 or a substantially similar assay format, the IC 50 value can be calculated as the concentration of antibody required to activate FXIa-mediated signal transduction to the observed half-maximal signal. Thus, according to certain embodiments, the present disclosure encompasses anti-FXI antibodies that inhibit FXIa-mediated activation of human FX in plasma by at least about 25% at an IC 50Human FXIa-mediated activation of human FX in plasma is at least about 25%: 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, as measured using the assay format described in Example 4 herein or substantially similar assays.

[0191] The present disclosure encompasses anti-FXI antibodies that preferentially bind to the catalytic domain (CAT), as demonstrated by direct binding to the 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 of FXI (e.g., the A2 domain).

[0192] The present disclosure encompasses anti-FXI antibodies that prolong the activated partial thromboplastin time (aPTT) (which is a measure of intrinsic pathway thrombosis) in human plasma, without a measurable effect on the prothrombin time (PT) (which is a measure of extrinsic pathway thrombosis). In one embodiment, aPTT is measured in pooled human plasma treated with ellagic acid using a hemostasis analyzer, and PT is measured in pooled human plasma treated with tissue factor, as exemplified in Examples 6 and 9. 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 antibodies prolong aPTT by about two-fold at the following concentrations without doubling PT: 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 - 100 nM, 1 nM - 100 nM, 1 nM - 50 nM, 100 pM - 50 nM, 5 nM - 50 nM, 5 nM - 40 nM, 5 nM - 15 nM, 10 nM - 20 nM, 15 nM - 25 nM, 20 nM - 30 nM, 25 nM - 35 nM, 30 nM - 40 nM, 35 nM - 45 nM, 40 nM - 50 nM, 45 nM - 55 nM, 50 nM - 60 nM, 55 nM - 65 nM, 60 nM - 100 nM, 65 nM - 75 nM, 70 nM - 80 nM, 75 nM - 85 nM, 80 nM - 90 nM, 85 nM - 95 nM, 90 nM - 100 nM or 95 nM - 105 nM.

[0193] The present disclosure encompasses anti-FXI antibodies that inhibit thrombin generation in vitro achieved through the intrinsic pathway (intrinsic thrombin) in human plasma, with little to no effect on thrombin generation achieved through the extrinsic pathway (extrinsic thrombin). In one embodiment, pathway-specific thrombin generation is determined in vitro by thrombin generation assays using calibrated automated thrombograms, as illustrated in Examples 6 and 9. Here, thrombin generation curves are generated, and peak thrombin concentrations in ellagic acid-treated plasma and tissue factor-treated plasma are determined with and without the anti-FXI antibody. Thus, in one embodiment, the anti-FXI antibody inhibits the generation of intrinsic thrombin at the following concentrations: 0.1 nM - 100 nM, 1 nM - 100 nM, 5 nM - 500 nM, 5 nM - 100 nM, 10 nM - 100 nM, 10 nM - 50 nM, 5 nM - 15 nM, 10 nM - 20 nM, 25 nM - 35 nM, 30 nM - 40 nM, 35 nM - 45 nM, 40 nM - 50 nM, 45 nM - 55 nM, 50 nM - 60 nM, 55 nM - 65 nM, 60 nM - 65 nM, ≥20 nM, ≥25 nM, ≥30 nM, ≥35 nM, ≥40 nM, ≥45 nM, ≥50 nM, ≥55 nM, ≥5 nM, ≥10 nM, or ≥15 nM. Here, at any concentration up to 500 nM, the anti-FXI antibody has no effect on the generation of extrinsic thrombin.

[0194] The present disclosure encompasses anti-FXI antibodies that increase the activated partial thromboplastin time (aPTT) in primates in vivo by at least two-fold, with immeasurable effect on the prothrombin time (PT). Here, an anti-FXI antibody is administered to a primate, plasma is obtained from the primate, the plasma is contacted with ellagic acid or tissue factor, and then the aPTT or PT is determined separately in an assay as illustrated in Example 7. In one embodiment, the primate is a human. In one embodiment, the primate is a monkey.

[0195] In one embodiment, the anti-FXI antibody is administered at the following doses: 0.01 mg / kg - 20 mg / kg, 0.1 mg / kg - 10 mg / kg, 1 mg / kg - 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.

[0196] In one embodiment, relative to no anti-FXI treatment, treatment with an anti-FXI antibody prolongs the aPTT 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.

[0197] In one embodiment, after administration of a dose of the anti-FXI antibody, the anti-FXI-mediated aPTT prolongation effect persists in the subject 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.

[0198] The present disclosure encompasses anti-FXI antibodies that inhibit in vivo the peak thrombin activity of the intrinsic pathway in primates without measurably affecting the peak thrombin activity of the extrinsic pathway. Here, an anti-FXI antibody is administered to a primate, plasma is obtained from the primate, the plasma is contacted with ellagic acid or tissue factor, and then the production of intrinsic thrombin or extrinsic thrombin is determined separately 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.

[0199] In one embodiment, the anti-FXI antibody is administered at the following doses: 0.01 mg / kg - 20 mg / kg, 0.1 mg / kg - 10 mg / kg, 1 mg / kg - 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.

[0200] In one embodiment, relative to no anti-FXI treatment, the peak intrinsic thrombin (i.e., thrombin generated by ellagic acid) activity in the anti-FXI treatment is inhibited by 1% - 100%, 5% - 95%, 10% - 90%, 20% - 80%, 1% - 10%, 5% - 20%, 10% - 30%, 15% - 40%, 20% - 50%, 25% - 60%, 5% - 15%, 10% - 20%, 15% - 25%, 20% - 30%, 25% - 35%, 30% - 40%, 35% - 45%, 40% - 50%, 45% - 55%, 50% - 60%, 55% - 65%, 60% - 70%, 65% - 75%, 70% - 80%, 75% - 85%, 80% - 90%, 85% - 95%, 90% - 100%, 95% - 105% or ≥ 100%.

[0201] In one embodiment, after a subject receives a certain dose of the anti-FXI antibody, the inhibition of the anti-FXI-mediated peak intrinsic thrombin activity persists in the subject 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.

[0202] When disclosed in terms of "measured by surface plasmon resonance", the binding characteristics of the antibodies of the present disclosure (e.g., any of the binding characteristics mentioned above herein) mean using a surface plasmon resonance instrument (e.g., the instrument, GE Healthcare) to measure the relevant binding characteristics regarding the interaction between the antibody and the antigen using the standard Biacore assay conditions as shown in Example 3 herein or substantially similar assay formats. In certain embodiments, the binding parameters are measured at 25°C, while in other embodiments, the binding parameters are measured at 37°C.

[0203] The present disclosure encompasses an antibody or an antigen-binding fragment thereof that specifically binds to FXI, the antibody or antigen-binding fragment thereof comprising an HCVR and / or an LCVR, the 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.

[0204] The antibodies of the present disclosure can have one or more of the above-described biological properties or any combination thereof. The foregoing list of biological properties of the antibodies of the present disclosure is not intended to be exhaustive. By reading the present disclosure, including the working examples herein, other biological properties of the antibodies of the present disclosure will be apparent to those of ordinary skill in the art.

[0205] Epitope mapping and related techniques

[0206] The epitope bound by the antibodies of the present disclosure can consist of a single continuous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the FXI protein. Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) of FXI. In some embodiments, the epitope is located on or near the surface of FXI, such as in a domain that interacts with any one of its ligands (e.g., FXIIa, thrombin, and FIX). In other embodiments, the epitope is located on or near a surface of FXI that does not interact with the FXI ligand, such as at a position on the surface of FXI where, when the antibody binds to such an epitope, the antibody does not interfere with the interaction between FXI and its ligand.

[0207] Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays (such as in Antibodies ( Antibodies)》, cross-blocking assays as described in Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine-scanning mutagenesis analysis, peptide blotting analysis (Reineke, 2004, Methods in Molecular Biology 248:443-463), and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociating the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids that interact with the antibody. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0208] 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 including any of the amino acid sequences listed 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 (e.g., antibodies including any of the amino acid sequences listed in Tables 1A-1C herein) for binding to FXI.

