RU2847356C2 - Antibodies to factor xi and methods of their application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method involves administering to a subject suffering from thromboembolic disease or at risk of developing it an effective amount of a pharmaceutical composition containing an antibody or its antigen-binding fragment that binds human factor XI and activated factor XI. The use of said antibody or its antigen-binding fragment for the manufacture of a medicament for the treatment of thromboembolic disease is also proposed.

EFFECT: effective treatment of thromboembolic disease with a reduced risk of bleeding.

28 cl, 16 dwg, 12 tbl, 11 ex

Description

This application claims priority from U.S. Provisional Patent Application No. 62/184,955, filed June 26, 2015, and U.S. Provisional Patent Application No. 62/341,568, filed May 25, 2016, each of which is incorporated herein by reference in its entirety.

This application contains a sequence listing that was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 23, 2016, is named "PAT056955-WO-PCT_SL.txt" and is 45,685 bytes in size.

PREREQUISITES FOR THE CREATION OF THE INVENTION

Thrombosis is the formation of blood clots in blood vessels as a result of a combination of hereditary and acquired risk factors, known as thrombophilia or hypercoagulable states. Damage to vessel walls, blood stasis, increased platelet reactivity, and activation of coagulation factors are some of the main hallmarks of thrombosis. Thrombosis can occur in both the venous and arterial circulations and can lead to deep vein thrombosis (DVT), pulmonary embolism, and stroke. If thrombosis occurs in the arterial circulation, ischemia may occur in downstream areas, leading to acute coronary syndromes (ACS), ischemic stroke, and acute limb ischemia. Thrombus formation in the venous circulation typically leads to deep vein thrombosis, pulmonary embolism, and chronic thromboembolic pulmonary hypertension. Blood clots can also form in the left atrial appendage in patients with atrial fibrillation (AF), and ruptured thrombi can lead to potentially severe complications, including thromboembolic stroke and systemic embolism. The use of all currently known antithrombotic agents, including low molecular weight heparin (LMWH), thrombin inhibitors, and factor Xa (FXa) inhibitors, is associated with a significant risk of bleeding (Weitz J.I. (2010) Thromb. Haemost. 103, 62). The development of an antithrombotic agent that does not affect hemostasis and, therefore, does not lead to complications such as bleeding, would be highly desirable.

Modern anticoagulants are either injected or taken orally. The injectable anticoagulant LMWH is widely used and has an improved therapeutic profile compared to previously used unfractionated heparin. For the past several decades, warfarin has been the most commonly used oral anticoagulant. Warfarin has a narrow therapeutic window, requiring frequent monitoring of coagulation status, and it interacts with other medications in various ways. Recently, direct FXa and thrombin inhibitors, taken orally, have entered the anticoagulant market and are increasingly used.

All LMWH, FXa inhibitors, and thrombin inhibitors are effective in preventing postoperative venous thromboembolic disease (VTE), treating spontaneous DVT and pulmonary embolism, and preventing stroke in atrial fibrillation. However, these anticoagulants also cause bleeding complications, typically similar to those seen with older medications such as warfarin and unfractionated heparin. In the ADVANCE-2 clinical trial, the FXa inhibitor apixaban (Eliquis) was compared with the LMWH enoxaparin in patients undergoing total knee replacement. While apixaban was more effective than enoxaparin in preventing VTE in the short term, both agents were associated with a significant risk of bleeding. Clinically significant bleeding occurred in 4% of patients receiving apixaban and 5% of patients receiving enoxaparin (Lassen, MR, et al. (2009) N. Engl. J. Med. 361, 594).

In the RE-LY clinical trial, the direct thrombin inhibitor dabigatran (Pradaxa) was compared with warfarin in patients with atrial fibrillation at risk of stroke (Connolly, SJ, et al. (2009) N. Engl. J. Med. 361, 1139). Chronic dabigatran therapy was associated with a significantly lower risk of stroke or systemic embolism. However, major bleeding complications occurred in 3.1% of patients receiving 150 mg daily of dabigatran and in 3.4% of patients receiving warfarin (p=0.31).

Atrial fibrillation (AF) remains the most common cardiac arrhythmia in clinical practice, accounting for approximately one-third of hospitalizations for heart rhythm disorders. It currently affects an estimated 6 million patients in Europe and approximately 2.3 million in the United States, and these rates continue to rise rapidly due to the increasing aging population. It is estimated that approximately 5% of people over the age of 65 and 10% of people over the age of 80 will develop AF, but the increase in AF incidence goes beyond what can be explained by age alone. The prevalence of risk factors for AF, such as hypertension, congestive heart failure, left ventricular hypertrophy, coronary artery disease, and diabetes, as well as obstructive sleep apnea, is also increasing. Consequently, the number of people with AF is projected to increase two- to threefold over the next three decades in Western populations. (Kannel and Benjamin (2008) Med Clin North Am. 2008; 92: 17-40; Bunch, et al. (2012) J Innovations of Card Rhythm Manag 2012; 3: 855-63).

The fundamental risk of AF increases four- to five-fold in embolic stroke. The inherent risk of AF-associated stroke increases sharply with age, reaching 23.5% at 80–89 years of age. AF is associated with a doubling of mortality in both men and women (Kannel and Benjamin, 2008). AF is also independently associated with cognitive decline and all forms of dementia (Marzona et al. (2012) CMAJ 2012; 184: 329–36; Geita et al. 2013; Bunch et al. 2012).

Most patients with AF require lifelong anticoagulation to prevent cardioembolic stroke and systemic embolism. The CHA2DS2-VASc risk score is a validated and widely used stratification tool for predicting the risk of thromboembolism in patients with atrial fibrillation and for identifying patients who will benefit from anticoagulation (LIP 2011; Camm et al. (2012) Eur Heart J 2012; 33: 2719-2747); accumulating evidence shows that CHA2DS2-VASc is at least as accurate as, or possibly more accurate than, scores such as CHADS2 for identifying patients who will develop stroke and thromboembolism, and is certainly more accurate for identifying patients with AF who are “truly low risk.” It is estimated that 85-90% of patients with AF will require anticoagulant therapy.

In a meta-analysis including the results of six clinical trials that assessed the effect of vitamin K antagonists (VKAs) on reducing the risk of stroke and systemic embolism, a significant reduction in the risk of stroke (relative risk reduction for stroke 67%) was observed. All-cause mortality was significantly reduced (26%) relative to control with an adjusted dose of VKAs (Hart, Pearce, and Aguilar (2007) Ann Intern Med 2007; 146: 857–867). A target international normalized ratio (INR) of 2 to 3 is associated with the best benefit-to-risk balance (Hylek et al. (2003) N Engl J Med; 349: 1019–1026) and is widely accepted for use in international and national guidelines.

In recent years, new oral anticoagulants (NOACs), also called direct oral anticoagulants (DOACs), have been approved for use and introduced into clinical practice. These drugs are at least as effective as, or more effective than, warfarin in reducing the incidence of thromboembolic disease (Connolly et al. (2009) N Engl J Med; 361: 1139–51; Connolly et al. (2011) N Engl J Med; 364: 806–17; Patel et al. (2011) N Engl J Med 2011; 365: 883–91). NOACs also greatly reduce the incidence of the most serious complications associated with warfarin, namely, hemorrhagic stroke and intracranial bleeding. Major bleeding events occurred in similar or slightly lower numbers than with warfarin therapy. Furthermore, NOACs have fewer drug interactions than warfarin and can be used without routine monitoring, which is expected to facilitate their use in routine clinical practice.

Despite recent improvements, the risk of bleeding remains high with anticoagulant use. For example, the annual incidence of major and clinically relevant non-major bleeding was 14.9%, and the annual incidence of major bleeding was 3.6% in patients receiving rivaroxaban in the ROCKET study (Patel et al. 2011). The annual incidence of major bleeding was >5% in patients at high bleeding risk, defined as a bleeding risk score HAS ≥3 (Gallego, et al. (2012) Carc Arrhythm Electrophysiol.; 5: 312–318). Major bleeding is a particularly relevant clinical outcome; for example, in the ROCKET study, after major bleeding episodes, the rate of death from any cause was 20.4% in the rivaroxaban group and 26.1% in the warfarin group. Following major bleeding episodes, stroke and systemic embolism occurred in 4.7% and 5.4% of patients in the rivaroxaban and warfarin groups, respectively (Piccini et al. (2014) Eur Heart J; 35: 1873-80). Major bleeding episodes also significantly impact the length of hospital stay, the need for blood transfusions, and the use of available resources. The risk of bleeding is also the main reason for withholding anticoagulants in patients who require them. According to the European Heart Study of Atrial Fibrillation, which included data from 182 hospitals in 35 countries on 5333 outpatients and hospitalized patients with AF, only 67% of patients requiring an oral anticoagulant were taking one at discharge (Nieuwlaat, et al. (2005) Eur Heart J; 26, 2422-2434).

Thus, there is a high unmet medical need for safer treatments that can reduce thromboembolic complications of AF, such as stroke, systemic embolism, cognitive decline, and mortality, with comparable efficacy to existing treatments but with a lower risk of bleeding.

ESSENCE OF THE INVENTION

The present invention relates to monoclonal antibodies that bind to human blood coagulation factors XI and XIa (activated factor XI) (hereinafter sometimes abbreviated as "FXI", "FXIa" and similar terms) and pharmaceutical compositions containing them, as well as to methods of treatment involving their administration. The development of an antithrombotic agent that is effective in the prevention and treatment of thrombosis or a thromboembolic disease/disorder (e.g., thrombotic stroke, atrial fibrillation, stroke prevention in atrial fibrillation (SPIA), deep vein thrombosis, venous thromboembolism, pulmonary embolism, acute coronary syndromes (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism), but carries no or only a minimal risk of bleeding, would satisfy the existing significant medical need.

In particular aspects, antibodies (e.g., human, chimeric, humanized monoclonal antibodies) provided herein bind with similar high affinity to the catalytic domain (CD) of human factors FXIa and FXI, and induce the formation of an inactive conformation of the protease domain of FXIa.

The isolated anti-FXI and/or anti-FXIa antibodies described herein, such as the full-length dual-binding site IgGs described herein, bind FXI and/or FXIa with an equilibrium dissociation constant ( _KD ) of less than or equal to 100 pM. For example, the isolated antibodies described herein can bind human FXI and/or FXIa with a _KD of less than or equal to 100 pM, less than or equal to 50 pM, less than or equal to 45 pM, less than or equal to 40 pM, less than or equal to 35 pM, less than or equal to 20 pM, or less than or equal to 10 pM. More specifically, the isolated antibodies described herein can also bind human FXI and/or FXIa with a _KD of less than or equal to 34 pM when measured by a surface plasmon resonance (SPR) method, such as in the BIACORE™ assay, or less than or equal to 4 pM when measured in an equilibrium solution titration (EST) assay; and can also bind cynomolgus monkey FXI and/or FXIa with a _KD of less than or equal to 53 pM when measured in the BIACORE™ assay or less than or equal to 4 pM when measured in the SST assay. In particular aspects, the isolated antibodies described herein (e.g., NOV1401) bind human FXI and FXIa with an apparent _KD of less than or equal to about 5 pM (e.g., 4.7 pM) and 2 pM (e.g., 1.3 pM), respectively, for example, when measured in a solution equilibrium titration (TEP) assay. In particular embodiments, the anti-FXI/FXIa antibodies described herein bind cynomolgus monkey FXI/FXIa with an apparent _KD of about 12.5 (±6.6) pM for FXIa and about 5.0 (±0.7) pM when measured in a TPP assay (see, for example, Example 2). In particular embodiments, the anti-FXI/FXIa antibodies described herein bind rabbit FXI and/or FXIa with a _KD of about 20 (±2) nM. In certain aspects, the anti-FXI/FXIa antibodies described herein bind human, cynomolgus monkey, and rabbit FXI and/or FXIa, but do not specifically bind mouse or rat FXI.

The isolated anti-FXI and/or anti-FXIa antigen-binding fragments described herein, such as Fab fragments and other fragments containing a single binding site, bind FXI and/or FXIa with an equilibrium dissociation constant ( _KD ) of less than or equal to 10 nM. For example, the isolated antigen-binding fragments described herein can bind human FXI and/or FXIa with a _KD of less than or equal to 10 nM, less than or equal to 5 nM, less than or equal to 1 nM, less than or equal to 500 pM, less than or equal to 305 pM, or less than or equal to 62 pM. More particularly, the isolated antigen-binding fragments described herein can also bind human FXI and/or FXIa with a _KD of less than or equal to 305 pM.

The present invention relates to an isolated antibody, or antigen-binding fragments thereof, that binds human, rabbit, and cynomolgus macaque FXIa. The invention also relates to an isolated antibody, or antigen-binding fragments thereof, that binds within the catalytic domain of FXI and/or FXIa, in particular to the surface of the active site region.

The present invention also relates to an isolated antibody, or antigen-binding fragments thereof, that binds FXI and/or FXIa and also competes for binding with an antibody listed in Table 1 (e.g., NOV1401). As used herein, the term "competition" between antibodies and/or their antigen-binding fragments means that both antibodies (or their binding fragments) bind the same or an overlapping epitope of FXI and/or FXIa (e.g., as determined in a competition binding assay using any methods well known to those skilled in the art). As described herein, an antibody or antigen-binding fragment thereof does not "compete" with an antibody or antigen-binding fragment of the invention against FXI and/or FXIa (e.g., NOV1401 or NOV1090) unless said competing antibody or antigen-binding fragment thereof binds the same epitope of FXI and/or FXIa, or an overlapping epitope of FXI and/or FXIa, as the antibody or antigen-binding fragment of the invention. As described herein, a competing antibody or antigen-binding fragment thereof does not include one that (i) sterically blocks an antibody or antigen-binding fragment of the invention from binding to its target (e.g., if said competing antibody binds to an adjacent, non-overlapping epitope of FXI and/or FXIa and physically interferes with the binding of the antibody or antigen-binding fragment of the invention to its target); and/or (ii) binds another, non-overlapping epitope of FXI and/or FXIa and causes a conformational change in the FXI and/or FXIa protein such that said protein can no longer be bound by the antibody, or antigen-binding fragment, of the invention against FXI and/or FXIa in the same way as in the absence of said conformational change.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and/or FXIa and also compete with an antibody listed in Table 1 for binding to the majority of amino acids in the epitope(s) that the said antibody from Table 1 binds. In another embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and/or FXIa and also compete with an antibody listed in Table 1 for binding to all of the epitope(s) that the said antibody from Table 1 binds.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind active FXI (FXIa) and, upon binding to the catalytic domain of active FXI (FXIa), cause a conformational change in FXIa to an inactive conformation. In another embodiment, the isolated antibodies, or antigen-binding fragments thereof, further cause a change in which the 4 N-terminal residues, loops 145, 188, and 220 of the inactive conformation are shifted and/or disordered relative to the active conformation.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI (e.g., human FXI) and, upon binding to FXI, prevent the FXI catalytic domain from adopting an active conformation in which loops 145, 188, and 220 are ordered as in the structure of the FXIa catalytic domain.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and, upon binding FXI, prevent the FXI catalytic domain from adopting an active conformation in which the 4 N-terminal residues, loops 145, 188, and 220, are ordered as in the structure of the FXIa catalytic domain.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and, upon binding FXI, prevent the catalytic domain of FXI from adopting an active conformation by causing a conformational change in the zymogen structure resulting in an inhibited conformation of FXI similar to that observed upon binding to FXIa.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and/or FXIa, and upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, cause a displacement and/or disorder of loops 145, 188, and 220 compared to the structure of the catalytic domain of non-complexed active factor XI (FXIa).

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and/or FXIa, and upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, cause a displacement and/or disorder of the 4 N-terminal residues, loops 145, 188, and 220, compared to the structure of the catalytic domain of non-complexed active factor XI (FXIa).

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind active FXI (FXIa) and cause a conformational change in the catalytic domain of FXI (FXIa) to an inactive conformation in which loops 145, 188, and 220 are shifted and/or disordered relative to the active conformation.

In one embodiment, the isolated antibodies, or antigen-binding fragments thereof, bind FXI and prevent the catalytic domain from adopting an active conformation by inducing a conformational change in the zymogen structure, thereby resulting in an inhibited conformation of FXI similar to that observed upon binding to FXIa.

The present invention also relates to an isolated antibody, or antigen-binding fragments thereof, that binds the same epitope as the antibody listed in Table 1 (e.g., NOV1401).

The binding affinity of the isolated antibodies and antigen-binding fragments described herein can be determined using a solution equilibrium titration (SUT) method. SUT methods are known in the art and are described in more detail below. Alternatively, the binding affinity of the isolated antibodies or fragments described herein can be determined using surface plasmon resonance, such as in BIACORE™ assays. BIACORE™ kinetic analysis methods are known in the art and are described in more detail below.

The isolated anti-FXI and/or FXIa antibodies and antigen-binding fragments described herein can be used to inhibit direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin, and/or binding to platelet receptors, and thus can prevent activation of the intrinsic and/or common pathways of blood coagulation.

The isolated anti-FXI and/or anti-FXIa antibodies and antigen-binding fragments described herein can be used to inhibit the direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin with an IC ₅₀ value of less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 35 nM, less than or equal to 25 nM, less than or equal to 10 nM, or less than or equal to 5.2 nM. More specifically, the isolated antibody, or antigen-binding fragments thereof, described herein can inhibit direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin with an IC ₅₀ value of less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 35 nM, less than or equal to 25 nM, less than or equal to 10 nM, or less than or equal to 5.2 nM. More specifically, the isolated antibody, or antigen-binding fragments thereof, described herein can inhibit direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin with an IC ₅₀ value of less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 35 nM, less than or equal to 25 nM, less than or equal to 20 nM, or less than or equal to 18 nM. More particularly, the isolated antibody, or antigen-binding fragments thereof, described herein can inhibit the direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin with an _IC50 value of less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 35 nM, less than or equal to 25 nM, less than or equal to 10 nM, or less than or equal to 5 nM. In a specific embodiment, the anti-FXI/FXIa antibody, or antigen-binding fragment thereof, described herein inhibits FXIa-mediated activation of its natural substrate FIX with an _IC50 value of less than or equal to 2 nM, such as 1.8 nM.