[0209] By using conventional methods known in the art and exemplified herein, it can be readily determined whether an antibody binds to the same epitope as a reference anti-FXI antibody or competes with the reference anti-FXI antibody for binding. 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 allowed to bind to the FXI protein. Next, the ability of the test antibody to bind to the FXI molecule is evaluated. If the test antibody is able to bind to FXI after saturation binding with the reference anti-FXI antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-FXI antibody. On the other hand, if the test antibody is not able to bind to the anti-FXI molecule after saturation binding with the reference anti-FXI antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-FXI antibody of the present disclosure. Additional conventional experiments (e.g., peptide mutagenesis and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether it is due to steric hindrance (or another phenomenon) causing the observed lack of binding. Such 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, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies are considered to have "overlapping epitopes".

[0210] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-FXI antibody, the above binding method is performed in two orientations: In the first orientation, the reference antibody is allowed to bind to the FXI protein under saturating conditions, and then the binding of the test antibody to the FXI molecule is evaluated. In the second orientation, the test antibody is allowed to bind to the FXI molecule under saturating conditions, and then the binding of the reference antibody to the FXI molecule is evaluated. If only the first (saturating) antibody is able to bind to the FXI molecule in both orientations, it is concluded that the test antibody and the reference antibody compete for binding to FXI (see, for example, the assay formats described in the examples herein, where the FXI protein is captured on the sensor tip and the FXI-coated sensor tip is treated sequentially and in two binding orders with the reference antibody and the test anti-FXI antibody). As will be understood by one of ordinary skill in the art, an antibody that competes for binding to a reference antibody may not necessarily 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).

[0211] Preparation of Human Antibodies

[0212] The anti-FXI antibodies of the present disclosure can 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 can be used in the context of the present disclosure to prepare human antibodies that specifically bind to human FXI.

[0213] Using technology (see, for example, US 6,596,541, Regeneron Pharmaceuticals, ) or any other known method for generating monoclonal antibodies, high-affinity chimeric antibodies against allergens that have human variable regions and murine constant regions are initially isolated. technology involves generating transgenic mice that have a genome comprising human heavy and light chain variable regions operably linked to the endogenous murine constant region locus such that the mice produce antibodies comprising human variable regions and murine constant regions in response to antigen stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. Then, the DNA is expressed in cells capable of expressing fully human antibodies.

[0214] Typically, attacking with the antigen of interest Mice, and lymphocytes (such as B cells) are recovered from the antibody-expressing mice. The lymphocytes can be fused with a myeloma cell line to produce an immortalized hybridoma cell line, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. The DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, the DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be directly isolated from the antigen-specific lymphocytes.

[0215] As described in the experimental section below, high-affinity chimeric antibodies with human variable regions and murine constant regions are characterized and selected to obtain desired properties, including affinity, selectivity, epitope, etc. Then the murine constant regions are replaced with the desired human constant regions to produce the fully human antibodies of the present disclosure, such as wild-type or modified IgG1 or IgG4. Although the constant regions selected can vary according to the specific use, the high-affinity antigen-binding and target-specificity properties reside in the variable regions.

[0216] In certain embodiments, it may be desirable to test the 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 may bind only to human FXI but will not cross-react with murine or rat FXI. Any method known to those skilled in the art can be used to produce such FXI humanized mice and rats.

[0217] Generally, when measured by binding to an antigen immobilized on a solid phase or in a liquid phase, the antibodies of the present disclosure have very high affinity, typically having a K -12 of about 10 -9 to about 10 D M.

[0218] Bioequivalents

[0219] The anti-FXI antibodies and antibody fragments of the present disclosure encompass proteins having an amino acid sequence different from the described antibodies but retaining the ability to bind to human FXI. Such variant antibodies and antibody fragments include one or more additions, deletions, or substitutions of amino acids compared to the parental sequence, but exhibit biological activity that is substantially equivalent to the biological activity of the described antibodies. Similarly, the DNA sequences encoding the anti-FXI antibodies of the present disclosure encompass sequences that include one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences but encode anti-FXI antibodies or antibody fragments that are substantially bioequivalent to the anti-FXI antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid and DNA sequences were discussed above.

[0220] Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives with absorption rates and extents of absorption that do not show significant differences when administered at the same molar dose (single or multiple doses) under similar experimental conditions. If some antibodies are equivalent in their extent of absorption but not in their absorption rate, the antibodies will be considered equivalents or pharmaceutical alternatives and may be considered bioequivalent because such differences in absorption rate that are intentional and reflected in the labeling are not necessary, for example, for achieving effective body drug concentrations upon long-term use and are considered medically insignificant for the particular drug product under study.

[0221] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0222] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can switch between a reference product and a biological product one or more times without an increased risk of expected side effects (including clinically significant changes in immunogenicity or decreased efficacy) compared to a continuous therapy where there is no switching between the reference product and the biological product.

[0223] In one embodiment, two antigen-binding proteins are bioequivalent if both act through one or more common mechanisms of action to such an extent that the mechanisms are known for one or more conditions of use.

[0224] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example: (a) in vivo testing in humans or other mammals, where the concentration of the antibody or its metabolites is measured over time in blood, plasma, serum, or other biological fluids; (b) in vitro testing that is related to and can reasonably predict in vivo bioavailability data in humans; (c) in vivo testing in humans or other mammals, where the appropriate acute pharmacological effect of the antibody (or its target) is measured over time; and (d) in well-controlled clinical trials to determine the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0225] Bioequivalent variants of the anti-FXI antibodies of the present disclosure can be constructed, for example, by making various substitutions to residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges upon refolding. In other contexts, bioequivalent antibodies can include anti-FXI antibody variants that include amino acid changes that modify the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0226] Species selectivity and species cross-reactivity

[0227] 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 encompasses anti-FXI antibodies that bind to human FXI and bind to FXI from one or more non-human species. For example, the anti-FXI antibodies of the present disclosure can bind to human FXI and, optionally, can bind or not bind to one or more of the following: 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.

[0228] Multispecific antibodies

[0229] The antibodies of the present disclosure can be monospecific or multispecific (e.g., bispecific). Multispecific antibodies can be specific for different epitopes of one target polypeptide or can contain antigen-binding domains that are specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, Journal of Immunology 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-FXI antibodies of the present disclosure can be linked or co-expressed with another functional molecule (e.g., another peptide or protein). For example, the antibody or a fragment thereof can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent association, or otherwise) 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.

[0230] The present disclosure encompasses bispecific antibodies, wherein one arm of the immunoglobulin binds to human FXI and the other arm of the immunoglobulin is specific for a second antigen. The FXI-binding arm can include any HCVR / LCVR or CDR amino acid sequence listed in Tables 1A-1C herein.

[0231] Exemplary bispecific antibody formats that can be used in the context of the present disclosure involve the use of a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, wherein the first and second Ig C H 3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A and the second Ig C H 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (by IMGT exon numbering; H435R, by EU numbering). The second C H 3 can further include the Y96F modification (by IMGT; Y436F, by EU). Additional modifications that can be found within the second C H 3 include: in the case of an IgG1 antibody, D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I, by EU); in the case of an IgG2 antibody, N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I, by EU); and in the case of an IgG4 antibody, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I, by EU). Variations of the above bispecific antibody formats are contemplated within the scope of the present disclosure.

[0232] Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or bispecific antibody 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, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats (see, e.g., Klein et al. 2012, Monoclonal Antibodies (mAbs) 4:6, 1-11 and references cited therein for a review of the foregoing formats). Peptide / nucleic acid conjugates can also be used to construct bispecific antibodies, e.g., where unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates, which are then self-assembled into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., Journal of the American Chemical Society (J. Am. Chem. Soc.) [e-Edition: Dec. 4, 2012]).

[0233] Therapeutic Formulations and Administration

[0234] 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 together with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN TM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowaxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0235] The dosage of the antibody administered to a patient can vary depending on the patient's age and body size, the target disease, the condition, the route of administration, etc. The preferred dosage is usually calculated based on body weight or body surface area. In adult patients, it may be advantageous to administer the antibody of the present disclosure intravenously in a single dose of generally about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. The effective dosage and schedule for administering the anti-FXI antibody can be determined empirically; for example, the patient's progress can be monitored by regular evaluations and the dosage adjusted accordingly. In addition, interspecies scaling of the dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0236] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J Biol Chem 262:4429-4432). The methods of introduction include but are not limited to intravitreal, intraocular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or skin mucosa linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and can be administered together with other bioactive agents. The administration can be systemic or local.

[0237] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Additionally, with respect to subcutaneous delivery, pen delivery devices are amenable to delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is prefilled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition in the reservoir has been emptied, the entire device is discarded.

[0238] Many reusable pen delivery devices and autoinjector delivery devices are applied to subcutaneously deliver the pharmaceutical compositions of the present disclosure. Examples include but are not limited to AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM , OPTIPEN PRO TM , OPTIPEN STARLET TM and OPTICLIK TM(Sanofi - Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR TM Pen (Sanofi - Aventis), FLEXPEN TM (Novo Nordisk), and the KWIKPEN TM (Eli Lilly and Company), SURECLICK TM Auto - Injector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and the HUMIRA TM Pen (Abbott Labs, Abbott Park IL), to name a few.

[0239] In certain cases, the pharmaceutical composition can be delivered in a controlled - release system. 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 Controlled Release: Medical Applications, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, Florida. In yet another embodiment, the controlled - release system can be placed near the target of the composition, so that only a fraction of the systemic dose is required (see, for example, Goodson, 1984, Controlled Release: Medical Applications, supra, Vol. 2, pp. 115 - 138). Other controlled - release systems are discussed in the review by Langer, 1990, Science 249:1527 - 1533.

[0240] Injectable preparations may include dosage forms for intravenous, intravitreal, intraocular, subcutaneous, intradermal, and intramuscular injection, infusion, etc. These injectable preparations can be prepared by publicly known methods. For example, an injectable preparation can be prepared, for example, by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or an oily medium commonly used for injection. As the aqueous medium for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared is preferably filled in appropriate ampoules.

[0241] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are formulated into dosage forms in unit doses suitable for accommodating a certain dose of the active ingredient. Such unit-dose dosage forms include, for example, tablets, pills, capsules, injectables (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained is usually about 5 mg to about 500 mg per unit-dose dosage form; especially in the injectable form, it is preferred that the content of the above-mentioned antibody is about 5 mg to about 100 mg, and for other dosage forms, about 10 mg to about 250 mg.

[0242] Therapeutic use of antibodies

[0243] The present disclosure includes methods of administering to a subject in need a therapeutic composition comprising an anti-FXI antibody (e.g., an anti-FXI antibody comprising any of the HCVR / LCVR or CDR sequences listed in Tables 1A-1C herein). The therapeutic composition can include any one or more of the anti-FXI antibodies or antigen-binding fragments thereof disclosed herein and a pharmaceutically acceptable carrier or diluent.

[0244] The antibodies of the present disclosure are particularly useful for treating, preventing, and / or ameliorating 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, especially thrombosis of the intrinsic pathway, while minimizing the negative impact on hemostasis and clot formation achieved through the extrinsic pathway.

[0245] The present disclosure includes methods of treating or preventing thrombosis by administering an anti-FXI antibody or an antigen-binding fragment thereof to a patient in need thereof, as disclosed elsewhere herein.

[0246] In one embodiment, the anti-FXI antibodies of the present disclosure can provide methods for treating or preventing thrombosis associated with any one or more of the following: Factor V Leiden; prothrombin gene mutations; deficiencies in natural proteins that prevent blood clotting (such as antithrombin, protein C, and protein S); elevated levels of homocysteine; elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia); elevated levels of Factor VIII, Factor IX, and / or Factor XI; abnormal fibrinolytic systems, including hypoplasminogenemia, dysplasminogenemia, 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; central venous catheter placement; obesity; pregnancy; use of supplemental estrogen, including oral contraceptives (birth control pills); hormone replacement therapy; prolonged bed rest or immobility; heart attack; congestive heart failure; stroke, and other diseases that result in reduced activity; heparin-induced thrombocytopenia (reduction in platelets in the blood due to heparin or low molecular weight heparin preparations); long-haul air travel; antiphospholipid antibody syndrome; deep vein thrombosis or pulmonary embolism; myeloproliferative disorders, such as polycythemia vera or essential thrombocythemia; paroxysmal nocturnal hemoglobinuria; inflammatory bowel syndrome; HIV / AIDS; nephrotic syndrome; COVID-19 infection or spike protein immune effects, etc.

[0247] In the context of the treatment methods described herein, the anti-FXI antibodies can be administered as a monotherapy (i.e., as the sole therapeutic agent) or in combination with one or more additional therapeutic agents.

[0248] Combination Therapies and Formulations

[0249] The present disclosure encompasses compositions and therapeutic formulations comprising combinations of any of the anti-FXI antibodies described herein with one or more additional therapeutically active components, as well as methods of treatment comprising administering such combinations to a subject in need thereof.

[0250] The anti-FXI antibodies of the present disclosure may be formulated with one or more drugs used to treat: factor V leiden; prothrombin gene mutations; defects in natural proteins that prevent clotting (such as antithrombin, protein C, and protein S); elevated levels of homocysteine; elevated levels of fibrinogen or dysfunctional fibrinogen (dysfibrinogenemia); elevated levels of factor VIII, factor IX, and / or factor XI; abnormal fibrinolytic system, including hypoplasminogenemia, dysplasminogenemia, 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); trauma or surgery during pregnancy; placement of a central venous catheter; obesity; pregnancy; use of supplemental estrogen, including oral contraceptives (birth control pills); hormone replacement therapy; prolonged bed rest or immobility; heart attack; congestive heart failure; stroke and other conditions that lead to reduced activity; heparin-induced thrombocytopenia (a decrease in platelets in the blood due to heparin or low molecular weight heparin preparations); long-distance airplane travel; antiphospholipid antibody syndrome; deep vein thrombosis or pulmonary embolism; myeloproliferative disorders such as polycythemia vera or essential thrombocythemia; nocturnal hemoglobinuria; inflammatory bowel syndrome; HIV / AIDS; nephrotic syndrome; COVID-19 infection or immune effects of the spike protein, etc.

[0251] The anti-FXI antibodies of the present disclosure may also be administered and / or co-formulated with antiviral drugs, 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.

[0252] The additional therapeutically active component, e.g., any of the agents listed above, or derivatives thereof, may be administered just prior to, simultaneously with, or shortly after administration of an anti-FXI antibody of the disclosure; (for purposes of the present disclosure, such administration regimens are considered to be administration of an anti-FXI antibody in combination with an additional therapeutically active component). The present disclosure encompasses pharmaceutical compositions in which an anti-FXI antibody of the disclosure is co-formulated with one or more of the additional therapeutically active components as described elsewhere herein.

[0253] Administration regimen

[0254] In 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 additional therapeutic active agent mentioned herein) can be administered to a subject over a defined time course. Methods according to this aspect of the present disclosure include sequentially administering multiple doses of the anti-FXI antibody of the present disclosure to a subject. As used herein, "sequentially administering" means that each dose of the anti-FXI antibody is administered to the subject at different time points, e.g., on different dates separated by a predetermined interval (e.g., hours, days, weeks, or months). The present disclosure encompasses methods that include sequentially administering a single initial dose of an anti-FXI antibody to a patient, followed by one or more second doses of the anti-FXI antibody, and optionally followed by one or more third doses of the anti-FXI antibody.

[0255] The terms "initial dose," "second dose," and "third dose" refer to the time sequence of administering the anti-FXI antibody of the present disclosure. Thus, an "initial dose" is the dose administered at the start of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is the dose administered after the initial dose; and a "third dose" is the dose administered after the second dose. The initial dose, second dose, and third dose can all contain the same amount of the anti-FXI antibody, but typically may differ from each other in terms of the frequency of administration. However, in certain embodiments, during the course of treatment, the amounts of the anti-FXI antibody contained in the initial dose, second dose, and / or third dose differ from each other (e.g., adjusted up or down as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0256] In certain exemplary embodiments of the present disclosure, 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、111 / 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 1 / 2 、24, 24 1 / 2 、25, 25 1 / 2 、26, 26 1 / 2 or more) weekly administrations of each second and / or third dose. As used herein, the phrase "immediately following the previous dose" means a dose of the anti-FXI antibody administered to a patient prior to the administration of the next dose in sequence in a series of administrations, without an intervening dose.

[0257] The method according to this aspect of the present disclosure may include administering to a patient any number of second and / or third doses of the anti-FXI antibody. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) third doses are administered to the patient. The administration regimen may be carried out indefinitely during the lifetime of a particular subject, or until such treatment is no longer therapeutically required or beneficial.

[0258] In embodiments involving multiple second doses, each second dose can be administered at the same frequency as the other second doses. For example, each second dose can be administered to the patient 1 to 2 weeks or 1 to 2 months after the previous dose. Similarly, in embodiments involving multiple third doses, each third dose can be administered at the same frequency as the other third doses. For example, each third dose can be administered to the patient 2 to 12 weeks after the previous dose. In certain embodiments of the present disclosure, during the course of the treatment regimen, the frequency of administering the second and / or third doses to the patient can vary. The doctor can also adjust the administration frequency during the course of treatment, depending on the needs of the individual patient after clinical examination.