Isolated anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, can be used to inhibit (e.g., block activation) of the intrinsic and/or common pathways of blood coagulation, for example, by inhibiting FXI and/or FXIa-mediated activation of FIX. Isolated anti-FXI/FXIa antibodies, or antigen-binding fragments thereof, can thus be used to prevent blood clotting or the propagation of blood clotting. Isolated antibodies, or antigen-binding fragments thereof, can be used to prevent, treat, or ameliorate blood clotting disorders such as deep vein thrombosis and stroke (e.g., ischemic stroke) by inhibiting FXI-mediated activation of FIX.

In specific embodiments, anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, are capable of increasing the clotting time (e.g., the time to the onset of blood clot formation) in human plasma in a concentration-dependent manner, as determined in an aPTT assay, for example, as described in the Examples section. In a specific embodiment, the clotting time (aPTT) is doubled compared to baseline at a total concentration of the anti-FXI antibody (e.g., NOV1401) in the range of 10 nM to 20 nM, such as about 14 nM or 15 nM, as determined in an aPTT assay. In particular embodiments, the anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, are capable of increasing clotting time in human plasma in a concentration-dependent manner with an IC ₅₀ value in the range of 5 nM to 20 nM, such as about 13 nM, when determined in an aPTT assay, such as described in the Examples section.

In specific embodiments, the anti-FXI and/or anti-FXIa antibodies described herein, or antigen-binding fragments thereof, are capable of increasing clotting time (e.g., time to onset of blood clot formation) in human plasma by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, or 2-fold, e.g., in a concentration-dependent manner, when determined in an aPTT assay, e.g., as described in the Examples section. In specific embodiments, the anti-FXI and/or anti-FXIa antibodies described herein, or antigen-binding fragments thereof, are capable of increasing clotting time (e.g., time to onset of blood clot formation) in human plasma by at least 1.4-fold, 1.5-fold, 1.6-fold, or 1.7-fold, as determined in an aPTT assay, such as described in the Examples section.

In particular aspects, the anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, described herein are capable of reducing the amount of thrombin in a concentration-dependent manner in a human plasma thrombin generation assay (TGA), which measures the effect of FXIa inhibition on the thrombin→FXIa positive feedback loop in the presence of very low concentrations of tissue factor (TF). In particular embodiments, the anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, described herein are capable of reducing the amount of thrombin in a human plasma thrombin generation assay (TGA), with an IC ₅₀ in the range of 10 nM to 30 nM, such as about 20 nM or 24 nM, and a residual thrombin concentration of about 159 nM.

In particular aspects, the present invention provides antibodies (e.g., antibodies in Table 1, such as NOV1401, or antibodies comprising HCDR regions 1-3 and LCDR regions 1-3 of NOV1401), or antigen-binding fragments thereof, that specifically bind the catalytic domain of human FXI and/or FXIa and that have a terminal half-life (t _½ ) of total antibody in cynomolgus monkeys of about 14-15 days. In particular embodiments, such anti-FXI/FXIa antibodies exhibit an absolute bioavailability upon subcutaneous (s.c.) administration of about 61-66%.

In a specific embodiment, an antibody, or antigen-binding fragment thereof, of the present invention (e.g., an antibody listed in Table 1, such as NOV1401), that specifically binds human FXI and/or FXIa has one or more (e.g., two or three or four or five or six or seven), or all, of the following characteristics:

(i) specifically binds the catalytic domain (CD) of human FXI and FXIa, e.g., with apparent K _D of approximately 1-2 pM and 4-5 pM, respectively;

(ii) increases blood clotting time as assessed by the activated partial thromboplastin time (aPTT) assay;

(iii) inhibits thrombin generation in human plasma by inhibiting FXI activation by activated factor XII (FXIIa) and thrombin, respectively;

(iv) exhibits antithrombotic and anticoagulant activity in FXI-/- mice administered with human FXI;

(v) reduces or prolongs the decrease in free FXI levels (FXI _c ), for example, in cynomolgus monkeys;

(vi) has a terminal half-life of total antibody of approximately 14-15 days, e.g., in cynomolgus macaques;

(vii) specifically binds human and monkey FXI and/or FXIa, but does not bind specifically mouse or rat FXI and/or FXIa; and

(viii) contacts one or more (e.g., two, three, four, five, six, or seven or more), or some or all, of the following residues of human FXI (Swissprot numbering): Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472-Glu476, Tyr521-Lys527, Arg548, His552, Ser575, Ser594-Glu597, and Arg602-Arg604.

The isolated anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, described herein can be monoclonal antibodies, human or humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, Fv fragments, F(ab') ₂ fragments, or scFv fragments, and/or IgG isotypes (e.g., IgG1, such as human IgG1). In specific embodiments, the anti-FXI and/or anti-FXIa antibodies described herein are recombinant human antibodies. In specific embodiments, the anti-FXI and/or anti-FXIa antibodies described herein are human IgG1/lambda (λ) antibodies. In specific embodiments, the anti-FXI and/or anti-FXIa antibodies described herein are human IgG1/lambda (λ) antibodies comprising an Fc domain engineered to reduce the potential for effector function (e.g., ADCC and/or CDC), such as a human Fc domain comprising the D265A and/or P329A substitutions.

The isolated anti-FXI and/or anti-FXIa antibodies, or antigen-binding fragments thereof, described herein may also comprise a framework in which an amino acid substitution occurs compared to the corresponding human germline VH or VL sequences.

Another aspect of the invention relates to an isolated antibody, or antigen-binding fragments thereof, having the full-length heavy and light chain sequences of the Fab fragments shown in Table 1. More specifically, the isolated antibody, or antigen-binding fragments thereof, may have the heavy and light chain sequences of NOV1090 and NOV1401.

A further aspect of the invention relates to an isolated antibody, or antigen-binding fragments thereof, having the heavy and light chain variable domain sequences of the Fab fragments shown in Table 1. More particularly, the isolated antibody, or antigen-binding fragment thereof, may have the heavy and light chain variable domain sequences of NOV1090 and NOV1401.

Another aspect of the invention relates to an isolated antibody, or antigen-binding fragments thereof, having the sequences of the heavy chain variable domain CDRs (i.e., HCDR1, HCDR2, and HCDR3) and the light chain variable domain CDRs (i.e., LCDR1, LCDR2, and LCDR3) of the antibodies listed in Table 1, such as the Kabat CDRs, the IMGT CDRs, the Chothia CDRs, or combined CDRs. More particularly, the isolated antibody, or antigen-binding fragment thereof, can have the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of NOV1090 and NOV1401, for example, listed in Table 1, such as the Kabat CDRs, the IMGT CDRs, the Chothia CDRs, or combined CDRs.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, comprising a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 3 and 23; a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 4 and 24; and a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 5 and 25, wherein the isolated antibody, or antigen-binding fragments thereof, binds human FXI and/or FXIa. In another aspect, such an isolated antibody, or antigen-binding fragments thereof, further comprises a light chain CDR1 selected from the group consisting of SEQ ID NOs: 13 and 33; a light chain CDR2 selected from the group consisting of SEQ ID NOs: 14 and 34; and a light chain CDR3 selected from the group consisting of SEQ ID NOs: 15 and 35.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, comprising a light chain CDR1 selected from the group consisting of SEQ ID NOs: 13 and 33; a light chain CDR2 selected from the group consisting of SEQ ID NOs: 14 and 34; and a light chain CDR3 selected from the group consisting of SEQ ID NOs: 15 and 35, wherein the isolated antibody, or antigen-binding fragments thereof, binds human FXI and/or FXIa.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, binding FXI and/or FXIa, which has HCDR1, HCDR2 and HCDR3, as well as LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise SEQ ID NOs: 3, 4 and 5, and LCDR1, LCDR2, LCDR3 comprise SEQ ID NOs: 13, 14 and 15; or HCDR1, HCDR2 and HCDR3 comprise SEQ ID NOs: 23, 24 and 25, and LCDR1, LCDR2, LCDR3 comprise SEQ ID NOs: 33, 34 and 35.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, binding FXI and/or FXIa, which has HCDR1, HCDR2 and HCDR3, as well as LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 comprise SEQ ID NOs: 43, 44 and 45, respectively, and LCDR1, LCDR2, LCDR3 comprise SEQ ID NOs: 47, 37 and 15, respectively.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, binding FXI and/or FXIa, which has HCDR1, HCDR2 and HCDR3, as well as LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 contain SEQ ID NOs: 46, 4 and 5, respectively, and LCDR1, LCDR2, LCDR3 contain SEQ ID NOs: 33, 14 and 15, respectively.

The invention also relates to an antibody or antigen-binding fragment having HCDR1, HCDR2 and HCDR3 of the heavy chain variable domain with SEQ ID NOs: 9 and 29, and LCDR1, LCDR2 and LCDR3 of the light chain variable domain with SEQ ID NOs: 19 and 39 as defined by Chothia. In another aspect of the invention, the antibody or antigen-binding fragment may have HCDR1, HCDR2 and HCDR3 of the heavy chain variable domain with the sequences of SEQ ID NOs: 9 and 29, and LCDR1, LCDR2 and LCDR3 of the light chain variable domain with the sequences of SEQ ID NOs: 19 and 39 as defined by Kabat.

The invention also relates to an antibody or antigen-binding fragment having HCDR1, HCDR2 and HCDR3 of the heavy chain variable domain with SEQ ID NOs: 9 and 29 and LCDR1, LCDR2 and LCDR3 of the light chain variable domain with SEQ ID NOs: 19 and 39 as defined by IMGT. In another aspect of the invention, the antibody or antigen-binding fragment may have HCDR1, HCDR2 and HCDR3 of the heavy chain variable domain with the sequences of SEQ ID NOs: 9 and 29 and LCDR1, LCDR2 and LCDR3 of the light chain variable domain with the sequences of SEQ ID NOs: 19 and 39 as defined by the combined system.

In one aspect of the invention, an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain sequence selected from the group consisting of SEQ ID NOs: 9 and 29. The isolated antibody or antigen-binding fragment may also comprise a light chain variable domain sequence, wherein the heavy chain variable domain and the light chain variable domain together form an antigen-binding site for FXIa. In particular, the light chain variable domain sequence may be selected from SEQ ID NOs: 19 and 39, wherein said isolated antibody or antigen-binding fragments thereof bind FXI and/or FXIa.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, comprising a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 19 and 39, wherein said isolated antibody, or antigen-binding fragments thereof, binds human FXI and/or FXIa. The isolated antibody, or antigen-binding fragment, may further comprise a heavy chain variable domain sequence, wherein the light chain variable domain and the heavy chain variable domain together form an antigen-binding site for FXI and/or FXIa.

In particular, an isolated antibody, or antigen-binding fragments thereof, that binds FXI and/or FXIa may have heavy and light chain variable domains comprising the sequences of SEQ ID NOs: 9 and 19; or 19 and 39, respectively.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, comprising a heavy chain variable domain having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 9 and 29, wherein said antibody binds FXI and/or FXIa. In one aspect, the isolated antibody, or antigen-binding fragments thereof, further comprises a light chain variable domain having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 19 and 39. In a further aspect of the invention, the isolated antibody, or antigen-binding fragment, has HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined by Kabat and set forth in Table 1. In a specific embodiment, the isolated antibody, or antigen-binding fragment, has HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined by Chothia, IMGT, or a combined system as set forth in Table 1.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, which has a light chain variable domain having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 19 and 39, wherein said antibody binds FXI and/or FXIa.

In another aspect of the invention, an isolated antibody, or antigen-binding fragments thereof, that binds FXI and/or FXIa, may have a heavy chain comprising the sequence of SEQ ID NOs: 11 or 31. The isolated antibody may also have a light chain that can, in combination with the heavy chain, form an antigen-binding site for human FXI and/or FXIa. In particular, the light chain may have a sequence comprising SEQ ID NOs: 21 or 41. In particular, an isolated antibody, or antigen-binding fragments thereof, that binds FXI and/or FXIa, may have a heavy chain and a light chain comprising the sequences of SEQ ID NOs: 11 and 21; or 31 and 41, respectively.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, which comprises a heavy chain having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 11 or 31, wherein said antibody binds FXI and/or FXIa. In one aspect, the isolated antibody, or antigen-binding fragments thereof, also comprises a light chain having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 21 or 41.

The invention also relates to an isolated antibody, or antigen-binding fragments thereof, comprising a light chain having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 21 or 41, wherein said antibody binds FXI and/or FXIa.

The invention also relates to compositions comprising the isolated antibody or antigen-binding fragments thereof described herein, as well as to compositions of the antibody in combination with a pharmaceutically acceptable carrier. In particular, the invention also relates to pharmaceutical compositions comprising the antibody or antigen-binding fragments thereof from Table 1, such as, for example, the antibody NOV1090 and NOV1401. The invention also relates to pharmaceutical compositions comprising a combination of two or more isolated antibodies or antigen-binding fragments thereof from Table 1.

The invention also relates to an isolated nucleic acid sequence encoding a heavy chain variable domain having a sequence selected from SEQ ID NOs: 9 and 29. In particular, the nucleic acid sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 10 and 30. In a further aspect of the invention, the sequence is SEQ ID NOs: 10 or 30.

The invention also relates to an isolated nucleic acid sequence encoding a light chain variable domain having a sequence selected from SEQ ID NOs: 20 and 40. In particular, the nucleic acid sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 20 and 40. In a further aspect of the invention, the sequence is SEQ ID NOs: 20 and 40.

The invention also relates to an isolated nucleic acid comprising a sequence encoding a polypeptide comprising a light chain variable domain having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 20 and 40.

The invention also relates to a vector comprising one or more nucleic acid molecules described herein.

The invention also relates to an isolated host cell comprising a recombinant DNA sequence encoding the heavy chain of the antibody described above and a second recombinant DNA sequence encoding the light chain of the antibody described above, wherein said DNA sequences are operably linked to a promoter and are capable of expression in the host cell. It is envisaged that the antibody may be a human monoclonal antibody. It is also envisaged that the host cell is a cell of a non-human mammal.

The invention also relates to a method for reducing the expression of FXI and/or FXIa, and/or activation of the intrinsic and/or common pathways of blood coagulation activation, comprising the step of contacting a cell with an effective amount of a composition containing the isolated antibody, or its antigen-binding fragments, described herein.

The invention also relates to a method for inhibiting the binding of FXI and/or FXIa to FIX, comprising the step of contacting a cell with an effective amount of a composition containing the isolated antibody, or antigen-binding fragments thereof, described herein.

It is envisaged that the cell is a human cell. It is also envisaged that the cell is located in the body of a subject. In one embodiment, it is envisaged that the cell is a platelet. It is also envisaged that the subject is a human.

The invention also relates to a method for treating, mitigating, or preventing a thromboembolic disease in a subject, comprising the step of administering to the subject an effective amount of a composition comprising the antibody, or antigen-binding fragments thereof, described herein. In one aspect, the thromboembolic disease is a thrombotic disease (e.g., thrombosis, thrombotic stroke, atrial fibrillation, atrial fibrillation stroke prevention (AFSP), deep vein thrombosis, venous thromboembolism, and pulmonary embolism). It is envisaged that the subject is a human.

Any of the above-mentioned isolated antibodies, or antigen-binding fragments thereof, may be a monoclonal antibody, or antigen-binding fragments thereof.

Non-limiting embodiments of the invention are described in the following paragraphs:

1. An isolated anti-FXI and/or anti-FXIa antibody, or fragment thereof, that binds within the catalytic domain of FXI and/or FXIa.

2. An isolated antibody, or fragment thereof, that binds one or more anti-FXI and/or FXIa epitopes, wherein the epitope comprises two or more amino acid residues from Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

3. The isolated antibody or fragment of claim 2, wherein the epitope comprises four or more amino acid residues from Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

4. The isolated antibody or fragment of claim 2, wherein the epitope comprises six or more amino acid residues from Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

5. The isolated antibody or fragment of claim 2, wherein the epitope comprises eight or more amino acid residues from Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

6. The isolated antibody or fragment of claim 2, wherein the epitope comprises residues Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

7. The isolated antibody or fragment of claim 2, wherein the epitope comprises the amino acid residues Pro410, Arg413, Lys527 and one or more amino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

8. The isolated antibody or fragment of claim 2, wherein the epitope comprises the amino acid residues Pro410, Arg413, Lys527 and four or more amino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

9. The isolated antibody or fragment of claim 2, wherein the epitope comprises the amino acid residues Pro410, Arg413, Lys527 and six or more amino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603 and Arg604.

10. An isolated anti-FXI and/or anti-FXIa antibody, or fragment thereof, that binds within the catalytic domain of FXI and/or FXIa, wherein said antibody or fragment blocks the binding of FXI and/or FXIa to one or more of factor IX, factor XIIa, and thrombin.

11. The isolated antibody or fragment of claim 10, wherein said antibody or fragment blocks the binding of FXI and/or FXIa to one or more of factor IX, factor XIIa or thrombin, and other components of the blood coagulation pathway.

12. The isolated antibody or fragment of claim 1, wherein said antibody or fragment blocks the binding of one or more of FIX, FXI, and FXIa to platelet receptors.