[0259] The present disclosure encompasses an administration regimen in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administering two or more maintenance doses to the patient on a less frequent basis. For example, according to this aspect of the present disclosure, if the loading doses are administered at a monthly frequency, the maintenance doses can be administered to the patient once every six weeks, once every two months, once every three months, etc.

[0260] Diagnostic uses of the antibody

[0261] 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, the anti-FXI antibody or a fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression of FXI (e.g., overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for FXI can include, for example, contacting a sample obtained from a patient with an anti-FXI antibody of the present disclosure, wherein the anti-FXI antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-FXI antibody can be combined with a secondary antibody that is itself detectably labeled for diagnostic applications. The detectable label or reporter molecule can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure FXI in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0262] Samples that can be used in the FXI diagnostic assays according to the present disclosure include any tissue or fluid sample obtainable from a patient that contains a detectable amount of FXI protein or fragment thereof under normal or pathological conditions. Typically, the FXI level in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal FXI levels or activity) is measured to initially establish a baseline or standard level of FXI. This FXI baseline level can then be compared to the FXI level measured in a sample obtained from an individual suspected of having an FXI-related disease or condition.

[0263] Examples

[0264] The following examples are presented in order to provide a complete disclosure and description to those of ordinary skill in the art of how to make and use the methods and compositions of the present disclosure, and are not intended to limit the scope that the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, room temperature is about 25 °C, and pressures are atmospheric or near atmospheric.

[0265] Example 1: Generation of Human Antibodies Against the CAT Domain of FXI

[0266] Human antibodies against the CAT domain of FXI were generated in mice comprising DNA encoding the variable region of the human immunoglobulin heavy chain and the variable region of the κ light chain. In one embodiment, in mice. In one embodiment, mice were immunized with human FXI (VI). The antibody immune response was monitored by FXI-specific immunoassays. For example, the specific antibody titer of serum against purified full-length FXI was determined. Antibody-producing clones were isolated using both B cell sorting technology (BST) and the hybridoma method. For example, when the desired immune response was reached, spleen cells 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 the cell lines producing FXI-specific antibodies.

[0267] Anti-FXI antibodies were also isolated directly from antigen-positive mouse B cells without fusion to myeloma cells, as described in U.S. Patent 7,582,298, which is specifically incorporated by reference in its entirety. Using this method, several fully human anti-FXI antibodies (i.e., antibodies having human variable domains and human constant domains) were obtained.

[0268] The biological properties of exemplary antibodies, controls, and comparators generated by the methods according to this example are described in detail in the examples listed below.

[0269] Example 2: Heavy and light chain region sequences

[0270] Table 1A lists the amino acid sequence identifiers of the heavy and light chain regions of exemplary anti-FXI antibodies of the present disclosure. Tables 1B and 1C list the nucleic acid (DNA) and amino acid (PEP) sequence identifiers of the heavy and light chain regions of the antibodies of the present disclosure.

[0271] Table 1A: Anti-FXI antibody sequences of exemplary antibody REGN7508

[0272]

[0273] Table 1B: Heavy chain sequences (SEQ ID NO) of exemplary antibodies REGN7508 and REGN9932

[0274] VH CDR1 CDR2 CDR3 HC Antibody DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP REGN7508 1 2 3 4 5 6 7 8 17 18 REGN9932 21 22 23 24 25 26 27 28 37 38

[0275] Table 1C: Light chain sequences (SEQ ID NO) of exemplary antibodies REGN7508 and REGN9932

[0276] VK CDR1 CDR2 CDR3 LC Antibody DNA PEP DNA PEP DNA PEP DNA PEP DNA PEP REGN7508 9 10 11 12 13 14 15 16 19 20 REGN9932 29 30 31 32 33 34 35 36 39 40

[0277] Exemplary full-length anti-FXI antibodies contain a fully human Fcγ4 heavy chain (i.e., IgG4 Fc) and a fully human light chain sequence. However, as will be understood by those of ordinary skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4, etc.), but in any case, the variable domains (including the CDRs) will remain the same, and regardless of the nature of the Fc domain, the binding characteristics of the antigen are expected to be the same or substantially similar.

[0278] Example 3: Biacore binding kinetics of anti-FXI monoclonal antibodies binding to different FXI reagents measured at 25 °C and 37 °C

[0279] The purpose of this experiment was to determine the kinetics and specificity of the binding of hFXI(ERL), hFXIa(ERL), hFXI.mmh(REGN3848), and mfFXI.mmh(REGN3883) to anti-FXI mAb and comparator mAb at 25 °C and 37 °C. The comparator mAb (REGN6166 or COMP6166) can be found, for example, in U.S. Patent 10,465,011.

[0280] Materials

[0281] Instruments used: Biacore 8k and T200-RED

[0282] Temperature: 25 °C and 37 °C

[0283] Running buffer: HBS-P and 300 mM NaCl, pH 7.4

[0284] Sensor type: anti-human Fc mAb (REGN2567)

[0285] Flow rate / time: 30 μL / min hFXI association - 180 s hFXI dissociation 600 s

[0286] Method

[0287] Approximately 27.1–41.9 RU of anti-FXImAb was captured on anti-hFc mAb (REGN2567) on the CM5 sensor surface. Next, stock solutions of 30 nM hFXI(ERL), hFXIa(ERL), hFXI.mmh(REGN3848), and mfFXI.mmh(REGN3883) were prepared and serially diluted 3-fold to prepare 10 nM, 3.3 nM, and 1.1 nM solutions. Then, all FXI solutions were injected at 30 μL / min on a Biacore 8K for 180 s, and dissociation was monitored for 10 min.

[0288] Results

[0289] Table 2: Kinetics of hFXI(ERL) binding to α-FXI mAb and comparator mAbs at 25 °C and 37 °C

[0290]

[0291] NB = No binding

[0292] At 25 °C and 37 °C, anti-FXImAb bound to human FXI(ERL) with K D values in the ranges of 4.54 pM - 119 pM and 3.09 pM – 36.5 pM, respectively.

[0293] Table 3: Kinetics of hFXIa(ERL) binding to α-FXI mAb and comparator mAbs at 25 °C and 37 °C

[0294]

[0295] NB = No binding

[0296] At 25 °C and 37 °C, anti-FXImAb bound to human FXIa(ERL) with K D values of 31.1 pM and 25.6 pM, respectively.

[0297] Table 4: Kinetics of binding of hFXI.mmh to α-FXI mAb and comparator mAbs at 25 °C and 37 °C

[0298]

[0299] At 25 °C and 37 °C, the anti-FXI mAbs bound to human FXI.mmh (REGN3848) with K D values of 5.34 pM – 116 pM and 32.6 pM – 387 pM, respectively.

[0300] Table 5: Kinetics of binding of mfFXI.mmh to α-FXI mAb and comparator mAbs at 25 °C and 37 °C

[0301]

[0302] At 25 °C and 37 °C, the anti-FXI mAbs bound to monkey FXI.mmh (REGN3883) with K D values of 4.81 pM – 64.4 pM and 43.5 pM – 268 pM, respectively.

[0303] Example 4: Activated partial thromboplastin time bioassay

[0304] Experimental procedure:

[0305] The BIOPHEN Factor XIa Kit (HYPHEN BioMed, Neuville-sur-Oise, FR), catalog number 220412) was used to evaluate the ability of the anti-FXI antibodies of the present disclosure to inhibit the activity of the zymogen factor XI (FXI) or pre-activated FXIa that results in the production of active factor Xa (FXa). Inhibition by the antibodies of the present disclosure was determined by measuring the decrease in the amount of chromogenic substrate converted by FXa (BIOPHEN kit component R3). All reagents in the BIOPHEN kit were used in the assay, except for reagent 1B (human factor IX) and the FXIa calibrator (Cal).

[0306] To test the dose-dependent activity of FXI or FXIa, normal human plasma, along with a plasma-free control, was initially diluted to 0.65% plasma in the provided Tris-BSA buffer (used as a dilution buffer for the assay) and then serially diluted down to 0.021% plasma. Normal human plasma was also diluted to 0.13% or 0.15% plasma. Antibodies (anti-FXI, control, and comparator) were serially diluted from a starting concentration of 500 nM or 300 nM to a concentration of 5.1 pM in separate buffer samples. To inhibit zymogen FXI, the anti-FXI antibody was pre-incubated with the diluted plasma at 25 °C for 30 minutes and then incubated with 0.32 μM aPTT-XL ellagic acid at 25 °C for an additional 30 minutes. To inhibit active FXIa, the diluted plasma was pre-activated with 0.32 μM aPTT-XL ellagic acid at 25 °C for 30 minutes and subsequently incubated with the anti-FXI antibody at 25 °C for 30 minutes.

[0307] After incubation of the plasma with ellagic acid and the antibody, reagent 1A (human FX, FVIII:C, fibrin polymerization inhibitor) was added and incubated at 37 °C for 5 minutes. Then, reagent 2 (thrombin, phospholipid, and calcium) was 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 at a wavelength of 405 nm on a FLEXSTATION 3 microplate reader (Molecular Devices, Sunnyvale, CA). The results were analyzed using nonlinear regression (4-parameter logistic) with Prism 6 software (GraphPad, La Jolla, CA) to obtain EC 50 and IC 50 values. The percent inhibition was calculated based on Equation 2 below:

[0308] Equation 1:

[0309] In this equation, “Absorbance 稀释血浆 ” refers to the absorbance measurement at 405 nm of diluted plasma (0.13% or 0.15% plasma) that has been activated with 0.32 μM aPTT-XL ellagic acid to cleave FXI to FXIa without the addition of any antibody. “Absorbance 抑制 ” refers to the minimum absorbance measurement at 405 nm from the dose response of a specific antibody with diluted plasma activated with 0.32 μM ellagic acid. “Absorbance 无血浆对照" refers to the absorbance measurement result of the Tris-BSA buffer alone at 405 nm in the absence of any plasma.

[0310] Summary of tabular data:

[0311] Table 6: Inhibition of FXI activation and FXIa or pre-activated FXIa activity by anti-FXI / FXIa antibodies in the aPTT-XL ellagic acid assay.

[0312]

[0313] Results summary and conclusion:

[0314] As shown in Table 6, the anti-FXI / FXIa antibody showed inhibition of FXI in diluted normal plasma, with an IC50 value of 190 pM and a maximum inhibition range of 87%. The anti-FXI / FXIa antibody of the present disclosure also inhibited FXIa in diluted plasma, with an IC50 value greater than 10 nM and a maximum inhibition of 35%. The comparator mAb showed inhibition of FXI, with an IC50 value of 38 pM and a maximum inhibition of 108%. The comparator mAb also showed inhibition of FXIa, with an IC50 value of 480 pM and a maximum inhibition of 95%. The isotype control mAb did not show inhibition of FXIa, but showed inhibition of FXI at high antibody concentrations, with an IC50 value in the range of >100 nM - 120 nM, where the maximum inhibition was in the range of 58% - 102% inhibition.

[0315] Example 5: Complex formation

[0316] 5.1: Size analysis of the complex formed between human coagulation factor XI and the subject mAb [REGN7508]

[0317] Experimental conditions

[0318] Sample Preparation

[0319] Solutions of anti-hFXI mAb with hFXI were prepared at equimolar ratios and allowed to incubate at room temperature for 2 hours. After incubation, the complex solution was injected into the column.

[0320] SEC - MALS Conditions

[0321] The samples were fractionated on a tandem Waters ACQUITY UPLC 200SEC column (1.7 μm, 4.6 mm × 150 mm) that was pre-equilibrated in 10 mM sodium phosphate, 500 mM sodium chloride (pH 7.0) at a flow rate of 0.3 mL / min.

[0322] Three online detectors monitored the protein species eluting from the column: an absorbance detector (280 nm), a multi-angle light scattering (MALS) detector, and a refractive index detector.

[0323] Data Analysis

[0324] The molar mass was determined by protein conjugate analysis of the free ligand and free drug samples. The complex samples used the modified dn / dc and UV values determined from the protein conjugate analysis of the free samples.

[0325] Using Astra TM software version 7.3.1.9 for data analysis.

[0326] Results

[0327] Overall, as Figure 1 seen, REGN7528 and REGN7531 showed the clearest evidence of extensive "paper-dolling".

[0328] In contrast, and as Figure 2 seen, REGN7503 and REGN7505 appeared to form smaller, less heterogeneous complexes; only the subject mAb [REGN7508] showed the presence of a 1:1 complex with FXI.

[0329] Table 7: Total average Mw (kDa) from SEC-MALS

[0330]

[0331] Discussion

[0332] 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 in order to eliminate mAbs showing extensive "paper-dolling" and provide additional characterization to support time-sensitive decisions.

[0333] SEC-MALS analysis was chosen for convenience, but due to the limited resolution range of the column, detailed interpretation of stoichiometry was prohibited; only qualitative comparisons could be made from this data.

[0334] Overall, among the panel of mAbs, REGN7528 (catalytic domain) and REGN7531 (A3 / A4 domain) showed the clearest evidence of extensive "paper-dolling".

[0335] In contrast, REGN7503 (A3 / A4 domain) and REGN7505 (A3 / A4 domain) appear to form more homogeneously distributed complexes with a smaller total average molar mass; however, the subject mAb [REGN7508] (catalytic domain) is the only mAb in this group that shows a significant 1:1 complex with FXI as previously observed by A4F-MALS.

[0336] 5.2 Size analysis of the complex formed between human coagulation factor XI and the subject mAb [REGN7508]

[0337] Experimental conditions

[0338] Sample Preparation

[0339] Samples were prepared in 1X DPBS (pH 7.4) and allowed to incubate for 2 hours at room temperature before fractionation of the total protein by A4F-MALLS (PostNova).

[0340] A4F - MALLS Conditions

[0341] 7 mg of the complex or 4 mg of the mAb or ligand was injected onto an A4F short channel equipped with a 350W spacer and a 10 kDa regenerated cellulose membrane with a mobile phase of 10 mM sodium phosphate, 500 mM NaCl, pH 7.0, and separated using the following gradient.

[0342] Flow rate:

[0343] ● Channel flow: 1.0 mL / min

[0344] ● Focusing flow: 1.0 mL / min for 4 minutes.

[0345] ● Cross flow: 3.0 mL / min to 0 mL / min linear gradient over 45 minutes, followed by 0 mL / min for 10 minutes.

[0346] Data Analysis

[0347] Molar mass was determined by protein conjugate analysis of free ligand and free antibody samples. Complex samples used the modified dn / dc and UV values determined from protein conjugate analysis of free samples.

[0348] Results

[0349] Table 8: Subject mAb:FXI complex and theoretical molar mass

[0350]

[0351] 1 Unequal ratios (such as 1:2 and 2:1) cannot be distinguished because they will have similar MW

[0352] As seen in Table 8 (above) and Figure 3 as seen herein, the subject mAb [REGN7508] (catalytic domain) favors lower order complexes, where the major species represent 1:1 and 2:2 discrete complexes with hFXI.

[0353] Table 9: mAb:FXI Complexes and Theoretical Molecular Weights

[0354]

[0355] 1 Unequal ratios (such as 1:2 and 2:1) cannot be distinguished because they will have similar MW

[0356] As seen in Table 9 (above) and Figure 4 as seen herein, [REGN9932] (catalytic domain) forms the highest proportion of lower order complexes, where the major species represent 1:1 and 2:2 discrete complexes with hFXI.

[0357] As Figure 5 seen herein, the molar mass and distribution of the complexes formed between the subject mAb [REGN7508] (catalytic domain) and hFXI are comparable to those previously observed with [REGN9932] (catalytic domain).

[0358] Discussion

[0359] Asymmetric flow field-flow fractionation coupled to multi-angle laser light scattering (A4F-MALLS) was used to evaluate the relative size distribution of the complexes formed between human coagulation factor XI (hFXI, from Enzyme Research Laboratory) and several anti-hFXI mAbs (subject mAbs [REGN7508] and [REGN9932]).

[0360] The subject mAb [REGN7508] (catalytic domain) favors lower order complexes, where the major species represent 1:1 and 2:2 discrete complexes with hFXI when mixed at different molar ratios.

[0361] The molar mass and size distribution observed for the complexes formed between the subject mAb [REGN7508] (catalytic domain) and hFXI are comparable to those previously observed with [REGN9932] (catalytic domain).