13. An isolated antibody or fragment according to claim 1, wherein said antibody or fragment prevents activation of the intrinsic or common pathways of blood coagulation.

14. An isolated antibody, or fragment thereof, that binds human FXI and/or FXIa protein with a K _D less than or equal to 34 nM when measured in the BIACORE™ assay, or less than or equal to 4 pM when measured in the equilibrium solution titration (EST) assay.

15. An isolated antibody or fragment according to claim 1, wherein said antibody or fragment comprises at least one complementarity determining region having at least 90% identity with at least one of the CDR regions listed in Table 1.

16. An isolated antibody or fragment according to claim 1, wherein said antibody or fragment comprises CDR1, CDR2 and CDR3 from Table 1.

17. An isolated variant of an antibody or fragment according to claim 1, wherein said antibody or fragment comprises CDR1, CDR2 and CDR3 from Table 1, and wherein the variant has at least one to four amino acid changes in one of the CDR1, CDR2 or CDR3 regions.

18. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain CDR3 selected from the group consisting of SEQ ID NO: 5 and 25.

20. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a VH selected from the group consisting of SEQ ID NOs: 9 and 29, or an amino acid sequence having 90% identity thereto; and a VL selected from the group consisting of SEQ ID NOs: 19 and 39, or an amino acid sequence having 90% identity thereto.

21. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a VH selected from the group consisting of SEQ ID NOs: 9 and 29, or an amino acid sequence having 95% identity thereto; and a VL selected from the group consisting of SEQ ID NOs: 19 and 39, or an amino acid sequence having 95% identity thereto.

22. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a VH selected from the group consisting of SEQ ID NOs: 9 and 29, or an amino acid sequence having 97% identity thereto; and a VL selected from the group consisting of SEQ ID NOs: 19 and 39, or an amino acid sequence having 97% identity thereto.

23. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 9 and 29.

24. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a light chain variable region sequence selected from the group consisting of SEQ ID NO: 19 and 39.

25. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 9 and 29; and a light chain variable region sequence selected from the group consisting of SEQ ID NOs: 19 and 39.

26. An isolated antibody or fragment according to claim 1, which is selected from the group consisting of an antibody or fragment comprising the heavy chain variable region sequence of SEQ ID NO: 9 and the light chain variable region sequence of SEQ ID NO: 19, and an antibody or fragment comprising the heavy chain variable region sequence of SEQ ID NO: 29 and the light chain variable region sequence of SEQ ID NO: 39.

27. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region CDR1 selected from the group consisting of SEQ ID NO: 46; a CDR2 selected from the group consisting of SEQ ID NO: 4; a CDR3 selected from the group consisting of SEQ ID NO: 5; a light chain variable region CDR1 selected from the group consisting of SEQ ID NO: 33; a CDR2 selected from the group consisting of SEQ ID NO: 14; and a CDR3 selected from the group consisting of SEQ ID NO: 15.

28. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region CDR1 selected from the group consisting of SEQ ID NOs: 3 and 23; a CDR2 selected from the group consisting of SEQ ID NOs: 4 and 24; a CDR3 selected from the group consisting of 5 and 25; a light chain variable region CDR1 selected from the group consisting of SEQ ID NOs: 13 and 33; a CDR2 selected from the group consisting of SEQ ID NOs: 14 and 34; and a CDR3 selected from the group consisting of SEQ ID NOs: 15 and 35.

29. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises a heavy chain variable region CDR1 selected from the group consisting of SEQ ID NOs: 6 and 26; a CDR2 selected from the group consisting of SEQ ID NOs: 7 and 27; a CDR3 selected from the group consisting of 8 and 28; a light chain variable region CDR1 selected from the group consisting of SEQ ID NOs: 16 and 36; a CDR2 selected from the group consisting of SEQ ID NOs: 17 and 37; and a CDR3 selected from the group consisting of SEQ ID NOs: 18 and 38.

30. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises CDR1 of the variable region of the heavy chain of SEQ ID NO: 3; CDR2 of the variable region of the heavy chain of SEQ ID NO: 4; CDR3 of the variable region of the heavy chain of SEQ ID NO: 5; CDR1 of the variable region of the light chain of SEQ ID NO: 13; CDR2 of the variable region of the light chain of SEQ ID NO: 14 and CDR3 of the variable region of the light chain of SEQ ID NO: 15.

31. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises CDR1 of the variable region of the heavy chain of SEQ ID NO: 23; CDR2 of the variable region of the heavy chain of SEQ ID NO: 24; CDR3 of the variable region of the heavy chain of SEQ ID NO: 25; CDR1 of the variable region of the light chain of SEQ ID NO: 33; CDR2 of the variable region of the light chain of SEQ ID NO: 34 and CDR3 of the variable region of the light chain of SEQ ID NO: 35.

32. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises CDR1 of the variable region of the heavy chain of SEQ ID NO: 6; CDR2 of the variable region of the heavy chain of SEQ ID NO: 7; CDR3 of the variable region of the heavy chain of SEQ ID NO: 8; CDR1 of the variable region of the light chain of SEQ ID NO: 16; CDR2 of the variable region of the light chain of SEQ ID NO: 17 and CDR3 of the variable region of the light chain of SEQ ID NO: 18.

33. The isolated antibody or fragment of claim 1, wherein the antibody or fragment comprises CDR1 of the variable region of the heavy chain of SEQ ID NO: 26; CDR2 of the variable region of the heavy chain of SEQ ID NO: 27; CDR3 of the variable region of the heavy chain of SEQ ID NO: 28; CDR1 of the variable region of the light chain of SEQ ID NO: 36; CDR2 of the variable region of the light chain of SEQ ID NO: 37 and CDR3 of the variable region of the light chain of SEQ ID NO: 38.

34. A pharmaceutical composition comprising an antibody, or a fragment thereof, according to one of the preceding claims and a pharmaceutically acceptable carrier.

35. The isolated antibody or fragment of claim 1, wherein the antibody or fragment binds the same epitope as the isolated antibody or fragment of any of the preceding claims.

36. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment competes for binding of human FXI and/or FXIa protein with an isolated antibody or fragment according to any of the preceding claims.

37. The isolated antibody or fragment of claim 1, wherein the antibody or fragment is selected from the group consisting of NOV1090 and NOV1401.

38. A method for treating a thromboembolic disease, comprising administering to a subject suffering from a thromboembolic disease an effective amount of a pharmaceutical composition comprising an antibody, or fragment, according to any of the preceding claims.

39. The method of claim 38, wherein the subject suffers from one or more of ischemic stroke associated with atrial fibrillation and deep vein thrombosis.

40. The method according to claim 38, characterized in that the subject suffers from ischemic stroke associated with atrial fibrillation.

41. A method for treating a thromboembolic disease, comprising administering to a subject suffering from a thromboembolic disease an effective amount of a pharmaceutical composition comprising an antibody, or fragment, according to any of the preceding claims in combination with statin therapy.

42. A medicinal product containing an antibody according to any of the preceding claims.

43. A nucleic acid encoding one or more antibodies according to any of the preceding claims.

44. A vector containing a nucleic acid according to claim 43.

45. A host cell containing the vector according to claim 44.

46. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment, when binding to the catalytic domain of active FXI (FXIa), causes a conformational change in FXIa to an inactive conformation in which the 4 N-terminal residues, loops 145, 188 and 220, are shifted and/or disordered compared to the active conformation.

47. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment, when bound to FXI, prevents the FXI catalytic domain from adopting an active conformation in which loops 145, 188 and 220 are ordered as in the structure of the FXIa catalytic domain.

48. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment, when bound to FXI, prevents the FXI catalytic domain from adopting an active conformation in which the 4 N-terminal residues, loops 145, 188 and 220, are ordered as in the structure of the FXIa catalytic domain.

49. The isolated antibody or fragment of claim 1, wherein the antibody or fragment, when bound to FXI, prevents the catalytic domain of FXI from adopting an active conformation, causing conformational changes in the structure of the zymogen, subsequently leading to an inhibited conformation of FXI, close to that observed upon binding to FXIa.

50. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment, upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, causes a displacement and/or disorder of loops 145, 188 and 220 compared to the structure of the catalytic domain of the active factor XI (FXIa) not in the complex.

51. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment, upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, causes a displacement and/or disorder of the 4 N-terminal residues, loops 145, 188 and 220, compared to the structure of the catalytic domain of non-complexed active factor XI (FXIa).

52. An isolated antibody or fragment according to claim 1, wherein the antibody or fragment binds active FXI (FXIa) and causes a conformational change in the catalytic domain of FXI (FXIa) to an inactive conformation in which loops 145, 188, and 220 are shifted and/or disordered compared to the active conformation.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

The terms "FXI protein," "FXI antigen," and "FXI" are used interchangeably and refer to the factor XI protein in various species. Factor XI is a coagulation factor XI in mammalian plasma, a glycoprotein present in human plasma at a concentration of 25-30 nM as a zymogen, which is then converted by limited proteolysis to an active serine protease and participates in the intrinsic or general pathways of coagulation activation.

The terms "FXIa protein,""FXIaantigen," and "FXIa" are used interchangeably and refer to the activated FXI protein across species. The zymogen factor XI is converted to its active form, factor XIa (FXIa), either through the contact phase of coagulation or through thrombin-mediated activation on the platelet surface. During this activation of factor XI, an internal peptide bond is cleaved in each of its two chains, yielding activated factor XIa, a serine protease consisting of two heavy and two light chains linked by disulfide bonds. This serine protease, FXIa, converts factor IX to IXa, which subsequently activates factor X (Xa). Xa can then mediate the activation of factor II/thrombin. For example, human FXI has the sequence shown in Table 1 (SEQ ID NO:1) and has been described in previous reports and in the literature (Mandle RJ Jr, et al. (1979) Blood; 54(4): 850; reference sequence in NCBI: AAA51985).

In the context of the present invention, the terms "FXI" and "FXIa" (and the like) include mutants and variants of the natural protein FXI and FXIa, respectively, which have substantially the same amino acid sequence as the natural primary structure (amino acid sequence) given in the above-mentioned literature sources.

As used herein, the terms "catalytic domain," "serine protease catalytic domain," and similar terms refer to amino acids Ile370 to Val607, measured from the Glu1 residue at the N-terminus of the mature protein present in the circulatory system. They can also be described as residues 388-625 at the C-terminus of FXI. As used herein, the term "active site" refers to the catalytic triad consisting of amino acids His413, Asp462, and Se557. (Bane and Gailani (2014) Drug Disc. 19(9)).

The term "approximately" when applied to a numerical value x means, for example, x ± 10%.

As used herein, the term "antibody" refers to a whole antibody and any antigen-binding fragment (i.e., an "antigen-binding portion"), or a single chain thereof. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (referred to herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (referred to herein as VL) and a light chain constant region. The light chain constant region consists of a single domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant regions of antibodies can mediate the binding of immunoglobulin to tissues or host factors, including various immune system cells (e.g., effector cells) and the first component (Clq) of the classical complement system.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody means one or more fragments of an intact antibody that retain the ability to specifically bind a particular antigen (e.g., factor XIa (FXIa)). The antigen-binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody include the Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; the F(ab) ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; the Fd fragment, consisting of the VH and CH1 domains; the Fv fragment, consisting of the VL and VH domains of one arm of the antibody; single-domain antibody (dAb) (Ward et al. , 1989 Nature 341:544–546), which consists of a VH domain or a VL domain, as well as a distinct complementarity-determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined recombinantly by an artificial peptide linker, which allows them to be produced as a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. , 1988 Science 242: 423–426; and Huston et al. , 1988 Proc. Natl. Acad. Sci. 85: 5879–5883). Such single-chain antibodies contain one or more antigen-binding portions, or fragments, of the antibody. These antibody fragments are produced using conventional techniques known to those skilled in the art and the fragments are screened for suitability in the same manner as intact antibodies.

Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126–1136). Antigen-binding portions of antibodies can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide-based monobodies).

Antigen-binding fragments can be incorporated into single-chain molecules containing a pair of tandem Fv segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al. , 1995 Protein Eng. 8(10): 1057-1062; and U.S. Patent No. 5,641,870).

As used herein, the term "affinity" refers to the strength of interaction between an antibody and an antigen at individual antigenic sites. At each antigenic site, the variable region of the antibody's "arm" interacts through weak non-covalent forces with the antigen at numerous points; the more interactions, the stronger the affinity. As used herein, the term "high affinity" with respect to an antibody or its antigen-binding fragments (e.g., a Fab fragment) generally refers to an antibody or antigen-binding fragment having a K _D of 10 ^-9 M or less (e.g., a K _D of 10 ^-10 M or less, a K _D of 10 ^-11 M or less, a K _D of 10 ^-12 M or less, a K _D of 10 ^-13 M or less, a K _D of 10 ^-14 M or less, and so on).

The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act similarly to natural amino acids. Natural amino acids include those encoded by the genetic code, as well as amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as a natural amino acid, that is, an alpha carbon atom bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as natural amino acids. Amino acid mimetics are chemical compounds that have a structure that differs from the general chemical structure of amino acids, but which act similarly to natural amino acids.

As used herein, the term "binding specificity" means the ability of a particular binding site of an antibody to interact with only one antigenic determinant.

The term "specifically (or selectively) binds" when used with respect to an antibody (e.g., an FXI and/or FXIa-binding antibody) means a binding reaction that is mediated by the presence of a recognizable antigen (e.g., human FXI and/or FXIa or cynomolgus monkey FXI and/or FXIa) in a heterogeneous population of proteins and other biological molecules. The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen."

The term "FXI and/or FXIa-mediated" refers to the fact that FXI and/or FXIa mediate the intrinsic and/or common pathways of coagulation activation through direct or indirect activation of factor IX (also known as FIX), factor X (FX), and/or thrombin, and/or through binding to platelet receptors.

The term "hemostasis" refers to the primary mechanisms that stop blood flow at sites of injury and restore vascular patency during wound healing, respectively. In normal hemostasis and pathological thrombosis, three mechanisms are activated simultaneously: primary hemostasis, which involves the interaction of activated platelets with the vascular wall, fibrin formation, and a process called fibrinolysis.

The terms "blood coagulation and the coagulation cascade," "the coagulation cascade model," and similar terms refer to the protein system that stabilizes the clot that forms to seal a wound. The coagulation activation pathway is a proteolytic cascade. Each enzyme in this pathway is present in plasma as a zymogen (in an inactive form), which, upon activation, undergoes proteolytic cleavage, releasing the active factor from the precursor molecule. The coagulation cascade operates through a series of positive and negative feedback loops that control the activation process. The ultimate goal of the activation pathway is the formation of thrombin, which can then convert soluble fibrinogen into fibrin, which forms a clot.

The process of thrombin generation can be divided into three phases: the intrinsic and extrinsic pathways, which provide alternative routes to the formation of the active clotting factor FXa (activated factor-X), and the final common pathway, which leads to thrombin generation (Hoffman MM and Monroe DM (2005) Curr Hematol Rep. 4: 391-396; Johne J, et al. (2006) Biol Chem. 387: 173-178).

"Platelet aggregation" refers to the process by which, when a blood vessel ruptures, substances not normally in direct contact with the bloodstream are released. These substances (primarily collagen and von Willebrand factor) allow platelets to adhere to the damaged surface. Once a platelet adheres to the surface, it releases chemicals that attract additional platelets to the damaged area, a phenomenon known as platelet aggregation. These two processes are the first responses aimed at stopping bleeding.

As used herein, the term "thromboembolic disease" or similar terms refers to any condition or disease in which the intrinsic and/or common pathways of coagulation are aberrantly activated or are not naturally deactivated (e.g., without the use of therapeutic agents). These conditions include, but are not limited to, thrombotic stroke, atrial fibrillation, stroke prevention in atrial fibrillation (SPAF), deep vein thrombosis, venous thromboembolism, and pulmonary embolism. They may also include conditions associated with catheter insertion (e.g., the Hickman catheter in cancer patients), in which catheters become clogged with clots, and with extracorporeal membrane oxygenation (ECMO), in which clots form in the tubing.