[0362] Example 6: Thrombin Generation Assay (TGA)

[0363] The TGA was performed to measure the following endpoints:

[0364] ● Lag time (minutes), clotting time

[0365] ● Thrombin peak (nM)

[0366] ● ttPeak: Time to peak (minutes)

[0367] ● ETP: Endogenous thrombin potential (nM * minutes) (ETP; area under the curve)

[0368] ● Velocity: Peak (nM) / ttPeak (minutes)

[0369] 6.1: TGA profile in pooled human plasma

[0370] Results

[0371] Table 10: Anti-FXI / FXIa test profile in pooled human plasma

[0372]

[0373] NC = No change

[0374] 6.2: TGA profile in cynomolgus monkey plasma (female)

[0375] The comparison of the effects of anti-FXI mAb on the intrinsic pathway thrombin generation in cynomolgus monkey plasma (female) can be seen in Figure 6A , 6B , 6C.

[0376] The comparison of the effects of anti-FXI mAb on the intrinsic pathway thrombin generation in cynomolgus monkey plasma (female) can be seen in Figure 7A , 7B , 7C.

[0377] 6.3: TGA profile in pooled female plasma

[0378] The comparison of the effects of anti-FXImAb on the intrinsic pathway thrombin generation in pooled female plasma can be seen in Figure 8A , 8B , 8C.

[0379] The comparison of the effects of anti-FXImAb on the intrinsic pathway thrombin generation in pooled female plasma can be seen in Figure 9A , 9B , 9C.

[0380] 6.4: Comparison of Thrombin Generation by Anti-FXI / FXIa mAbs in Six Single Donors

[0381] The comparison of the effect of the anti-FXI mAb [REGN9932] on intrinsic pathway thrombin generation in six single donors can be seen in Figure 10A 、 10B 、10C.

[0382] The comparison of the effect of the subject anti-FXI mAb [REGN7508] on intrinsic pathway thrombin generation in six single donors can be seen in Figure 11A 、 11B 、11C.

[0383] The comparison of the effect of the anti-FXI mAb [REGN9932] on extrinsic pathway thrombin generation in six single donors can be seen in Figure 12A 、 12B 、12C.

[0384] The comparison of the effect of the subject anti-FXI mAb [REGN7508] on extrinsic pathway thrombin generation in six single donors can be seen in Figure 13A 、 13B 、13C.

[0385] Example 7: Pharmacokinetic Study of the Subject Anti-FXI Antibody Drug Substance in Cynomolgus Monkeys

[0386] The purpose 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.

[0387] Plasma was collected at the following time points: before dosing, 5 minutes, 6 hours, day 1, day 2, day 3, day 5, day 7, day 14, day 21, day 28, day 35, day 42, day 49, and day 56.

[0388] The measured endpoints were as follows:

[0389] 1) Target level (total)

[0390] 2) hFc level (total)

[0391] 3) aPTT / PT

[0392] 4) TGA-EA / TF

[0393] 5) Modified FXIa Activity Assay (Biophen)

[0394] 6) CBC (starting at 72 hours)

[0395] Table 11: Summary of Doses and Dose Groups

[0396]

[0397] *REGN9932 will be used as a "bridge" from a previous PK / PD study

[0398] Example 8: Determine the kinetic and equilibrium binding parameters of the subject's anti-FXI antibody drug substance interacting with FXI from human, cynomolgus monkey, rabbit, and mouse, and FXIa from human

[0399] 8.1: Experimental Design

[0400] Surface plasmon resonance (SPR) experiments were performed on a Biacore instrument to determine the binding affinity of REGN7508 for plasma-derived human FXI and FXIa proteins and recombinant mmH-tagged forms of human, cynomolgus monkey, rabbit, and mouse FXI proteins. At pH 7.4 and 25 °C (for all FXI and FXIa proteins) or 37 °C (for plasma-derived human FXI / FXIa only), different concentrations of plasma-derived and recombinant FXI or FXIa proteins were injected onto REGN7508 captured on the sensor surface, followed by a dissociation phase. Changes in the binding signal were recorded, specific binding signals were calculated, and kinetic binding parameters were determined by fitting the data to a 1:1 binding model with mass transport limitations

[0401] Covalent coupling of anti-human FcG antibody to the surface of the sensor chip

[0402] Mouse anti-human FcG monoclonal antibody (anti-hFcG) was immobilized on the surface of the sensor chip using standard amine coupling chemistry. Filtered and degassed HBS-P (10 mM HEPES, 300 mM NaCl, 0.05% (v / v) polysorbate 20, pH 7.4) was used as the running buffer for the coupling procedure at a flow rate of 10 μL / min. The sensor surface was activated by injecting a 1:1 (by volume) mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.1 M N-hydroxysuccinimide onto the chip for 7 minutes. After surface activation, anti-hFcG (20 μg / mL) prepared in 10 mM sodium acetate (pH 5.0) was injected onto the activated chip surface for 7 minutes. The remaining reactive 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. Then, before performing kinetic binding experiments, the sensor chip surface was treated by injecting 20 mM phosphoric acid at least 10 times, 12 seconds each time, to remove all uncoupled residual proteins and washed with the running buffer HBS-P

[0403] Kinetics of the binding interaction of REGN7508 with FXI and FXIa proteins

[0404] The binding of FXI and FXIa proteins to REGN7508 (Lot 9048800001 [all FXI / FXIa proteins] and Lot REGN7508-L2 [human FXI / FXIa only]) was measured at pH 7.4 and 25 °C or 37 °C using HBS-P as the running buffer. REGN7508 was captured by surface-coupled anti-hFcG until a signal of 59 - 101 RU was reached. Plasma-derived and recombinant FXI and FXIa proteins were serially diluted 2-fold to concentrations in the 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 were injected separately onto the captured REGN7508 surface at a flow rate of 50 μL / min for 1 minute (25 °C) or 0.5 minute (37 °C), followed by a 20-minute dissociation phase, and the resulting changes in the binding signal were recorded. Each concentration was tested in duplicate.

[0405] Specific binding signals were obtained by a double-reference procedure and were plotted as SPR sensorgrams. Double-reference was performed as follows: First, the signal of each injection on the reference surface (anti-hFcG) was subtracted from the signal on the experimental surface (anti-hFcG captured REGN7508), thereby removing the effects caused by refractive index changes. Additionally, running buffer injections were performed to allow subtraction of the signal changes caused by dissociation of the captured REGN7508 from the coupled anti-hFcG surface. Kinetic parameters were obtained by globally fitting these specific binding signals to a 1:1 binding model with mass transport limitations. The equilibrium dissociation constant (KD) was calculated as the ratio of the dissociation rate constant to the association rate constant (KD = kd / ka). The dissociation half-life (t 1 / 2 ) was calculated by dividing 0.693 (natural logarithm of 2) by the experimentally determined kd.

[0406] 8.2: Results

[0407] Binding parameters for the interaction of REGN7508 with FXI and FXIa

[0408] The kinetic binding parameters of REGN7508 with FXI proteins from human, cynomolgus monkey, rabbit, and mouse, and with human 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 were performed at 25 °C and pH 7.4 using injections of a range of concentrations of FXI or FXIa proteins onto a captured REGN7508 sensor surface. The calculated kinetic binding parameters are summarized in Table 12.

[0409] In SPR experiments conducted at 25 °C and pH 7.4, REGN7508 bound to plasma-derived hFXI and hFXIa, as well as recombinant human FXI (hFXI.mmH) with picomolar affinity.

[0410] REGN7508 also bound to recombinant cynomolgus monkey FXI (MfFXI.mmH) with picomolar affinity. Up to the highest concentrations tested (250 nM or 50 nM, respectively), REGN7508 did not bind to recombinant rabbit (rbFXI.mmH) or mouse (mFXI.mmH) proteins, indicating binding specificity for human and cynomolgus monkey FXI.

[0411] The kinetic binding parameters of individual batches of REGN7508 with human FXI and FXIa were also determined. REGN7508 with lot number 9048800001, described above and used in the toxicology study, and REGN7508 with lot number REGN7508-L2, used in several non-clinical in vitro and in vivo pharmacology studies, showed similar binding affinities for human FXI and FXIa.

[0412] Table 12: Summary of kinetic binding parameters of REGN7508 interaction with FXI and FXIa at 25 °C and pH 7.4

[0413]

[0414] ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant; t1 / 2: dissociation half-life; NB, no detectable binding was observed under the assay conditions used.

[0415] The equilibrium dissociation constants (KD) of REGN7508 for plasma-derived human FXI and FXIa were 3.00 pM 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 pM and 3.73 pM, respectively, but did not show 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).

[0416] Example 9: In Vitro Functional Characterization of REGN7508

[0417] 9.1: Experimental Design

[0418] The ability of REGN7508 to block the coagulation pathway in human and cynomolgus monkey donor plasma was evaluated in vitro using coagulation assays and thrombin generation assays (TGA). The effects on the intrinsic coagulation pathway were measured based on aPTT and thrombin generation induced by EA, while the effects on the extrinsic coagulation pathway were measured by PT and thrombin generation induced by TF( Figure 14 ).

[0419] The aPTT test evaluates all the clotting factors in the intrinsic and common pathways of the coagulation cascade by measuring the time to form a clot after addition of calcium and EA. The PT test evaluates all the clotting factors in the extrinsic and common pathways of the coagulation cascade after addition of calcium and TF.

[0420] TGA measurements induced by EA measure the rate and amount of thrombin generated through the intrinsic and common pathways. TGA measurements induced by TF measure the rate and amount of thrombin generated through the extrinsic and common pathways.

[0421] aPTT assay (for intrinsic pathway activity)

[0422] The aPTT in human or cynomolgus monkey donor plasma in the presence of REGN7508 or IgG4P isotype control was determined using a STart4 Hemostasis Analyzer. Plasma samples (50 μL) were added to In cuvettes and incubated together with 2-fold serial dilutions of REGN7508 (9 nM to 1.2 μM) or IgG4P isotype control (19 nM to 1.2 μM) at 37 °C for 5 minutes; an antibody-free control cuvette containing PBS in place of the antibody was also included for baseline measurements. A second set of REGN7508 concentrations was also tested at 4 nM increments from 4 nM to 28 nM and 31 nM. 50 μL of aPTT-XL EA was added for a 5-minute incubation, followed by the addition of 50 μL of 20 mM calcium chloride to initiate the reaction. The measured clotting time of each test sample was normalized to the plasma clotting time of the antibody-free control. The mean change relative to the antibody-free control for each concentration (run in duplicate) was plotted against the antibody concentration.

[0423] The concentration at which aPTT doubles (doubling time) is C2xt and this concentration was determined in the second set of experiments where the antibody concentration range (4 nM to 31 nM) was tested in smaller 4 nM increments. In the aPTT clotting time, values in seconds were generated relative to the baseline (i.e., antibody-free control or PBS only value), which corresponds 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 will be placed at twice the baseline value in seconds or for mAb values, corresponding to a 2.0-fold change in aPTT, relative to the baseline in GraphPad Prism.

[0424] PT assay (for extrinsic pathway activity)

[0425] The PT in human or cynomolgus monkey donor plasma in the presence of REGN7508 or IgG4P isotype control was determined using a STart4 Hemostasis Analyzer. Plasma samples (50 μL) were added to cuvettes and incubated together with REGN7508 or IgG4P isotype control (600 nM and 1.2 μM) at 37 °C for 5 minutes; an antibody-free control plasma sample containing PBS in place of the antibody was also included for baseline measurements. TriniCLOT PT Excel S (TF and calcium, 100 μL) was added to initiate the reaction. The measured clotting time of each test sample was normalized to the plasma clotting time of the antibody-free control. The mean change relative to the antibody-free control for each concentration (run in duplicate) was plotted against the antibody concentration.

[0426] Thrombin generation assay

[0427] Using Calibrated Automated The platform determines the thrombin generation curves of REGN7508 or IgG4P isotype control in human or cynomolgus monkey donor plasma. Thrombin activity is measured by monitoring the cleavage of a fluorogenic substrate and comparing it to the constant, known thrombin activity in non - clotting samples evaluated in parallel. Plasma samples (55 μL) are added to the wells of an Immulon II HB U - bottom microplate and incubated with 2 - fold serial dilutions of REGN7508 or IgG4P isotype control in the range of 16 nM to 500 nM at 37 °C for 30 minutes; antibody - free control wells containing PBS in place of the antibody are also included for baseline measurements. A second set of REGN7508 concentrations for intrinsic pathway activity only is also tested at 4 nM increments from 4 nM to 31 nM; the IgG4P isotype control is tested at 31 nM.

[0428] Thrombin generation is then induced by adding 15 μL of aPTT - XL EA (intrinsic pathway activity) pre - diluted in MP reagent or 15 μL of PPP reagent low TF (extrinsic pathway activity). After incubation at 37 °C for 45 minutes, immediately before continuous 90 - minute readings in an Immulon II HB U - bottom microplate, 15 μL of FluoCa buffer containing pre - warmed Fluo substrate is added to the wells. The measured real - time thrombin concentration values recorded within the first 60 minutes are plotted against time to generate a thrombinogram curve ( Figure 15 ) for each antibody concentration tested. The lag time, peak thrombin, and endogenous thrombin potential are determined from each thrombinogram.

[0429] 9.2: Results

[0430] 9.2.1: Role of REGN7508 in clotting assays using human or cynomolgus monkey donor plasma

[0431] Clotting assays using human plasma

[0432] REGN7508 increases aPTT in a concentration - dependent manner relative to baseline (antibody - free); where aPTT in human plasma increases up to 3.8 - fold at concentrations of 9 nM to 1.2 μM ( Figure 16A and Table 13) and at concentrations of 4 nM to 31 nM ( Figure 16B and Table 14) in the second set of experiments. When tested using a range of antibody concentrations with smaller increments (4 nM to 31 nM) in the second set of experiments, doubling of aPTT relative to baseline is estimated to occur at 16 nM in human plasma. At the highest antibody concentration tested (1.2 μM), in human ( Figure 16CNo change in PT relative to baseline was observed in plasma (and Table 13). For the IgG4P isotype control, no change in aPTT or PT relative to baseline was observed in human plasma up to the highest antibody concentration tested (1.2 μM).

[0433] Table 13: Summary of aPTT (9 nM to 1.2 μM) and PT (600 nM and 1.2 μM) in human donor plasma

[0434]

[0435] a Relative to baseline (i.e., no antibody); NT, not tested

[0436] Table 14: Summary of aPTT (4 nM to 31 nM) in human donor plasma

[0437]

[0438] a Relative to baseline (i.e., no antibody); NT, not tested

[0439] Coagulation assays performed with cynomolgus monkey plasma

[0440] REGN7508 increased aPTT relative to baseline (no antibody) in a concentration-dependent manner; wherein aPTT in cynomolgus monkey plasma increased up to 2.8-fold at concentrations of 9 nM to 1.2 μM ( Figure 17A and Table 15) and at a second set of concentrations tested from 4 nM to 31 nM ( Figure 17B and Table 16). In a second experiment, using a range of antibody concentrations with smaller increments (4 nM to 31 nM), doubling of 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 cynomolgus monkey ( Figure 17C and Table 15) plasma. For the IgG4P isotype control, no change in aPTT or PT relative to baseline was observed in cynomolgus monkey plasma up to the highest antibody concentration tested (1.2 μM).

[0441] Table 15: Summary of aPTT (9 nM and 1.2 μM) and PT (600 nM and 1.2 μM) in cynomolgus monkey donor plasma

[0442]

[0443] a Relative to baseline (i.e., no antibody); NT, not tested

[0444] Table 16: Summary of aPTT (4 nM to 31 nM) in cynomolgus monkey donor plasma

[0445]

[0446] a Relative to baseline (i.e., no antibody); NT, not tested

[0447] 9.2.2 Effect of REGN7508 in TGAs with human or cynomolgus monkey donor plasma

[0448] TGAs with human plasma

[0449] When thrombin generation is induced by EA through the intrinsic pathway in human plasma, REGN7508 increases the lag time of thrombin generation up to 6.6-fold relative to baseline (i.e., no antibody), reduces the peak thrombin level to 1% relative to baseline, and reduces the endogenous thrombin potential to 2% relative to baseline. REGN7508 exerts these effects in a concentration-dependent manner, with maximal effects achieved at concentrations ≥125 nM ( Figure 18A and Table 17). Using an IgG4P isotype control, no increase in the lag time of thrombin generation was observed up to the highest antibody concentration tested (500 nM), and only minor decreases in the peak thrombin level and endogenous thrombin potential relative to baseline were observed; these effects were not concentration-dependent ( Figure 18B and Table 17).