As used herein, the term "thromboembolic" or similar terms may also refer to any number of the following conditions for the prevention or treatment of which the anti-FXI and/or anti-FXIa Abs, or antigen-binding fragments thereof, of the invention can be used:

- thromboembolism in subjects with suspected or confirmed cardiac arrhythmia such as paroxysmal, persistent or permanent atrial fibrillation or atrial flutter;

- prevention of stroke in atrial fibrillation (PAFI), one of the subgroups of patients are patients with AF undergoing percutaneous coronary intervention (PCI);

- treatment of acute venous thromboembolic events (VTE) and extended secondary prevention of VTE in patients with a high risk of bleeding;

- cerebral and cardiovascular events in secondary prevention after transient ischemic attack (TIA) or non-disabling stroke and prevention of thromboembolic events in heart failure with sinus rhythm;

- left atrial clot formation and thromboembolism in subjects undergoing cardioversion for cardiac arrhythmia;

- thrombosis before, during and after the ablation procedure due to cardiac arrhythmia;

- venous thrombosis, this includes, but is not limited to, treatment and secondary prevention of deep and superficial vein thrombosis in the lower extremities or upper extremities, thrombosis in the abdominal and thoracic veins, sinus thrombosis and jugular vein thrombosis;

- thrombosis on any artificial surface in the veins, such as a catheter or pacemaker wires;

- pulmonary embolism in patients with or without venous thrombosis;

- chronic thromboembolic pulmonary hypertension (CTEPH);

- arterial thrombosis at the site of a ruptured atherosclerotic plaque, thrombosis at an intra-arterial graft or catheter, and thrombosis in apparently normal arteries, including, but not limited to, acute coronary syndromes, ST-segment elevated myocardial infarction, non-ST-segment elevated myocardial infarction, unstable angina, stent thrombosis, thrombosis of any artificial surface in the arterial circulation, and pulmonary artery thrombosis in subjects with and without pulmonary hypertension;

- thrombosis and thromboembolism in patients undergoing percutaneous coronary intervention (PCI);

- cardioembolic and cryptogenic strokes;

- thrombosis in patients with invasive and non-invasive malignant neoplasms;

- thrombosis in the cavitary catheter;

- thrombosis and thromboembolism in patients in serious condition;

- cardiac thrombosis and thromboembolism, including, but not limited to, cardiac thrombosis after myocardial infarction, cardiac thrombosis associated with conditions such as cardiac aneurysm, myocardial fibrosis, cardiac enlargement and heart failure, myocarditis and artificial surfaces in the heart;

- thromboembolism in patients with valvular heart disease, with or without atrial fibrillation;

- thromboembolism in mechanical or biological valve prostheses;

- thromboembolism in patients with natural or artificial heart patches, arterial or venous conduits after heart surgery for simple or complex heart defects;

- venous thrombosis and thromboembolism after knee replacement surgery, hip replacement surgery, as well as orthopedic surgery, thoracic or abdominal surgery;

- arterial or venous thrombosis after neurosurgery, including intracranial surgery and spinal cord surgery;

- congenital or acquired thrombophilia, including, but not limited to, factor V Leiden mutation, prothrombin mutation, antithrombin III deficiency, protein C and protein S deficiency, factor XIII mutation, familial dysfibrinogenemia, congenital plasminogen deficiency, elevated factor XI levels, sickle cell disease, antiphospholipid syndrome, autoimmune disease, chronic bowel disease, nephrotic syndrome, hemolytic uremia, myeloproliferative disease, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria and heparin-induced thrombopenia;

- thrombosis and thromboembolism in chronic renal failure; and

- thrombosis and thromboembolism in patients undergoing hemodialysis and in patients undergoing extracorporeal membrane oxygenation.

The term "chimeric antibody" means an antibody molecule in which (a) the constant region, or a portion thereof, is altered, substituted, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, with a different effector function, and/or from a different species, or an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, substituted, or replaced with a variable region having a different or altered antigen specificity. For example, a murine antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. By replacing it with a human constant region, the chimeric antibody may retain its antigen recognition specificity while having less antigenicity in humans than the original murine antibody.

The term "conservatively modified variant" applies to both amino acid and nucleotide sequences. When applied to specific nucleic acid sequences, conservatively modified variants are nucleic acids that encode identical or substantially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, then substantially identical sequences. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode a particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be replaced with any of the corresponding listed codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. In this document, each nucleotide sequence encoding a polypeptide also includes any possible silent variation of the nucleic acid. One skilled in the art understands that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be altered to produce a functionally identical molecule. Accordingly, each silent variation of the nucleic acid encoding the polypeptide is implied for each described sequence.

In the case of polypeptide sequences, "conservatively modified variants" include single substitutions, deletions, or additions to the polypeptide sequence resulting in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables representing functionally similar amino acids are well known in the art. Such conservatively modified variants complement, but do not exclude, the polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups include amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T) and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence.

The term "epitope" refers to a protein determinant capable of specific binding by an antibody. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge properties. Conformational and non-conformational epitopes differ in that binding to the former, but not the latter, is lost in the presence of denaturing solvents. Two antibodies are said to "compete" if one antibody is shown to bind the same epitope as the second antibody in a competitive binding assay using any method well known to those skilled in the art.

As used herein, the term "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from human antibody sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant variants of human germline sequences. The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro , or by somatic mutation in vivo ).

The term "human monoclonal antibody" refers to antibodies exhibiting a single binding specificity, in which the variable regions in both the framework and CDR regions are derived from human sequences. In one embodiment, human monoclonal antibodies are produced using phage display methods to screen libraries of human immunoglobulin genes.

A "humanized" antibody is one that retains the reactivity of a non-human antibody but is less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining portions of the antibody with their human immunoglobulin counterparts (i.e., the constant region as well as the variable region framework portions). See, for example, Morrison et al. , Proc. Natl. Acad. Sci. USA, 81: 6851–6855, 1984; Morrison and Oi, Adv. Immunol., 44: 65–92, 1988; Verhoeyen et al. , Science, 239: 1534–1536, 1988; Padlan, Molec. Immun., 28: 489–498, 1991; and Padlan, Molec. Immun., 31: 169-217, 1994. Other examples of human antibody genetic modification technologies include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

The terms "identical" or "percentage identity" in the context of two or more nucleotide or polypeptide sequences refer to two or more sequences, or subsequences, that are the same. Two sequences are "substantially identical" if the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region or, if not specified, over the entire sequence) when compared and aligned for maximum correspondence within a comparison window or specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length or more, preferably over a region that is 100-500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

When comparing sequences, one sequence typically serves as a reference sequence against which the test sequences are compared. Using a sequence comparison algorithm, information about the test and reference sequences is entered into the computer, subsequence coordinates are determined if necessary, and the sequence comparison algorithm program parameters are defined. The program's default parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequences relative to the reference sequence, based on the program parameters.

As used herein, the term "comparison window" means a segment of any number of contiguous positions selected from the group consisting of 20-600, typically from about 50 to about 200, more often from about 100 to about 150, in which a sequence can be compared with a reference sequence of the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison purposes are well known in the art. Optimal alignment of sequences for comparison purposes can be performed, for example, by the Smith-Waterman local homology algorithm (1970) Adv. Appl. Math. 2: 482c, by the Needleman-Wunsch homology region alignment algorithm, J. Mol. Biol. 48: 443, 1970, by the Pearson-Lipman similarity search method, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al. , Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389–3402; and Altschul et al. (1990) J. Mol. Biol. 215: 403–410, respectively. A program for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm primarily involves identifying high-similarity sequence pairs (HSPs) by identifying short words of length W in the search sequence that either match or satisfy some positive threshold T when aligned with a word of the same length in a sequence database. T is called the word similarity score threshold (Altschul et al. , supra). These initial hits, or word hits, are used as seeds to begin searching for longer HSPs containing them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward for a pair of residue matches; always >0) and N (penalty for residue mismatches; always <0). For amino acid sequences, a substitution matrix is used to calculate the cumulative score. Extension of word hits in each direction is stopped when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score reaches zero or below due to the accumulation of one or more negative residue alignment scores; or the end of any of the sequences is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses a default wordlength of 3 and an expectation (E) of 10, as well as the BLOSUM62 substitution matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989), alignments (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873–5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which is an indication of the likelihood that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also be determined using the Myers-Miller algorithm (Comput. Appl. Biosci., 4: 11-17, 1988), which is included in the ALIGN program (version 2.0), using the PAM120 residue substitution weight table, a gap extension penalty of 12, and a gap insertion penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman-Wunsch algorithm (J. Mol. Biol. 48: 444-453, 1970), which is included in the GAP program in the GCG software package (available online at gcg.com), using either the Blossom 62 matrix or the PAM250 matrix, a gap insertion penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap extension penalty of 1. 2, 3, 4, 5 or 6.

In addition to the percentage of sequence identity described above, another indication that the sequences of two nucleic acids or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to the second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleotide sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Another indication that two nucleotide sequences are substantially identical is that the same primers can be used to amplify the sequences.

The term "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds FXI and/or FXIa is substantially free of antibodies that specifically bind antigens other than FXI and/or FXIa). An isolated antibody that specifically binds FXI and/or FXIa may, however, have cross-reactivity with other antigens. Furthermore, an isolated antibody may be substantially free of other cellular material and/or chemical reagents.

The term "isotype" refers to the antibody class (e.g., IgM, IgE, IgG, such as IgG1 or IgG4) determined by the heavy chain constant region genes. An isotype also includes modified variants of one of these classes, in which modifications have been introduced to alter the function of the Fc region, for example, to enhance or reduce effector functions or binding to Fc receptors.

As used herein, the term "k _ass " or " _ka " refers to the rate of association in a particular antibody-antigen interaction, while the term "k _diss " or "k _d " refers to the rate of dissociation in a particular antibody-antigen interaction. As used herein, the term "K _D " refers to the dissociation constant, which is determined based on the ratio of k _d to _ka (i.e., k _d / _ka ) and is expressed as a molar concentration (M). K _D values for antibodies can be determined by methods well known in the art. Methods for determining the K _D of an antibody include measurement by surface plasmon resonance using a biosensor system such as the BIACORE™ system, or measurement of affinity in solution by the equilibrium solution titration (ESS) method.

As used herein, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides, as well as polymers thereof, in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, whether synthetic, natural, or non-natural, that have binding properties similar to those of a reference nucleic acid and that are metabolized similarly to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

Unless otherwise stated, a particular nucleotide sequence is meant to also include its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the specifically designated sequence. In particular, as described in detail below, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al. , J. Biol. Chem. 260: 2605–2608, 1985; and Rossolini et al. , Mol. Cell. Probes 8: 91–98, 1994).

The term "operably linked" refers to a functional association between two or more polynucleotide segments (e.g., DNA). Typically, the term refers to the functional association of a transcriptional regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in the corresponding host cell or other expression system. Typically, transcriptional regulatory sequences of a promoter that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, that is, they act in cis . However, some transcriptional regulatory sequences, such as enhancers, do not necessarily need to be physically adjacent or in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term "optimized" means that the nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the producing cell or organism, typically a eukaryotic cell such as a picaria cell, a Chinese hamster ovary (CHO) cell, or a human cell. The optimized nucleotide sequence is designed to fully or as closely as possible retain the amino acid sequence encoded by the original nucleotide sequence, also referred to as the "parent" sequence. The optimized sequences described herein were designed to contain codons preferred in mammalian cells. However, this document also provides for optimized expression of these sequences in other eukaryotic or prokaryotic cells. Amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

In this document, the terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acids, as well as to polymers of natural amino acids and polymers of non-natural amino acids. Unless otherwise specified, a given polypeptide sequence is understood to also include conservatively modified variants thereof.

As used herein, the term "recombinant human antibody" includes all human antibodies that are produced, expressed, created, or isolated by recombinant methods, such as antibodies obtained from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes, or isolated from a hybridoma derived from its cells; antibodies isolated from a host cell transformed to express a human antibody, such as from a transfectoma; antibodies isolated from a recombinant, combinatorial library of human antibodies, as well as antibodies produced, expressed, created, or isolated by any other methods that involve splicing all or part of a human immunoglobulin gene sequence with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in particular embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from, and related to, human germline VH and VL sequences, may not be naturally present in the human germline antibody repertoire in vivo .

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms should refer not only to the specific cell of the subject but also to the progeny of such a cell. Because some modifications may arise in subsequent generations due to either mutation or environmental factors, such progeny may not actually be identical to the parent cell, but they are still encompassed by the term "host cell" as used herein.

The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals), such as non-human primates (e.g., cynomolgus macaques), sheep, rabbits, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably herein. As used herein, the terms "cynomolgus macaque" or "cynomolgus macaque" refer to the cynomolgus macaque ( Macaca fascicularis ). In certain aspects, a patient or subject is a human.

As used herein, the term "therapy" or "treatment" of a disease or disorder (e.g., a thromboembolic disease) in one embodiment means ameliorating the disease or disorder (i.e., slowing or halting, or attenuating the progression of the disease, or at least one of its clinical symptoms). In another embodiment, "therapy" or "treatment" means ameliorating or reducing at least one physical parameter, including those that may not be noticeable to the patient. In another embodiment, "therapy" or "treatment" means modulating the disease or disorder, either physically (e.g., stabilizing a noticeable symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In another embodiment, "therapy" or "treatment" means preventing or delaying the onset or development, or progression of the disease or disorder.

“Prevention,” when applied to the conditions described herein, including, for example, thromboembolic disease, means any action that prevents or slows down the worsening of, for example, the parameters of thromboembolic disease as described below, in a patient at risk of such worsening.

The term "vector" refers to a polynucleotide molecule capable of transferring another polynucleotide to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as the adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are functionally linked. In this document, such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors"). Expression vectors used in recombinant DNA techniques are typically in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably herein, as the plasmid is the most commonly used form of vector. However, the invention also encompasses the use of other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

BRIEF DESCRIPTION OF DRAWINGS

Figures 1A-C show the effect of NOV1401 on FeCl3 _- induced thrombosis in FXI ^-/- mice administered with human FXI protein. NOV1401 inhibited thrombosis in a dose-dependent manner. The antibody prolonged aPTT to a similar extent as in untreated FXI ^-/- mice.

Figures 2A-B show the effect of multiple intravenous (IV) (A; N=2) or subcutaneous (SC) (B; N=2) doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg NOV1401 on aPTT (diamonds) and the relationship to total plasma NOV1401 levels (squares) in cynomolgus macaques. A single 3 mg/kg dose resulted in an approximately 2-fold increase in aPTT that was maintained for 5-6 weeks. All doses tested prolonged aPTT to a similar extent, and higher doses tested did not appear to prolong aPTT more than the 3 mg/kg dose.

Figures 3A-B show the effect of multiple intravenous (A; N=2) or subcutaneous (B; N=2) doses of 3 mg/kg, 10 mg/kg, and 30 mg/kg NOV1401 on plasma free FXI (squares) and the relationship with aPTT (diamonds) in cynomolgus macaques. A single 3 mg/kg dose resulted in a reduction in free FXI of approximately 90% over 5-6 weeks. All doses tested reduced free FXI to a similar extent, and higher doses tested did not appear to reduce free FXI to a greater extent than the 3 mg/kg dose.

Figures 4A-B show the X-ray structure of the Fab fragment of the NOV1401 antibody of the invention linked to FXI. Figure 4A shows the X-ray structure of the NOV1401 Fab-FXI CD complex. The FXI catalytic domain is depicted as a gray surface, Fab is depicted as a ribbon of light gray (light chain) and dark gray (heavy chain). Figure 4B shows the X-ray structure of the NOV1401 Fab-FXI CD complex superimposed on the FXI zymogen structure. The FXI catalytic domain is depicted as a gray ribbon. The Fab variable domains are depicted as a ribbon of light gray (VL) and dark gray (VH). Superimposed is an image of the zymogen structure, including the four apple-shaped domains as a dark gray ribbon at the base of the structure (PDB 2F83). The activating cleavage site (Ile370) is indicated.

Figures 5A-B show the structural changes of FXIa upon binding of the NOV1401 Fab fragment. Figure 5A shows the active site of FXIa prior to Fab binding. FXIa is depicted as a ribbon with a transparent surface. Sections of the structure that change conformation upon Fab binding are labeled (loop 145, loop 188, and loop 220). Subpockets S1 and S1' are indicated. Figure 5B shows the inactive conformation of FXI in complex with Fab (Fab not shown).

Figures 6A-Cshow the concentration-response curves for the anti-FXI/FXIa antibody. OnFigure 6AInhibition of factor XIa activity by NOV1401 is shown. Representative curve of inhibition of the enzymatic activity of full-length human FXIa versus the concentration of the NOV1401 antibody. The assay measured the cleavage of a fluorescently labeled peptide as described in Example 3. A logistic curve model was used to fit the nonlinear curve [y=A2+(A1-A2)/(1+(x/IC₅₀)^p), where y represents the % inhibition at inhibitor concentration x. A1 represents the lowest inhibition value and A2 the highest inhibition value. The exponent, p, is the Hill coefficient for this representative data set, from which the IC value was derived.₅₀160 p.m. OnFigure 6BThe aPTT curve is shown as a function of antibody concentration. A representative curve showing the increase in clotting time in the aPTT assay versus the concentration of the NOV1401 antibody using pooled human plasma. The assay measured the time to clotting after initiating the intrinsic clotting cascade in the presence of NOV1401 at different concentrations, as described in Example 4. The black line represents the fit using a logistic curve model. The dotted line corresponds to the baseline clotting time for pooled human plasma in the absence of NOV1401. The baseline clotting time is 32.3 seconds and is shown as the gray dashed line in the graph. The gray dotted line represents the antibody concentration at which the clotting time doubles compared to the baseline, i.e., reaches 2x the aPTT, which is 14 nM.Figure 6CA concentration-response curve of the antibody in the AOT assay is shown. A representative concentration-response curve of the inhibition of thrombin generation in the AOT assay for the NOV1401 antibody using pooled human plasma is shown. The assay measured the effects of different concentrations of NOV1401 on FXI-dependent thrombin generation through the so-called thrombin→FXIa positive feedback loop, which can be triggered by very low concentrations of tissue factor (TF), as described in Example 4. The black line represents a fit using a four-parameter dose-response model. The dotted line represents the residual thrombin concentration due to thrombin generation induced by small amounts of TF. The IC value₅₀24 nM and residual thrombin concentration of 159 nM (dotted line) were calculated from this dose-response curve.

Figures 7A-B show the effect of weekly s.c. administration of 10 mg/kg (N=3) and 100 mg/kg (N=5) NOV1401 for 13 weeks (14 doses) or i.v. administration of 50 mg/kg (N=3) for 4 weeks (5 doses) on aPTT and FXI activity (FXI:C). Figure 7A shows the effect on aPTT measured on study days 2, 23, and 79. aPTT increased 2.1- to 3-fold in all animals receiving NOV1401 and remained elevated throughout the dosing phase of the study. There was no dose dependence, and no dependence on mouse sex was observed. Figure 7B shows the effect on FXI:C measured on study days 2, 23, and 79 and expressed as a percentage of plasma FXI activity. FXI:C decreased in all animals receiving NOV1401 to levels of 5-12% and remained at these levels throughout the dosing phase of the study. There was no dose-dependent effect, and no gender-specific differences were observed.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of antibody molecules that specifically bind FXIa and inhibit its biological activity. The invention relates to both full-length IgG antibodies and their antigen-binding fragments, such as Fab fragments (e.g., antibodies NOV1090 and NOV1401).