[0450] When tested in smaller increments, a second set of experiments was performed using REGN7508 concentrations in the range of 4 nM to 31 nM to obtain greater resolution of the thrombin peak at concentrations <16 nM; however, no gradual dose response was observed. A sharp change in the PD effect was observed at REGN7508 concentrations >16 nM, followed by a plateau. In addition, similar concentration-dependent effects on EA-induced thrombin generation were observed under other identical assay conditions ( Figure 19 and Table 18).

[0451] When thrombin generation is induced by TF through the extrinsic pathway in human plasma, REGN7508 partially reduces the peak thrombin level and endogenous thrombin potential to 66% and 68% relative to baseline, respectively. REGN7508 exerts these effects in a concentration-dependent manner, with maximal effects achieved at concentrations ≥125 nM. Using 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). Using an IgG4P isotype control, no increase in the lag time of thrombin generation or decrease in the peak thrombin or endogenous thrombin potential was observed up to the highest antibody concentration tested (500 nM) ( Figure 18Dand Table 19).

[0452] Table 17: Summary of parameters for measuring the intrinsic pathway TGA (16 nM to 500 nM) in human donor plasma

[0453]

[0454] a Changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) were relative to the no-antibody control (i.e., PBS).

[0455] Table 18: Summary of parameters for measuring the intrinsic pathway TGA (4 nM to 31 nM) in human donor plasma

[0456]

[0457] a Changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) were relative to the no-antibody control (i.e., PBS). NT: Not tested

[0458] Table 19: Summary of parameters for measuring the extrinsic pathway TGA (16 nM to 500 nM) in human donor plasma

[0459]

[0460] a Changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) were relative to the no-antibody control (i.e., PBS).

[0461] TGA performed with cynomolgus monkey plasma

[0462] When thrombin generation was induced by EA through the intrinsic pathway in cynomolgus monkey plasma, REGN7508 increased the lag time of thrombin generation up to 2.8-fold relative to baseline (i.e., no antibody), decreased the peak thrombin level to 6% relative to baseline, and decreased the endogenous thrombin potential to 15% relative to baseline. REGN7508 exerted these effects in a concentration-dependent manner, with maximal effects achieved at concentrations ≥500 nM ( Figure 20A and Table 20). Using an IgG4P isotype control, no increase in the lag time of thrombin generation or decrease in peak thrombin or endogenous thrombin potential was observed up to the highest antibody concentration tested (500 nM) ( Figure 20B and Table 20).

[0463] When testing in smaller increments, a second set of experiments was conducted using REGN7508 concentrations in the range of 4 nM to 31 nM to obtain greater resolution of the thrombin curve at concentrations less than 16 nM. However, a gradual dose response was not observed because once target saturation was reached, the effect was abrupt. Additionally, a similar concentration-dependent effect on EA-induced thrombin generation was observed under other identical assay conditions ( Figure 21 and Table 21).

[0464] When thrombin generation was induced by TF via the extrinsic pathway in cynomolgus monkey plasma, REGN7508 reduced the peak thrombin level to 55% relative to baseline and the endogenous thrombin potential to 65% relative to baseline. REGN7508 exerted these effects in a concentration-dependent manner, with maximum effects achieved at concentrations ≥125 nM ( Figure 20C and Table 22). Using REGN7508, no concentration-dependent increase in the lag time of thrombin generation was observed up to the maximum antibody concentration tested (500 nM). For the IgG4P isotype control, no increase in the lag time of thrombin generation or reduction in peak thrombin or endogenous thrombin potential was observed up to the maximum antibody concentration tested (500 nM) ( Figure 20D and Table 22).

[0465] Table 20: Summary of parameters for measuring the intrinsic pathway TGA (16 nM to 500 nM) in cynomolgus monkey donor plasma

[0466]

[0467] a Changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) are relative to the antibody-free control (i.e., PBS).

[0468] Table 21: Summary of parameters for measuring the intrinsic pathway TGA (4 nM to 31 nM) in cynomolgus monkey donor plasma

[0469]

[0470] a Changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) are relative to the antibody-free control (i.e., PBS). NT: Not tested.

[0471] Table 22: Summary of parameters for measuring the extrinsic pathway (16 nM to 500 nM) in cynomolgus monkey donor plasma

[0472]

[0473] a The changes in lag time, peak thrombin (%), and endogenous thrombin potential (ETP%) were relative to the no-antibody control (i.e., PBS).

[0474] Conclusion

[0475] REGN7508 mediates complete blockade of the intrinsic coagulation pathway in human and cynomolgus monkey plasma in a concentration-dependent manner. REGN7508 also mediates concentration-dependent partial blockade of the extrinsic coagulation pathway in human and cynomolgus monkey plasma, although to a much lesser extent than its effect on the intrinsic pathway.

[0476] Example 10: Evaluation of the binding of REGN7508-FXI immune complex to C1q

[0477] Circulating immune complexes (CICs) are formed by the polymerization of antibodies with soluble antigens. The deposition of CICs in tissues and subsequent inflammatory responses can lead to tissue damage at the deposition sites. Large immune complexes can also activate the complement component C1q in serum (Rojko, 2014).

[0478] REGN7508 is unlikely to form immune complexes capable of binding to C1q because it contains a hinge-stabilized IgG4-derived fragment crystallizable (Fc) constant domain (referred to as IgG4P), and IgG4 does not bind to C1q like IgG1 (Patel, 2015). Nevertheless, an enzyme immunoassay (EIA) was performed to evaluate the potential of REGN7508-FXI and REGN7508-FXIa complexes to bind to C1q.

[0479] REGN7508-FXI and REGN7508-FXIa complexes did not exhibit detectable binding to C1q, which is consistent with the minimal effector function activity of IgG4-based antibodies.

[0480] Example 11: Evaluation of the pharmacokinetics and toxicokinetics of REGN7508 in cynomolgus monkeys

[0481] Characterization of the blockade of the coagulation pathway in cynomolgus monkeys mediated by REGN7508 was evaluated as part of a single-dose pharmacokinetic (PK) study. The results of these studies showed that REGN7508 mediated inhibition of the intrinsic coagulation pathway with minimal effect on the extrinsic coagulation pathway. The results of these studies further showed no tolerance issues following subcutaneous and intravenous administration of REGN7508.

[0482] Informal Sequence Listing

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Claims

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

20.

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

10.

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

20.

4. An isolated antibody or antigen-binding fragment thereof, said isolated antibody or antigen-binding fragment thereof binds to the catalytic (CAT) domain of coagulation factor XI (FXI), wherein said antibody or antigen-binding fragment thereof comprises a heavy chain region (HC) and a light chain region (LC), said HC comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18, said 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 according to claim 4, wherein said HC comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 18, and wherein said LC comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

20.

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

20.

7. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to human FXI with a K of less than about 5 pM, as measured by surface plasmon resonance at 25 °C or 37 °C. D as measured by surface plasmon resonance at 25 °C or 37 °C.

8. The isolated antibody or antigen-binding fragment according to claim 7, wherein the antibody or its antigen-binding fragment has a K that is selected from the group consisting of D for binding to human FXI: 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.

9. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, 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. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has an IC 50 that inhibits the activation of human coagulation factor X (FX).

11. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has an IC 50 that inhibits the activation of human coagulation factor X (FX).

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

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

14. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof inhibits FXIa-mediated thrombin activity by at least 5%, at least 10%, at least 15%, or 5% - 15%.

15. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof prolongs the aPTT in human plasma by at least two-fold at a concentration of ≤100 nM, ≤75 nM, or ≤50 nM.

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

17. An isolated antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof inhibits thrombin generation or activation 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 thrombin generation or activation via the extrinsic coagulation pathway.

18. An isolated antibody or antigen-binding fragment, which competes with the antibody or antigen-binding fragment according to any one of the preceding claims for binding.

19. An isolated antibody or antigen-binding fragment, which binds to the same epitope as the antibody or antigen-binding fragment according to any one of the preceding claims.

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

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

22. A vector comprising the nucleic acid molecule according to claim 21.

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

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

25. The method according to claim 24, wherein the biological activity is thrombosis, and wherein thrombosis is inhibited upon contact of FXI with the antibody or antigen-binding fragment thereof.

26. The method according to claim 25, wherein the contact results in an increase in aPTT or a decrease in thrombin activity in plasma.

27. A method of treating or preventing a disease or disorder in a subject associated with FXI activity or expression, or for alleviating at least one symptom associated with the disease or disorder associated with FXI activity or expression, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20.

28. The method according to claim 27, wherein the disease or disorder is a blood coagulation disease or disorder in the subject, or a disease or disorder with an increased risk of developing thrombosis.

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

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