Accordingly, the present invention relates to antibodies that specifically bind FXI and/or FXIa (e.g., human, rabbit, and cynomolgus monkey FXI and/or FXIa), pharmaceutical compositions, methods for producing, and methods for using such antibodies and compositions.

Factor XI

FXI plays an important role in both the intrinsic and extrinsic pathways of coagulation activation and in triggering the initiation and amplification phases of plasma hemostasis. Both factor XIIa and thrombin can activate FXI, resulting in persistent thrombin generation and inhibition of fibrinolysis. FXI plays a minor role in normal hemostasis in the setting of high tissue factor levels after vascular injury, yet it appears to play a key role in thrombosis. Severe factor XI deficiency is associated with a reduced incidence of ischemic stroke and venous thromboembolic events (Salomon et al. 2008; Salomon et al. (2011) Thromb Haemost.; 105: 269–73). Bleeding in individuals with severe factor XI deficiency is rare, typically mild, caused by injury, and primarily affects tissues with increased fibrinolytic activity, such as the oral mucosa, nasal mucosa, and urinary tract (Salomon et al. , 2011). Bleeding into critical organs, if it occurs, is extremely rare.

Blood coagulation is a sequential process during which coagulation factors interact and are activated, ultimately leading to fibrin formation and clot formation. In the classical cascade model of blood coagulation, fibrin formation can be initiated by two different pathways, the intrinsic and extrinsic pathways, respectively (Mackman, 2008).

In the extrinsic pathway, vascular damage allows extravascular tissue factor (TF) to interact with factor VII (FVII) and activate it, which subsequently leads to the activation of factor X and prothrombin. Active thrombin ultimately converts soluble fibrinogen into fibrin. The extrinsic pathway is essential for hemostasis; interference with the action of coagulation factors in this pathway is associated with the risk of bleeding.

In the intrinsic pathway, factor XII may be activated in some cases in a process called contact activation. The appearance of activated factor XIIa leads to the subsequent activation of factor XI and factor IX. Since factor IXa activates factor X, the extrinsic and intrinsic pathways converge (into a common pathway) at this stage. Thrombin activity is enhanced by increased thrombin generation via a positive feedback loop in which thrombin activates factor XI independently of factor XII. This positive feedback loop promotes continued thrombus growth but is only minimally involved in hemostasis, since strong activation by extravascular tissue factor is sufficient for clot formation. Thus, the intrinsic pathway is only marginally involved in hemostasis (Gailani and Renné (2007) Arterioscler Thromb Vasc Biol. 2007, 27(12): 2507–13, Müller, Gailiani, and Renné 2011).

Preclinical studies using various approaches to inhibit FXI or FXIa in different species have contributed to the importance of this therapeutic target. FXI-/- mice are resistant to experimental venous (Wang, et al. (2006) J Thromb Haemost; 4: 1982-8) and arterial (Wang, et al. (2005) J Thromb Haemost; 3: 695-702) thrombosis. Administration of an antibody (Ab 14E11) that blocks FXI activation by FXIIa to mice inhibited experimental thrombosis (Cheng, et al. (2010) Blood, 116: 3981-9) and reduced cerebral infarction size in a mouse model of ischemic stroke (Leung, et al. (2012) Transl Stroke Res 2012; 3: 381-9). Baboons administered an anti-FXI Ab that blocks binding and activation of FIX by FXIa showed reduced platelet-rich thrombi growth on collagen-coated vascular grafts (Tucker, et al. (2009) Blood 2009; 113: 936-44), and similar results were obtained with the 14E11 antibody in this model (Cheng, 2010). Excessive bleeding was not observed in any of these studies.

Blocking FXI synthesis with antisense oligonucleotides in mice (Zhang, et al. (2010) Blood 2010; 116: 4684-92), cynomolgus monkeys (Younis, et al. (2012) Blood 2012; 119: 2401-8), and baboons (Crosby, et al. (2013) Arterioscler Thromb Vasc Biol 2013; 33: 1670-8) resulted in antithrombotic and anticoagulant effects without excessive bleeding. Furthermore, similar effects were obtained by blocking FXIa with small molecule inhibitors in rat (Schumacher, et al. (2007) Eur J Pharmacol 2007; 570: 167-74) and rabbit (Wong, et al. (2011) J Thromb Thrombolysis 2011; 32: 129-37) models of venous and arterial thrombosis.

Patients with severe FXI deficiency rarely experience spontaneous bleeding and experience only moderate trauma-induced bleeding, except in tissues with high fibrinolytic activity. The rarity of severe FXI deficiency cases necessitates population-based studies to elucidate the thrombotic profile of these patients compared with the general population. Notably, reports of such studies have noted a reduced incidence of ischemic stroke (Salomon, 2008) and deep vein thrombosis (DVT) (Salomon, et al. (2011) Blood 2008; 111: 4113-17) in these patients. Thus, the incidence of ischemic stroke (N=1) in 115 patients with severe FXI deficiency was lower (p<0.003) than the expected incidence (N=8.6) in the general Israeli population, while the incidence of DVT (N=0) was lower (p<0.019) in patients with severe FXI deficiency than the expected incidence in the control population (N=4.7). Conversely, individuals with FXI levels above the 90th percentile have a two-fold higher risk of developing DVT (Meijers, et al. (2000) N Engl J Med. 2000; 342: 696–701).

Recently, patients undergoing total knee replacement, a procedure that predisposes to DVT, were treated with anti-FXI antisense agents or standard treatment (enoxaparin). The antisense group (300 mg) had a seven-fold reduction in the incidence of venous thrombosis and a nonsignificant reduction in the number of bleeding episodes compared with patients receiving standard treatment (Büller et al., (2014) N Engl J Med. 372(3): 232-40. doi: 10.1056/NEJMoa1405760. Epub 2014 Dec 7).

Taken together, the above results strongly support FXI as an important target for antithrombotic therapy.

Anti-FXIa antibodies and antigen-binding fragments

The present invention relates to antibodies that specifically bind FXI and/or FXIa. In some embodiments, the present invention relates to antibodies that specifically bind FXI and/or FXIa from humans, rabbits, and cynomolgus monkeys. Antibodies of the invention include, but are not limited to, human monoclonal antibodies and Fab fragments isolated as described in the Examples section.

The present invention relates to antibodies that specifically bind FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus macaque FXI and/or FXIa), wherein the antibodies comprise a VH domain having the amino acid sequence of SEQ ID NOs: 9 and 29. The present invention also relates to antibodies that specifically bind FXI and/or FXIa protein, wherein the antibodies comprise a VH CDR having the amino acid sequence of any of the VH CDRs listed in Table 1 below. In particular, the invention relates to antibodies that specifically bind FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus macaque FXI and/or FXIa), wherein the antibodies comprise (or, alternatively, consist of) one, two, three or more VH CDRs having the amino acid sequence of any of the VH CDRs listed in Table 1 below.

The present invention relates to antibodies that specifically bind FXIa protein, wherein said antibodies comprise a VL domain having the amino acid sequence of SEQ ID NOs: 19 or 39. The present invention also relates to antibodies that specifically bind FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus macaque FXI and/or FXIa), wherein the antibodies comprise a VL CDR having the amino acid sequence of any of the VL CDRs listed in Table 1 below. In particular, the invention relates to antibodies that specifically bind FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus macaque FXI and/or FXIa), wherein the antibodies comprise (or, alternatively, consist of) one, two, three or more VL CDRs having the amino acid sequence of any of the VL CDRs listed in Table 1 below.

Other antibodies of the invention comprise amino acid sequences that have been mutated but still have at least 60, 70, 80, 85, 90, or 95 percent identity of the CDR regions with the CDR regions whose sequences are listed in Table 1. In some embodiments, mutant amino acid sequences are provided in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the CDR regions compared to the CDR regions whose sequences are listed in Table 1.

The present invention also relates to nucleotide sequences encoding the VH, VL, full-length heavy chain, and full-length light chain of antibodies that specifically bind the FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus macaque FXI and/or FXIa). Such nucleotide sequences can be optimized for expression in mammalian cells (e.g., Table 1 provides optimized nucleotide sequences for the heavy chain and light chain of the antibodies of the invention).

Table 1. Examples of anti-FXIa antibodies, Fab fragments, and FXIa proteins. Description of the sequence Sequence designation (SEQ ID NO:) Amino acid or polynucleotide sequence Full-length human FXIa protein sequence (NCBI reference sequence: AAA51985) 1 miflyqvvhf ilftsvsgec vtqllkdtcf eggdittvft psakycqvvc tyhprcllft ftaespsedp trwftcvlkd svtetlprvn rtaaisgysf kqcshqisac nkdiyvdldm
kginynssva ksaqecqerc tddvhchfft yatrqfpsle hrnicllkht qtgtptritk ldkvvsgfsl kscalsnlac irdifpntvf adsnidsvma pdafvsgric thhpgclfft
ffsqewpkes qrnlcllkts esglpstrik kskalsgfsl qscrhsipvf chssfyhdtd flgeeldiva aksheacqkl ctnavrcqff tytpaqascn egkgkcylkl ssngsptkil
hgrggisgyt lrlckmdnec ttkikprivg gtasvrgewp wqvtlhttsp tqrhlcggsi ignqwiltaa hcfygvespk ilrvysgiln qseikedtsf fgvqeiiihd qykmaesgyd
iallklettv nytdsqrpic lpskgdrnvi ytdcwvtgwg yrklrdkiqn tlqkakiplv tneecqkryr ghkithkmic agyreggkda ckgdsggpls ckhnevwhlv gitswgegca
qrerpgvytn vveyvdwile ktqav Full-length nucleotide sequence of human FXIa (NCBI reference sequence: nm_000128.3) 2 aggcacacag gcaaaatcaa gttctacatc tgtccctgtg tatgtcactt gtttgaatac gaaataaaat taaaaaaata aattcagtgt attgagaaag caagcaattc tctcaaggta
tatttctgac atactaagat tttaacgact ttcacaaata tgctgtactg agagagaatg ttacataaca ttgagaacta gtacaagtaa atattaaagt gaagtgacca tttcctacac
aagctcattc agaggaggat gaagaccatt ttggaggaag aaaagcaccc ttattaagaa ttgcagcaag taagccaaca aggtcttttc aggatgattt tcttatatca agtggtacat
ttcattttat ttacttcagt ttctggtgaa tgtgtgactc agttgttgaa ggacacctgc tttgaaggag gggacattac tacggtcttc acaccaagcg ccaagtactg ccaggtagtc
tgcacttacc acccaagatg tttactcttc actttcacgg cggaatcacc atctgaggat cccacccgat ggtttacttg tgtcctgaaa gacagtgtta cagaaacact gccaagagtg
aataggacag cagcgatttc tgggtattct ttcaagcaat gctcacacca aataagcgct tgcaacaaag acatttatgt ggacctagac atgaagggca taaactataa cagctcagtt
gccaagagtg ctcaagaatg ccaagaaaga tgcacggatg acgtccactg ccactttttc acgtacgcca caaggcagtt tcccagcctg gagcatcgta acatttgtct actgaagcac
acccaaacag ggacaccaac cagaataacg aagctcgata aagtggtgtc tggattttca ctgaaatcct gtgcactttc taatctggct tgtattaggg acattttccc taatacggtg
tttgcagaca gcaacatcga cagtgtcatg gctcccgatg cttttgtctg tggccgaatc tgcactcatc atcccggttg cttgtttttt accttctttt cccaggaatg gcccaaagaa
tctcaaagaa atctttgtct ccttaaaaca tctgagagtg gattgcccag tacacgcatt aaaaagagca aagctctttc tggtttcagt ctacaaagct gcaggcacag catcccagtg
ttctgccatt cttcatttta ccatgacact gatttcttgg gagaagaact ggatattgtt gctgcaaaaa gtcacgaggc ctgccagaaa ctgtgcacca atgccgtccg ctgccagttt
tttacctata ccccagccca agcatcctgc aacgaaggga agggcaagtg ttacttaaag ctttcttcaa acggatctcc aactaaaata cttcacggga gaggaggcat ctctggatac
acattaaggt tgtgtaaaat ggataatgag tgtaccacca aaatcaagcc caggatcgtt ggaggaactg cgtctgttcg tggtgagtgg ccgtggcagg tgaccctgca cacaacctca
cccactcaga gacacctgtg tggaggctcc atcattggaa accagtggat attaacagcc gctcactgtt tctatggggt agagtcacct aagattttgc gtgtctacag tggcatttta
aatcaatctg aaataaaaga ggacacatct ttctttgggg ttcaagaaat aataatccat gatcagtata aaatggcaga aagcgggtat gatattgcct tgttgaaact ggaaaccaca
gtgaattaca cagattctca acgacccata tgcctgcctt ccaaaggaga tagaaatgta atatacactg attgctgggt gactggatgg gggtacagaa aactaagaga caaaatacaa
aatactctcc agaaagccaa gataccctta gtgaccaacg aagagtgcca gaagagatac agaggacata aaataaccca taagatgatc tgtgccggct acagggaagg agggaaggac
gcttgcaagg gagattcggg aggccctctg tcctgcaaac acaatgaggt ctggcatctg gtaggcatca cgagctgggg cgaaggctgt gctcaaaggg agcggccagg tgtttacacc
aacgtggtcg agtacgtgga ctggattctg gagaaaactc aagcagtgtg aatgggttcc caggggccat tggagtccct gaaggaccca ggatttgctg ggagagggtg ttgagttcac
tgtgccagca tgcttcctcc acagtaacac gctgaagggg cttggtgttt gtaagaaaat gctagaagaa aacaaactgt cacaagttgt tatgtccaaa actcccgttc tatgatcgtt
gtagtttgtt tgagcattca gtctctttgt ttttgatcac gcttctatgg agtccaagaa ttaccataag gcaatatttc tgaagattac tatataggca gatatagcag aaaataacca
agtagtggca gtggggatca ggcagaagaa ctggtaaaag aagccaccat aaatagattt gttcgatgaa agatgaaaac tggaagaaag gagaacaaag acagtcttca ccattttgca
ggaatctaca ctctgcctat gtgaacacat ttcttttgta aagaaagaaa ttgattgcat ttaatggcag attttcagaa tagtcaggaa ttcttgtcat ttccatttta aaatatatat
taaaaaaaat cagttcgagt agacacgagc taagagtgaa tgtgaagata acagaatttc tgtgtggaag aggattacaa gcagcaattt acctggaagt gataccttag gggcaatctt
gaagatacac tttcctgaaa aatgatttgt gatggattgt atatttattt aaaatatctt gggaggggag gctgatggag atagggagca tgctcaaacc tccctaagac aagctgctgc
tgtgactatg ggctcccaaa gagctagatc gtatatttat ttgacaaaaa tcaccataga ctgcatccat actacagaga aaaaacaatt agggcgcaaa tggatagtta cagtaaagtc
ttcagcaagc agctgcctgt attctaagca ctgggatttt ctgtttcgtg caaatattta tctcattatt gttgtgatct agttcaataa cctagaattt gaattgtcac cacatagctt
tcaatctgtg ccaacaacta tacaattcat caagtgtg NOV1090 HCDR1 (Kabat) 3 TAAMS HCDR2 (Kabat) 4 GISGSGSSTYYADSVKG HCDR3 (Kabat) 5 ELSYLYSGYYFDY HCDR1 (Chothia) 6 GFTFSTA HCDR2 (Chothia) 7 SGSGSS HCDR3 (Chothia) 8 ELSYLYSGYYFDY HCDR1 (IMGT) 43 GFTFSTAA HCDR2 (IMGT) 44 ISGSGSST HCDR3 (IMGT) 45 ARELSYLYSGYYFDY HCDR1 (combined) 46 GFTFSTAAMS HCDR2 (combined) 4 GISGSGSSTYYADSVKG HCDR3 (combined) 5 ELSYLYSGYYFDY VH 9 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSS DNA encoding VH 10 CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTACTGCTGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCGGTATCTCTGGTTCTGGTTCTTCTACCTACTATGCG GATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGGTGAACTGTCTTACCTGTACTCTGGTTACTACTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA Heavy chain 11 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DNA encoding the heavy chain 12 CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTACTGCTGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCGGTATCTCTGGTTCTGGTTCTT CTACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGAACTGTCTTACCTGTACTCTGGTTACTACTTCGATTACTGGGGC CAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGAC AAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCAGCGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA LCDR1 (Kabat) 13 SGSSSNIGSNDVS LCDR2 (Kabat) 14 KNYNRPS LCDR3 (Kabat) 15 SAWDQRQFDVV LCDR1 (Chothia) 16 SSSNIGSND LCDR2 (Chothia) 17 KNY LCDR3 (Chothia) 18 WDQRQFDV LCDR1 (IMGT) 47 SSNIGSND LCDR2 (IMGT) 37 KNY LCDR3 (IMGT) 15 SAWDQRQFDVV LCDR1 (combined) 33 SGSSSNIGSNDVS LCDR2 (combined) 14 KNYNRPS LCDR3 (combined) 15 SAWDQRQFDVV VL 19 DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCSAWDQRQFDVVFGGGTKLTVL DNA encoding VL 20 GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCTGACCATTAGCTGTAGCGGCAGCAGCAGCAACATTGGTTCTAACGACGTGTCTTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTACAAAAACTACAACCGC CCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCTCTGCTTGGGACCAGCGTCAGTTCGACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA Light chain 21 DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCSAWDQRQFDVVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA encoding the light chain 22 GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCTGACCATTAGCTGTAGCGGCAGCAGCAGCAACATTGGTTCTAACGACGTGTCTTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTACAAAAACTACAAC CGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCTCTGCTTGGGACCAGCGTCAGTTCGACGTTGTGTTTGGCGGCGGCACGAAGTTAACC GTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGA GTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA NOV1401 HCDR1 (Kabat) 23 TAAMS HCDR2 (Kabat) 24 GISGSGSSTYYADSVKG HCDR3 (Kabat) 25 ELSYLYSGYYFDY HCDR1 (Chothia) 26 GFTFSTA HCDR2 (Chothia) 27 SGSGSS HCDR3 (Chothia) 28 ELSYLYSGYYFDY HCDR1 (IMGT) 43 GFTFSTAA HCDR2 (IMGT) 44 ISGSGSST HCDR3 (IMGT) 45 ARELSYLYSGYYFDY HCDR1 (combined) 46 GFTFSTAAMS HCDR2 (combined) 4 GISGSGSSTYYADSVKG HCDR3 (combined) 5 ELSYLYSGYYFDY VH 29 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSS DNA encoding VH 30 CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCACCGCCGCTATGAGCTGGGTTCGACAGGCCCCAGGGAAAGGCCTCGAGTGGGTCTCAGGGATTAGCGGTAGCGGCTCTAGCACCTACTACGCC GATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCTGAGCTACCTGTATAGCGGCTACTACTTCGACTACTGGGGTCAAGGCACCCTGGTCACCGTGTCTAGC Heavy chain 31 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK DNA encoding the heavy chain 32 CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCACCGCCGCTATGAGCTGGGTTCGACAGGCCCCAGGGAAAGGCCTCGAGTGGGTCTCAGGGATTAGCGGTAGCGGCTCTA GCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCTGAGCTACCTGTATAGCGGCTACTACTTCGACTACTGGGGT CAAGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCT CTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGAC AAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGT ACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAG ACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACT ACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGCAAG LCDR1 (Kabat) 33 SGSSSNIGSNDVS LCDR2 (Kabat) 34 KNYNRPS LCDR3 (Kabat) 35 SAWDQRQFDVV LCDR1 (Chothia) 36 SSSNIGSND LCDR2 (Chothia) 37 KNY LCDR3 (Chothia) 38 WDQRQFDV LCDR1 (IMGT) 47 SSNIGSND LCDR2 (IMGT) 37 KNY LCDR3 (IMGT) 15 SAWDQRQFDVV LCDR1 (combined) 33 SGSSSNIGSNDVS LCDR2 (combined) 14 KNYNRPS LCDR3 (combined) 15 SAWDQRQFDVV VL 39 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCSAWDQRQFDVVFGGGTKLTVL DNA encoding VL 40 CAGTCAGTCCTGACTCAGCCCCCTAGCGCTAGTGGCACCCCTGGTCAAAGAGTGACTATTAGCTGTAGCGGCTCTAGCTCTAATATCGGCTCTAACGACGTCAGCTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTATAAGAACTATAATAGG CCTAGCGGCGTGCCCGATAGGTTTAGCGGATCTAAATCAGGGACTTCTGCTAGTCTGGCTATTAGCGGCCTGCAGTCAGAGGACGAGGCCGACTACTACTGTAGCGCCTGGGATCAGCGTCAGTTCGACGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTG Light chain 41 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCSAWDQRQFDVVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS DNA encoding the light chain 42 CAGTCAGTCCTGACTCAGCCCCCTAGCGCTAGTGGCACCCCTGGTCAAAGAGTGACTATTAGCTGTAGCGGCTCTAGCTCTAATATCGGCTCTAACGACGTCAGCTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTATAAGAACTATAAT AGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGATCTAAATCAGGGACTTCTGCTAGTCTGGCTATTAGCGGCCTGCAGTCAGAGGACGAGGCCGACTACTACTGTAGCGCCTGGGATCAGCGTCAGTTCGACGTGGTGTTCGGCGGAGGCACTAAGCTGACC GTGCTGGGTCAACCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGC GTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGCAGC

Other antibodies of the invention include those in which the amino acid sequences or nucleotide sequences encoding the amino acid sequences have been mutated but still have at least 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to the sequences listed in Table 1. Some embodiments include mutant amino acid sequences in which no more than 1, 2, 3, 4, or 5 amino acids have been mutated in the variable regions compared to the variable region sequences listed in Table 1, but they retain substantially the same antigen-binding activity.

Because each of these antibodies can bind FXI and/or FXIa, the VH, VL, full-length light chain, and full-length heavy chain sequences (amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other FXI and/or FXIa-binding antibodies of the invention. Such "mixed and matched" FXI and/or FXIa-binding antibodies can be tested in binding assays known in the art (e.g., ELISA and other assays described in the Examples section). When mixing and matching these chains, the VH sequence from a particular VH/VL pair should be replaced with a structurally similar VH sequence. Similarly, the full-length heavy chain sequence from a particular full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Similarly, the VL sequence from a particular VH/VL pair should be replaced with a structurally similar VL sequence. Similarly, the full-length light chain sequence from a particular full-length heavy chain/full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.

Accordingly, in one aspect, the invention provides an isolated antibody, or antigen-binding region thereof, having: a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29, and a light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 39, wherein the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa).

More particularly, in some aspects, the invention relates to an isolated antibody, or antigen-binding region thereof, having a heavy chain variable domain and a light chain variable domain comprising amino acid sequences selected from SEQ ID NOs: 9 and 29; or 19 and 39, respectively.

In a specific embodiment, the antibody, or antigen-binding fragment thereof, provided herein that specifically binds human FXI and/or FXIa comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19.

In a specific embodiment, the antibody, or antigen-binding fragment thereof, provided herein that specifically binds human FXI and/or FXIa comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39.

In another aspect, the invention provides (i) an isolated antibody having: a full-length heavy chain comprising an amino acid sequence that has been optimized for expression in mammalian cells selected from the group consisting of SEQ ID NOs: 11 or 31, and a full-length light chain comprising an amino acid sequence that has been optimized for expression in mammalian cells selected from the group consisting of SEQ ID NOs: 21 or 41; or (ii) a functional protein comprising an antigen-binding fragment thereof. More particularly, in some aspects, the invention relates to an isolated antibody, or an antigen-binding region thereof, having a heavy chain and a light chain comprising amino acid sequences selected from SEQ ID NOs: 11 and 31; or 19 and 39, respectively.

In a specific embodiment, the antibody, or antigen-binding fragment thereof, provided herein that specifically binds human FXI and/or FXIa comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

In a specific embodiment, the antibody, or antigen-binding fragment thereof, provided herein, that specifically binds human FXI and/or FXIa comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 41.

As used herein, the terms "complementarity-determining region" and "CDR" refer to the amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. Typically, there are three CDRs in each variable region of the heavy chain (HCDR1, HCDR2, HCDR3) and three CDRs in each variable region of the light chain (LCDR1, LCDR2, LCDR3).

The exact amino acid sequence boundaries of a particular CDR can be easily determined using any of the well-known systems, including those described in: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering system), Al-Lazikani et al. (1997) JMB 273, 927–948 (the “Chothia” numbering system), Lefranc et al. (2003) Dev. Comp. Immunol., 27, 55–77 (the “IMGT” numbering system), or a “combined” system.

For example, according to the Kabat system, the amino acid residues of the CDRs of the NOV1090 antibody in the variable domain of the heavy chain (VH) are 31-35 (HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3); and the amino acid residues of the CDRs in the variable domain of the light chain (VL) are 22-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3). According to the Chothia system, the amino acid residues of the CDRs in VH are 26-32 (HCDR1), 52-57 (HCDR2), and 99-111 (HCDR3); and the amino acid residues in VL are 25-33 (LCDR1), 51-53 (LCDR2), and 92-99 (LCDR3). When combining the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3) in the human VH region and amino acid residues 22-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3) in the human VL region. When combining the Kabat and Chothia CDR definitions, the "combined" CDRs consist of amino acid residues 26-35 (HCDR1), 50-66 (HCDR2), and 99-108 (HCDR3) in the human VH region and amino acid residues 24-38 (LCDR1), 54-60 (LCDR2), and 93-101 (LCDR3) in the human VL region. As another example, according to the IMGT system, the amino acid residues of the CDRs in the variable domain of the heavy chain (VH) have sequence numbers 26-33 (HCDR1), 51-58 (HCDR2), and 97-108 (HCDR3); and the amino acid residues of the CDRs in the variable domain of the light chain (VL) have sequence numbers 27-36 (LCDR1), 54-56 (LCDR2), and 93-101 (LCDR3). Table 1 provides exemplary sequences according to the Kabat, Chothia, combined, and IMGT systems for HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of anti-FXI/FXIa antibodies, such as NOV1090 and NOV1401. In another aspect, the present invention relates to FXIa-binding antibodies comprising the heavy chain and light chain CDR1, CDR2, and CDR3 regions listed in Table 1, or a combination thereof. The amino acid sequences of the VH CDR1 of the antibodies are set forth in SEQ ID NOs: 3 and 23. The amino acid sequences of the VH CDR2 of the antibodies are set forth in SEQ ID NOs: 4 and 24. The amino acid sequences of the VH CDR3 of the antibodies are set forth in SEQ ID NOs: 5 and 25. The amino acid sequences of the VL CDR1 of the antibodies are set forth in SEQ ID NOs: 13 and 33. The amino acid sequences of the VL CDR2 of the antibodies are set forth in SEQ ID NOs: 14 and 34. The amino acid sequences of the VL CDR3 of the antibodies are set forth in SEQ ID NOs: 15 and 35. These CDR regions are defined in accordance with the Kabat system.

Alternatively, when determined according to the Chothia system (Al-Lazikani et al. , (1997) JMB 273, 927-948), the amino acid sequences of the VH CDR1 antibodies are set forth in SEQ ID NOs: 6 and 26. The amino acid sequences of the VH CDR2 antibodies are set forth in SEQ ID NOs: 7 and 27. The amino acid sequences of the VH CDR3 antibodies are set forth in SEQ ID NOs: 8 and 28. The amino acid sequences of the VL CDR1 antibodies are set forth in SEQ ID NOs: 16 and 36. The amino acid sequences of the VL CDR2 antibodies are set forth in SEQ ID NOs: 17 and 37. The amino acid sequences of the VL CDR3 antibodies are set forth in SEQ ID NOs: 18 and 38.

Alternatively, when determined according to the combined system, the amino acid sequences of the VH CDR1 antibodies are set forth in SEQ ID NO: 46. The amino acid sequences of the VH CDR2 antibodies are set forth in SEQ ID NO: 4. The amino acid sequences of the VH CDR3 antibodies are set forth in SEQ ID NO: 5. The amino acid sequences of the VL CDR1 antibodies are set forth in SEQ ID NO: 33. The amino acid sequences of the VL CDR2 antibodies are set forth in SEQ ID NO: 14. The amino acid sequences of the VL CDR3 antibodies are set forth in SEQ ID NO: 15.

Alternatively, when determined according to the IMGT numbering system, the amino acid sequences of the VH CDR1 antibodies are set forth in SEQ ID NO: 43. The amino acid sequences of the VH CDR2 antibodies are set forth in SEQ ID NO: 44. The amino acid sequences of the VH CDR3 antibodies are set forth in SEQ ID NO: 45. The amino acid sequences of the VL CDR1 antibodies are set forth in SEQ ID NO: 47. The amino acid sequences of the VL CDR2 antibodies are set forth in SEQ ID NO: 37. The amino acid sequences of the VL CDR3 antibodies are set forth in SEQ ID NO: 15.

Since each of these antibodies can bind FXI and/or FXIa and the antigen-binding specificity is determined primarily by the CDR1, 2, and 3 regions, the VH CDR1, 2, and 3 sequences and the VL CDR1, 2, and 3 sequences can be "mixed and matched" (i.e., CDRs from different antibodies can be mixed and matched, although each antibody preferably comprises VH CDR1, 2, and 3 and VL CDR1, 2, and 3, to create other FXI and/or FXIa-binding molecules of the invention). Such "mixed and matched" FXI and/or FXIa-binding antibodies can be tested in binding assays known in the art and described in the Examples section (e.g., ELISA, SET, BIACORE™ assays). When combining and combining VH CDR sequences, the CDR1, CDR2 and/or CDR3 sequence from a particular VH sequence should be replaced by structurally similar CDR sequence(s). Similarly, when combining and combining VL CDR sequences, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence should be replaced by structurally similar CDR sequence(s). Those skilled in the art will appreciate that new VH and VL sequences can be created by replacing one or more VH and/or VL CDR region sequences with structurally similar sequences from the CDR sequences provided herein for the monoclonal antibodies of the present invention. In one embodiment, in addition to the above, the antigen-binding fragments of the antibodies described herein may comprise VH CDR1, 2 and 3 or VL CDRs 1, 2 and 3, wherein the fragment binds FXI and/or FXIa as a single variable domain.

In specific embodiments of the invention, the antibodies, or antigen-binding fragments thereof, may have the heavy and light chain sequences from the Fab shown in Table 1. More specifically, the antibody, or antigen-binding fragments thereof, may have the heavy and light chain sequence from NOV1090 and NOV1401.

In other embodiments, an antibody or antigen-binding fragment that specifically binds FXI and/or FXIa comprises a variable heavy chain CDR1, variable heavy chain CDR2, variable heavy chain CDR3, variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3, as determined according to the Kabat system and as listed in Table 1. In other embodiments, an antibody or antigen-binding fragment that specifically binds FXI and/or FXIa comprises a variable heavy chain CDR1, variable heavy chain CDR2, variable heavy chain CDR3, variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3, as determined according to the Chothia system and as listed in Table 1. In other embodiments, an antibody or antigen-binding fragment that specifically binds FXI and/or FXIa comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3, as determined according to the combined system and as listed in Table 1. In other embodiments, an antibody or antigen-binding fragment that specifically binds FXI and/or FXIa comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3, as determined according to the IMGT system and as listed in Table 1.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQ ID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chain variable region CDR1 of SEQ ID NO: 13; a light chain variable region CDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 of SEQ ID NO: 15.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 23; a heavy chain variable region CDR2 of SEQ ID NO: 24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 of SEQ ID NO: 35.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQ ID NO: 7; a heavy chain variable region CDR3 of SEQ ID NO: 8; a light chain variable region CDR1 of SEQ ID NO: 16; a light chain variable region CDR2 of SEQ ID NO: 17; and a light chain variable region CDR3 of SEQ ID NO: 18.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 26; a heavy chain variable region CDR2 of SEQ ID NO: 27; a heavy chain variable region CDR3 of SEQ ID NO: 28; a light chain variable region CDR1 of SEQ ID NO: 36; a light chain variable region CDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 of SEQ ID NO: 38.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQ ID NO: 44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a light chain variable region CDR1 of SEQ ID NO: 47; a light chain variable region CDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 of SEQ ID NO: 15.

In a specific embodiment, the invention relates to an antibody that specifically binds FXI and/or FXIa, which comprises a heavy chain variable region CDR1 of SEQ ID NO: 46; a heavy chain variable region CDR2 of SEQ ID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a light chain variable region CDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 of SEQ ID NO: 15.

In specific embodiments, the invention relates to antibodies or antigen-binding fragments that specifically bind FXI and/or FXIa, which are listed in Table 1. In a preferred embodiment, the antibody or antigen-binding fragment that binds FXI and/or FXIa is NOV1090 and NOV1401.

As described herein, a human antibody comprises variable regions of a heavy or light chain, or full-length heavy or light chains, that are a "product" or "derived from" a specific germline sequence if the variable regions or full-length chains of the antibody are obtained from a system that utilizes human germline immunoglobulin genes. Such systems include immunization of a transgenic mouse carrying human immunoglobulin genes with an antigen of interest or screening a library of human immunoglobulin genes displayed on phage with the antigen of interest. A human antibody that is a "product" of or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is most closely related (i.e., has the highest % identity) to the human antibody sequence.

A human antibody that is a "product" or "derived from" a specific human germline immunoglobulin sequence may have amino acid differences compared to the germline sequence, such as due to naturally occurring somatic mutations or intentional site-specific mutations. However, within the VH or VL framework regions, a selected human antibody typically shares at least 90% amino acid sequence identity with the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that distinguish the human antibody as uniquely human when compared to the amino acid sequences of germline immunoglobulins from other species (e.g., murine germline immunoglobulin sequences). In some cases, the human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.

Typically, a recombinant human antibody will have no more than 10 amino acid differences in the VH or VL framework regions from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may have no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene. Examples of human germline immunoglobulin genes include, but are not limited to, the germline variable domain fragments described below, as well as DP47 and DPK9.

Homologous antibodies

In another embodiment, the present invention provides an antibody, or antigen-binding fragment thereof, comprising amino acid sequences that are homologous to the sequences set forth in Table 1 (e.g., SEQ ID NOs: 29, 31, 39, or 41), wherein the antibody binds an FXI and/or FXIa protein (e.g., human, rabbit, and cynomolgus monkey FXIa) and retains the desired functional properties of the antibodies set forth in Table 1, such as NOV1090 and NOV1401. In particular aspects, such homologous antibodies retain the amino acid sequences of the CDRs set forth in Table 1 (e.g., Kabat CDRs, Chothia CDRs, IMGT CDRs, or combined CDRs).

For example, the invention relates to an isolated antibody, or a functional antigen-binding fragment thereof, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29; the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 and 39; and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa). In one embodiment, the isolated antibody, or functional antigen-binding fragment thereof, comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 19; and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa). In one embodiment, an isolated antibody, or a functional antigen-binding fragment thereof, comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 29; the light chain variable domain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 39; and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa). In some aspects of the invention, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences defined in accordance with the Kabat system, for example, SEQ ID NOs: 3, 4, 5, 13, 14 and 15, respectively. In some other aspects of the invention, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences defined in accordance with the Chothia system, for example, SEQ ID NOs: 6, 7, 8, 16, 17 and 18, respectively. In some other aspects, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined according to the combined system, for example, SEQ ID NOs: 46, 4, 5, 33, 14, and 15, respectively. In some other aspects, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined according to the IMGT system, for example, SEQ ID NOs: 43, 44, 45, 47, 37, and 15, respectively.

In other embodiments, the VH and/or VL amino acid sequences may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences shown in Table 1. In other embodiments, the VH and/or VL amino acid sequences may be identical except for an amino acid substitution at no more than 1, 2, 3, 4, or 5 amino acid positions. An antibody having VH and VL regions with a high degree (i.e., 80% or more) of identity to the VH and VL regions of the antibodies listed in Table 1 can be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the encoding nucleic acid molecules, SEQ ID NOs: 10 or 30 and SEQ ID NOs: 20 and 40, respectively, followed by testing the encoded altered antibody for retention of function using the functional assays described herein.

In other embodiments, the amino acid sequences of the full-length heavy chain and/or the full-length light chain may be 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 (e.g., SEQ ID NOs: 11 and/or 21, or 31 and/or 41). An antibody having a full-length heavy chain and a full-length light chain with a high degree (i.e., 80% or more) of identity to full-length heavy chains having any of SEQ ID NOs: 11 or 31 and full-length light chains having any of SEQ ID NOs: 21 or 41 can be produced by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding such polypeptides, followed by testing the encoded altered antibody for retention of function using the functional assays described herein.

In one aspect of the present invention, there is provided an isolated antibody, or a functional antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 31; the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 41, and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa). In one embodiment, the isolated antibody, or functional antigen-binding fragment thereof, comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 11; the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 21, and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus macaque FXIa). In one embodiment, the isolated antibody, or functional antigen-binding fragment thereof, comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 31; the light chain comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 41, and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus monkey FXIa). In some aspects of the invention, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences defined in accordance with the Kabat system, for example, SEQ ID NOs: 3, 4, 5, 13, 14 and 15, respectively. In some other aspects of the invention, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences defined in accordance with the Chothia system, for example, SEQ ID NOs: 6, 7, 8, 16, 17 and 18, respectively. In some other aspects, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined according to the combined system, for example, SEQ ID NOs: 46, 4, 5, 33, 14, and 15, respectively. In some other aspects, the heavy and light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences defined according to the IMGT system, for example, SEQ ID NOs: 43, 44, 45, 47, 37, and 15, respectively.

In other embodiments, the nucleotide sequences of the full-length heavy chain and/or the full-length light chain may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 (e.g., SEQ ID NOs: 12 and/or 22, or 32 and/or 42).

In other embodiments, the nucleotide sequences of the heavy chain variable regions and/or light chain variable regions may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in Table 1 (e.g., SEQ ID NOs: 10 and/or 20, or 30 and/or 40).

As used herein, the percent identity between two sequences depends on the number of identical positions in both sequences (i.e., the percent identity is equal to the number of identical positions/the total number of positions x 100), taking into account the number of deletions and the length of each deletion required to optimally align the two sequences. Sequence comparison and determination of the percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the present invention can also be used as a "target sequence" to search publicly available databases, for example, to identify related sequences. For example, such a search can be performed using the BLAST program (version 2.0) described in Altschul, et al. , 1990 J. Mol. Biol. 215: 403-10.

Antibodies with conservative modifications

In specific embodiments, an antibody of the invention comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences have the specific amino acid sequences of the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the FXIa-binding antibodies of the invention.

Accordingly, the invention relates to an isolated antibody, or an antigen-binding fragment thereof, consisting of a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein: the amino acid sequences of CDR1 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 3 and 23, as well as conservative modifications thereof; the amino acid sequences of CDR2 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 4 and 24, as well as conservative modifications thereof; the amino acid sequences of CDR3 of the heavy chain variable region are selected from the group consisting of SEQ ID NOs: 5 and 25, as well as conservative modifications thereof; the amino acid sequences of CDR1 of the variable region of the light chain are selected from the group consisting of SEQ ID NOs: 13 and 33, as well as conservative modifications thereof; the amino acid sequences of CDR2 of the variable region of the light chain are selected from the group consisting of SEQ ID NOs: 14 and 34, as well as conservative modifications thereof; the amino acid sequences of CDR3 of the variable region of the light chain are selected from the group consisting of SEQ ID NOs: 15 and 35, as well as conservative modifications thereof; wherein the antibody, or antigen-binding fragments thereof, specifically binds FXIa.

In one aspect, the present invention relates to an isolated antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein: the amino acid sequences of CDR1 of the heavy chain variable region are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; the amino acid sequences of CDR2 of the heavy chain variable region are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; the amino acid sequences of CDR3 of the heavy chain variable region are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; the amino acid sequences of CDR1 of the variable region of the light chain are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; the amino acid sequences of CDR2 of the variable region of the light chain are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; the amino acid sequences of CDR3 of the variable region of the light chain are selected from the group consisting of the sequences listed in Table 1, as well as conservative modifications thereof; wherein the antibody, or antigen-binding fragments thereof, specifically binds FXIa.

In other embodiments, an antibody of the invention is optimized for expression in a mammalian cell and has a full-length heavy chain sequence and a full-length light chain sequence, wherein one or more of these sequences comprise specific amino acid sequences of the antibodies described herein or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the FXIa-binding antibodies of the invention. Accordingly, the invention provides an isolated antibody optimized for expression in a mammalian cell, consisting of a full-length heavy chain and a full-length light chain, wherein the full-length heavy chain comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 11 or 31, as well as conservative modifications thereof; and the full-length light chain comprises amino acid sequences selected from the group consisting of SEQ ID NOs: 21 or 41, as well as conservative modifications thereof; and the antibody specifically binds FXI and/or FXIa (e.g., human, rabbit, and cynomolgus monkey FXIa).

Antibodies that bind the same epitope

The present invention provides antibodies that bind the same epitope as the FXI and/or FXIa binding antibodies listed in Table 1. Accordingly, additional antibodies can be identified based on their ability to compete (e.g., competitively inhibit binding, to a statistically significant extent, by binding to the same or an overlapping epitope) with other antibodies of the invention in FXI and/or FXIa binding assays (such as those described in the Examples section). The ability of a test antibody to inhibit binding of antibodies of the invention to an FXI and/or FXIa protein demonstrates that the test antibody can compete with the antibody for binding to FXI and/or FXIa; Such an antibody may, according to a non-limiting theory, bind the same or a related (e.g., structurally similar or spatially proximal) epitope on the FXI and/or FXIa protein as the antibody with which it competes. In a specific embodiment, an antibody that binds the same epitope on FXI and/or FXIa as the antibodies of the present invention is a human monoclonal antibody. Such monoclonal antibodies can be produced and isolated as described herein.

As described herein, an antibody "competes" for binding when the competing antibody binds the same epitope of FXI and/or FXIa as the antibody, or antigen-binding fragment, of the invention (e.g., NOV1401 or NOV1090) and inhibits the binding of FXI and/or FXIa by the antibody, or antigen-binding fragment, of the invention by more than 50% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) in the presence of an equimolar concentration of the competing antibody. This can be determined, for example, in a competitive binding assay using any methods well known to those skilled in the art.

As described herein, an antibody, or antigen-binding fragment thereof, does not "compete" with an anti-FXI and/or anti-FXIa antibody, or antigen-binding fragment, of the invention (e.g., NOV1401 or NOV1090), unless said competing antibody, or antigen-binding fragment thereof, binds the same epitope of FXI and/or FXIa, or an overlapping epitope of FXI and/or FXIa, as the antibody, or antigen-binding fragment, of the invention. As described herein, a competing antibody, or antigen-binding fragment thereof, does not include an antibody that (i) sterically blocks an antibody, or antigen-binding fragment, of the invention from binding to its target (e.g., if said competing antibody binds a nearby, non-overlapping epitope of FXI and/or FXIa and physically interferes with the binding of the antibody, or antigen-binding fragment, of the invention to its target); and/or (ii) binds another, non-overlapping epitope of FXI and/or FXIa and induces a conformational change in the FXI and/or FXIa protein such that said protein can no longer be bound by the anti-FXI and/or FXIa antibody, or antigen-binding fragment, of the invention in the same way as it would in the absence of said conformational change.

Engineered and modified antibodies

An antibody of the invention can also be produced using an antibody comprising one or more of the VH and/or VL sequences provided herein as a starting material for constructing a modified antibody, wherein the modified antibody can have altered properties compared to the original antibody. The antibody can be constructed by modifying one or more residues in one or both variable regions (i.e., VH and/or VL), for example, in one or more CDR regions and/or in one or more framework regions. Additionally or alternatively, the antibody can be constructed by modifying residues in the constant region(s), for example, to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. Consequently, amino acid sequences within the CDRs vary more among individual antibodies than sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific natural antibodies by constructing expression vectors containing CDR sequences from a particular natural antibody grafted onto framework sequences from another antibody with different properties (see, e.g., Riechmann, L. et al. , 1998 Nature 332: 323–327; Jones, P. et al. , 1986 Nature 321: 522–525; Queen, C. et al. , 1989 Proc. Natl. Acad., USA 86: 10029–10033; U.S. Patent No. 5,225,539 to Winter, and U.S. Patents Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370). authors Queen et al. )

Accordingly, another embodiment of the invention relates to an isolated antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 25, respectively; and a light chain variable region comprising CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 33; CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34; and CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 35, respectively. Thus, such antibodies contain CDR sequences from the VH and VL of monoclonal antibodies, although they may contain framework sequences different from those of these antibodies.

Such framework sequences can be obtained from publicly available DNA databases or published sources that contain germline antibody gene sequences. For example, germline DNA sequences for the human heavy and light chain variable region genes can be found in the VBase human germline gene sequence database (available online at mrc-cpe.cam.ac.uk/vbase) and in Kabat, EA, et al. , 1991 Sequences of Proteins of Immunological Interest, fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. , 1992 J. Mol. Biol. 227: 776–798; and Cox, JPL et al. , 1994 Eur. J Immunol. 24: 827–836; the contents of each of which are incorporated herein by reference in their entirety.

Exemplary framework sequences for use in the antibodies of the invention are those that have structural similarity to framework sequences used in selected antibodies of the invention, such as consensus sequences and/or framework sequences used in monoclonal antibodies of the invention. The VH CDR1, 2, and 3 and VL CDR1, 2, and 3 sequences may be grafted onto framework regions having a sequence identical to the germline immunoglobulin gene sequence from which the framework sequence is derived, or the CDR sequences may be grafted onto framework regions having one or more mutations compared to the germline sequences. For example, it has been found that in some cases it is advantageous to mutate residues in framework regions to maintain or enhance the antigen-binding ability of an antibody (see, e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762; and 6,180,370 to Queenet al.). Frameworks that can be used as scaffolds on which to generate the antibodies and antigen-binding fragments described herein include, but are not limited to, VH1A, VH1B, VH3, Vk1, Vl2, and Vk2. Additional frameworks are known in the art and can be found, for example, in the vBase database on the Internet at the address vbase.mrc-cpe.cam.ac.uk/index.php?&MMN_position=1:1.

Accordingly, one embodiment of the invention relates to isolated FXIa-binding antibodies, or antigen-binding fragments thereof, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 29, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions in the framework region of such sequences, and also comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 or 39, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions in the framework region of such sequences.

Another type of variable region modification is the introduction of mutations into amino acid residues in the VH and/or VL CDR1, CDR2, and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation." Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding or other functional property of interest can be assessed in vitro or in vivo assays, as described in the Examples section herein. Conservative modifications (as described above) can be made. Mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically no more than one, two, three, four, or five residues in the CDR region are altered.

Accordingly, in another embodiment, the invention provides isolated FXIa-binding antibodies, or antigen-binding fragments thereof, consisting of a heavy chain variable region comprising a VH CDR1 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 3 and 23; a VH CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 4 and 24; a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 25, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 5 and 25; a VL CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 33, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 13 and 33; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 34, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 14 and 34; and a VL CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 35, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 15 and 35.

Accordingly, in another embodiment, the invention provides isolated FXIa-binding antibodies, or antigen-binding fragments thereof, consisting of a heavy chain variable region comprising a VH CDR1 region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 6 and 26; a VH CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 27, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 7 and 27; a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 8 and 28; a VL CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 16 and 36; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 37, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 17 and 37; and a VL CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 38, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 18 and 38.

Grafting of antigen-binding domains onto alternative scaffolds or backbones

A wide variety of antibody/immunoglobulin scaffolds or frameworks can be used, provided that the resulting polypeptide contains at least one binding region that specifically binds FXIa. Such scaffolds or frameworks include the five major idiotypes of human immunoglobulins, or fragments thereof, and include immunoglobulins from other animal species, preferably with humanized aspects. Single-heavy-chain antibodies, such as those found in camelids, are of particular interest in this regard. Those skilled in the art continue to discover and develop new antibody scaffolds, frameworks, and fragments.

In one aspect, the invention relates to the production of non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds onto which the CDR regions of the invention can be grafted. Known or future-developed non-immunoglobulin scaffolds and frameworks can be used, provided they contain a binding region specific for the target FXI and/or FXIa protein. Known non-immunoglobulin scaffolds or backbones include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxibodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

Fibronectin scaffolds are based on the fibronectin type III domain (e.g., the tenth module of fibronectin type III (Fn3 domain 10)). The fibronectin type III domain has 7 or 8 beta strands distributed between two beta sheets, which themselves are folded against each other to form the core of the protein and also contain loops (analogous to CDR regions) that connect the beta strands to each other and are solvent-exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the protein boundary perpendicular to the direction of the beta strands (see US 6,818,418). These fibronectin-based scaffolds are not immunoglobulins, although their overall folding closely resembles that of the smallest functional fragment of an antibody, the heavy chain variable region, which contains the complete antigen-recognizing module in camel and llama IgG. This structure allows the non-immunoglobulin antibody to mimic antigen-binding properties similar in nature and affinity to those of antibodies. Such scaffolds can be used in a loop-shuffling strategy in vitro , analogous to the affinity maturation process of antibodies in vivo . These fibronectin-based molecules can be used as scaffolds, where the loop regions of the molecule can be replaced with the CDR regions of the invention using standard cloning methods.

Ankyrin technology is based on the use of proteins with repeating ankyrin modules as scaffolds for carrying variable regions that can be used to bind different targets. The ankyrin repeating module is a 33-amino acid polypeptide consisting of two antiparallel α-helices and a β-turn. Binding of the variable regions is primarily optimized using ribosome display.

Avimers are derived from a naturally occurring A-domain-containing protein, such as LRP-1. These domains are used in nature for protein-protein interactions, and over 250 human proteins have A-domains as their structural basis. Avimers are composed of a number of different A-domain monomers (2-10) linked via amino acid linkers. Avimers that are capable of binding a target antigen can be designed using methodology described, for example, in U.S. Patent Application Publications 20040175756, 20050053973, 20050048512, and 20060008844.

Affibody affinity ligands are small, simple proteins consisting of a three-helix bundle based on the scaffold of one of the IgG-binding domains of protein A. Protein A is a surface protein of the bacterium Staphylococcus aureus . This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (see, for example, US 5831012). Affibody molecules mimic antibodies; they have a molecular mass of 6 kDa, compared to the molecular mass of antibodies, which is 150 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies.

Anticalins are products developed by Pieris ProteoLab AG. They are derived from lipocains, a widespread group of small and stable proteins typically involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several types of natural lipocains are present in human tissues and body fluids. The protein structure resembles that of immunoglobulins, with hypervariable loops atop a rigid framework. However, unlike antibodies or their recombinant fragments, lipocains consist of a single polypeptide chain with 160-180 amino acid residues, being only slightly larger than a single immunoglobulin domain. The set of four loops that form the binding pocket exhibits marked structural flexibility and allows the presence of various side chains. The binding site can thus be modified using a proprietary method to recognize intended target molecules of varying shapes with high affinity and specificity. One of the lipocaine family proteins, bilin-binding protein (BBP) from Pieris brassicae , was used to develop anticalins by mutagenesis of a set of four loops. One example of a patent application describing anticalins is PCT publication no. WO 199916873.

Affiliate molecules are small non-immunoglobulin proteins engineered to exhibit specific affinities for proteins and small molecules. New affinity molecules can be rapidly selected from two libraries, each based on different human scaffold proteins. Affiliate molecules do not share any structural homology with immunoglobulin proteins. Two affinity scaffolds are currently used: one is gamma-crystallin, a human structural protein of the eye lens, and the other is a member of the ubiquitin protein superfamily. Both human scaffold proteins are very small, exhibit high thermal stability, and are almost completely resistant to changes in pH and denaturants. This high stability is primarily due to the proteins' extensive beta-sheet structure. Examples of proteins derived from gamma crystallin are described in WO 200104144 and examples of "ubiquitin-like" proteins are described in WO 2004106368.

Protein epitope mimetics (PEMs) are medium-sized cyclic peptide-like molecules (MW 1–2 kDa) that mimic beta-hairpin secondary structures of proteins, the major secondary structures involved in protein–protein interactions.

The present invention relates to fully human antibodies that specifically bind the FXIa protein. Compared to chimeric or humanized antibodies, the human FXIa-binding antibodies of the invention exhibit even reduced antigenicity when administered to human subjects.

Camelid antibodies

Antibody proteins obtained from members of the Bactrian and dromedary camels ( Camelus bactrianus and Calelus dromaderius ), including New World members of the family such as llamas ( Lama paccos , Lama glama , and Lama vicugna ), have been characterized in terms of size, structural complexity, and antigenicity to human subjects. Some IgG antibodies from members of this mammalian family have been found to be naturally devoid of light chains and thus have a structure distinct from the typical four-chain quaternary structure with two heavy and two light chains of antibodies from other animals. See PCT/EP93/02214 (WO 94/04678, publication date March 3, 1994).

The region of a camelid antibody that is a small single variable domain identified as VHH can be genetically engineered as a small protein with high affinity for a target, a low molecular weight antibody-derived protein known as a "camelid nanobody." See U.S. Patent No. 5,759,808, issued June 2, 1998; see also Stijlemans, B. et al. , 2004 J Biol Chem 279: 1256–1261; Dumoulin, M. et al. , 2003 Nature 424: 783–788; Pleschberger, M. et al., 2003 Bioconjugate Chem 14: 440–448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456–62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512–3520. Constructed libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other non-human antibodies, the amino acid sequence of a camelid antibody can be altered by recombinant methods to produce a sequence that more closely resembles the human sequence; that is, the nanobody can be “humanized.” In this way, the naturally low antigenicity of camelid antibodies for humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of this small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins. This means that camelid nanobodies are useful as reagents for detecting antigens that would otherwise remain undetectable using classical immunological methods, as well as potential therapeutic agents. Thus, another consequence of their small size is that camelid nanobodies can inhibit by binding to a specific site in the groove or narrow cleft of the target protein, thus serving a role that more closely resembles that of a classical small molecule drug than that of a classical antibody.

The low molecular weight and compact size also result in camelid nanobodies being extremely heat-stable, resistant to extreme pH and proteolytic degradation, and weakly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues and even cross the blood-brain barrier, potentially treating diseases affecting nervous tissue. Nanobodies can also facilitate drug transport across the blood-brain barrier. See US Patent Application 20040161738, published August 19, 2004. These properties, combined with low antigenicity in humans, suggest enormous therapeutic potential. Furthermore, these molecules can be fully expressed in prokaryotic cells such as E. coli , expressed as fusion proteins with bacteriophage, and are functional.

Accordingly, one of the features of the present invention is a camelid antibody or nanobody having a high affinity for FXI and/or FXIa. In specific embodiments of the present invention, the camelid antibody or nanobody is naturally produced in a camelid, i.e., produced in a camelid after immunization with FXI and/or FXIa, or a peptide fragment thereof, using the methods described herein for other antibodies. Alternatively, the FXI and/or FXIa-binding camelid nanobody is engineered, i.e., obtained by selection, for example, from a phage display library of appropriately mutated camelid nanobody proteins using panning methods with FXI and/or FXIa, and/or their domains, and/or peptide fragments, as a target, as described in the examples provided herein. The constructed nanobodies can be further modified using genetic engineering techniques to have a half-life in the recipient's body of between 45 minutes and two weeks. In a specific embodiment, the camelid antibody or nanobody is produced by grafting the CDR sequences of the heavy or light chain of the human antibodies of the invention onto the framework sequences of the nanobody or single-domain antibody, as described, for example, in PCT/EP93/02214.

Bispecific molecules and multivalent antibodies

In another aspect, the present invention relates to bispecific or multispecific molecules comprising an FXI and/or FXIa-binding antibody, or fragment thereof, of the invention. The antibody of the invention, or its antigen-binding fragments, can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand for a receptor), to produce a bispecific molecule that binds at least two different binding sites or two target molecules. In fact, the antibody of the invention can be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind more than two different binding sites and/or target molecules; such multispecific molecules are also encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention may be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to form the bispecific molecule.

Accordingly, the present invention includes bispecific molecules having at least one first binding specificity for FXI and/or FXIa and a second binding specificity for a second target epitope. For example, the second target epitope is a different epitope of FXI and/or FXIa than the first target epitope.

Furthermore, in an embodiment of the invention in which the bispecific molecule is multispecific, the molecule may further have a third binding specificity for an additional epitope, in addition to the first and second target epitopes.

In one embodiment, the bispecific molecules of the invention comprise, as a binding specificity, at least one antibody or fragment thereof, including, for example, Fab, Fab', F(ab') ₂ , Fv, or a single-chain Fv fragment. The antibody can also be a dimer of a light chain or a heavy chain, or any minimal fragment thereof, such as Fv, or a single-chain construct, as described in Ladner et al. U.S. Patent No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which the VH and VL domains are expressed on a single polypeptide chain and linked by a linker that is too short to permit pairing between the two domains on the same chain. The VH and VL domains pair with the complementary domains of another chain, resulting in two antigen-binding sites (see, e.g., Holliger et al. , 1993 Proc. Natl. Acad. Sci. USA 90: 6444–6448; Poljak et al. , 1994 Structure 2: 1121–1123). Diabodies can be produced by expressing two polypeptide chains with either the VHA-VLB and VHB-VLA (VH-VL configuration) or the VLA-VHB and VLB-VHA (VL-VH configuration) structure in the same cell. Most of them can be expressed in soluble form in bacteria. Single-chain diabodies (scDb) are produced by joining two diabody-forming polypeptide chains with a linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4): 128-30; Wu et al. , 1996 Immunotechnology, 2(1): 21-36). scDb can be expressed in bacteria in a soluble, active monomeric form (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34): 128–30; Wu et al. , 1996 Immunotechnology, 2(1): 21–36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83–105; Ridgway et al. , 1996 Protein Eng., 9(7): 617–21). The diabody can be fused with an Fc fragment to produce a “di-diabody” (see Lu et al. , 2004 J. Biol. Chem., 279(4): 2856–65).

Other antibodies that can be used in the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules can be produced by conjugating the constituent binding specificities using methods known in the art. For example, each binding specificity of a bispecific molecule can be created separately and then conjugated to one another. When the binding specificities are proteins or peptides, various coupling or cross-linking reagents can be used for covalent conjugation. Examples of cross-linking reagents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. , 1984 J. Exp. Med. 160: 1686; Liu, MA et al. , 1985 Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus, 1985 Behring Ins. Mitt. No. 78, 118-132; Brennan et al. , 1985 Science 229: 81-83 and Glennie et al. , 1987 J. Immunol. 139: 2367-2375. Conjugating reagents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificity is antibodies, they can be conjugated by sulfhydryl linkage of the C-terminal hinge regions of two heavy chains. In a specific embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, such as one, prior to conjugation.

Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') ₂ , or ligand x Fab fusion protein. The bispecific molecule of the invention may be a single-chain molecule comprising one single-chain antibody and a binding determinant, or a single-chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Patent No. 4,881,175; U.S. Patent No. 5,132,405; US patent number 5091513; US patent number 5476786; US patent number 5013653; US patent number 5258498 and US patent number 5482858.

Binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot analysis. Each of these assays typically detects the presence of the protein-antibody complexes of interest using a labeled reagent (e.g., an antibody) specific for the complex of interest.

In another aspect, the present invention relates to multivalent compounds comprising at least two identical or different antigen-binding fragments of the antibodies of the invention that bind FXIa. The antigen-binding fragments can be linked together by protein fusion or by covalent or non-covalent association. Alternatively, linking methods have been described for bispecific molecules. Tetravalent compounds can be prepared, for example, by cross-linking the antibodies of the invention with an antibody that binds constant regions of the antibodies of the invention, such as the Fc or hinge region.

Trimerized domains are described, for example, in Borean patent EP 1 012 280B1. Pentamerized modules are described, for example, in PCT/EP97/05897.

Antibodies with an extended half-life

The present invention relates to antibodies that specifically bind the FXIa protein and have an increased half-life in vivo .

Many factors can influence the half-life of a protein in vivo . For example, renal filtration, hepatic metabolism, degradation by proteolytic enzymes (proteases), and immune responses (e.g., protein neutralization by antibodies and uptake by macrophages and dendritic cells). Various strategies can be used to prolong the half-life of the antibodies of the present invention. For example, chemical linkage to polyethylene glycol (PEG), reCODE PEG, an antibody backbone, polysialic acid (PSA), hydroxyethyl starch (HES), albumin-binding ligands, and carbohydrate protecting groups; genetic fusion to proteins that bind to serum proteins such as albumin, IgG, FcRn, and transferrin; linkage (genetically or chemically) to other binding moieties that bind serum proteins, such as nanobodies, Fab fragments, DARPins, avimers, affibodies, and anticalins; genetic fusion to rPEG, albumin, albumin domain, albumin-binding proteins, and Fc fragment; or encapsulation in nanocarriers, sustained-release preparations, or medical devices.

To prolong serum circulation of antibodies in vivo, inert polymeric molecules such as high-molecular-weight PEG can be attached to an antibody or antibody fragment, with or without the addition of a multifunctional linker, either through site-specific conjugation of PEG to the N- or C-terminus of antibodies or through epsilon-amino groups located on lysine residues. PEGylation of an antibody typically involves reacting the antibody or antibody fragment with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions whereby one or more PEG groups are attached to the antibody or antibody fragment. PEGylation can be accomplished by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" encompasses any form of PEG used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In specific embodiments, the antibody to be PEGylated is an aglycosylated antibody. Derivatization with a linear or branched polymer is used, resulting in minimal loss of biological activity. The degree of conjugation can be carefully monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to antibodies. Unreacted PEG can be separated from the antibody-PEG conjugates by size-exclusion or ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding capacity, as well as in vivo efficacy, using methods well known to those skilled in the art, for example, using the immunoassays described herein. Methods for PEGylation of proteins are known in the art and can be applied to the antibodies of the invention. See, for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other PEGylation technologies include reductive chemical orthogonal targeting engineering (ReCODE PEG), which enables the incorporation of chemically precisely defined side chains into biosynthetic proteins through a reconstituted system involving tRNA synthetase and tRNA. This technology enables the incorporation of more than 30 new amino acids into biosynthetic proteins in E. coli , yeast, and mammalian cells. The tRNA incorporates a "non-native" amino acid at any location where an amber codon is located, converting the amber codon from a stop codon to a codon that signals the incorporation of a chemically precisely defined amino acid.

Recombinant PEGylation (rPEG) technology can also be used to increase serum half-life. This technology involves genetically fusing a 300-600 amino acid unstructured protein "tail" to an existing pharmaceutical protein. Because the apparent molecular weight of this unstructured protein chain is approximately 15 times greater than its actual molecular weight, the serum half-life of the protein is significantly increased. Unlike traditional PEGylation, which requires chemical conjugation and repeated purification, the manufacturing process is greatly simplified and the product is homogeneous.

Polysialylation is another technology that utilizes the natural polymer polysialic acid (PSA) to prolong the active state and enhance the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used to deliver protein and peptide therapeutics, polysialic acid provides a protective microenvironment through conjugation. This prolongs the active state of the therapeutic protein in the circulatory system and prevents its recognition by the immune system. The PSA polymer is naturally present in the human body. Some bacteria have evolved over millions of years to coat their walls with this polymer. These naturally polysialylated bacteria have become capable of evading the body's defenses through molecular mimicry. PSA, the basis of nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when bound to proteins, because it is chemically identical to PSA in the human body.

Another technology involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes. HES solutions are typically administered to restore blood volume and improve blood rheology. HESylation of the antibody increases the half-life in the circulation by increasing the stability of the molecule and by decreasing renal clearance, leading to increased biological activity. By varying various parameters, such as the molecular weight of HES, a wide variety of HES-antibody conjugates can be produced, tailored to specific applications.

Antibodies having an increased half-life in vivo can also be produced by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the constant domain of IgG or its FcRn binding fragment (preferably the Fc domain or the hinge region of the Fc domain). See, for example, International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375.

Additionally, antibodies can be conjugated to albumin (e.g., human serum albumin; HSA) to impart greater stability to the antibody, or antibody fragment, in vivo or to increase its half-life in vivo . These techniques are well known in the art, see, for example, International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413622. Furthermore, in the context of the bispecific antibody described above, the specificities of the antibody can be engineered such that one binding domain of the antibody binds FXIa, while a second binding domain of the antibody binds serum albumin, preferably HSA.

Half-life enhancement strategies are particularly useful for nanobodies, fibronectin-based binding compounds, and other antibodies or proteins for which an increased half-life in vivo is desired.

Antibody conjugates

The present invention relates to antibodies, or fragments thereof, that specifically bind the FXIa protein, which are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or fragment thereof, preferably a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce fusion proteins. In particular, the invention relates to fusion proteins comprising an antigen-binding fragment of an antibody described herein (e.g., a Fab fragment, an Fd fragment, an Fv fragment, an F(ab) ₂ fragment, a VH domain, a VH CDR, a VL domain, or a VL CDR) and a heterologous protein, polypeptide, or peptide. Methods of fusing or conjugating proteins, polypeptides, or peptides to an antibody or antibody fragment are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Patent Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al. , 1991, Proc. Natl. Acad. Sci. USA 88: 10535–10539; Zheng et al. , 1995, J. Immunol. 154: 5590–5600; and Vil et al. , 1992, Proc. Natl. Acad. Sci. USA 89: 11337–11341.

Additional fusion proteins can be generated using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activities of the antibodies, or fragments thereof, of the invention (e.g., to generate antibodies, or fragments thereof, with higher affinities and lower off-rates). See, generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al. , 1997, Curr. Opinion Biotechnol. 8: 724–33; Harayama, 1998, Trends Biotechnol. 16(2): 76–82; Hansson, et al. , 1999, J. Mol. Biol. 287: 265-76 and Lorenzo and Blasco, 1998, Biotechniques 24(2): 308-313 (the entire contents of all these patents and publications are incorporated herein by reference). Antibodies or fragments thereof, or encoded antibodies or fragments thereof, can be altered by subjecting them to random mutagenesis by lower-fidelity PCR, random nucleotide insertion, or other methods prior to recombination. A polynucleotide encoding an antibody, or fragment thereof, that specifically binds the FXIa protein can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

Additionally, antibodies or fragments thereof can be fused to marker sequences, such as a peptide, that facilitates purification. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 48), such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others; many of these sequences are commercially available. As described in Gentz et al. , 1989, Proc. Natl. Acad. Sci. USA 86: 821-824, for example, hexahistidine (SEQ ID NO: 48) allows for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza virus hemagglutinin protein (Wilson et al. , 1984, Cell 37:767), and the "flag" tag.

In other embodiments, the antibodies of the present invention, or fragments thereof, are conjugated to a diagnostic or detectable agent. Such antibodies may be useful for monitoring or predicting the onset, progression, and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the effectiveness of a particular therapy. Such diagnosis and detection can be accomplished by linking the antibody to detectable agents, including, but not limited to, various enzymes such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive materials such as, but not limited to, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, and ¹¹¹ In), technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga, ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, 159 Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr ^{, 105} Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn and ¹¹⁷ Tin; and positron-active metals for use in various positron emission tomography techniques, as well as non-radioactive paramagnetic metal ions.

The present invention also includes the use of antibodies, or fragments thereof, conjugated to a therapeutic agent. An antibody, or fragment thereof, can be conjugated to a therapeutic moiety such as a cytotoxin, such as a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, such as an alpha-particle emitter. A cytotoxin, or cytotoxic agent, includes any agent that is lethal to cells.

Furthermore, the antibody, or fragment thereof, can be conjugated to a therapeutic agent or drug that modifies a specific biological response. Therapeutic agents or drugs should not be understood as limited to classical chemical drugs. For example, the drug can be a protein, peptide, or polypeptide that has a desired biological activity. Such proteins can include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent, an anti-apoptotic agent, or a biological response modifier such as, for example, a lymphokine.

Furthermore, the antibody can be conjugated with therapeutic agents such as a radioactive metal ion, for example, alpha particle emitters such as ^213Bi , or macrocyclic chelators useful for conjugating radioactive metal ions, including but not limited to ^131In , ^131LU , ^131Y , ^131Ho , ^131Sm , to polypeptides. In particular embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are well known in the art and are described in Denardo et al. , 1998, Clin Cancer Res. 4(10): 2483-90; Peterson et al. , 1999, Bioconjug. Chem. 10(4): 553–7; and Zimmerman et al. , 1999, Nucl. Med. Biol. 26(8): 943–50, the entire contents of each of which are incorporated herein by reference.

Methods for conjugating therapeutic agents to antibodies are well known, see, for example, Arnon et al. , "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc. 1985); Hellstrom et al. , "Antibodies For Drug Delivery," in: Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623–53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in: Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475–506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. , 1982, Immunol. Rev. 62: 119-58.

Antibodies can also be bound to solid supports, which are particularly useful for immunoassays or target antigen purification. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Methods for producing antibodies

Nucleic acids encoding antibodies

The invention relates to substantially purified nucleic acid molecules encoding polypeptides comprising segments or domains of the FXIa-binding antibody chains described above. Some of the nucleic acids of the invention comprise a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 10 or 30 and/or a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 20 or 40. In a particular embodiment, the nucleic acid molecules are those set forth in Table 1. Some other nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (e.g., at least 65%, 80%, 95%, or 99%) to the nucleotide sequences set forth in Table 1. When expressed from appropriate expression vectors, the polypeptides encoded by these polynucleotides are capable of exhibiting the ability to bind an FXI and/or FXIa antigen.

The invention also relates to polynucleotides encoding at least one CDR region, and typically all three CDR regions, of the heavy or light chain of the FXIa-binding antibody described above. Certain other polynucleotides encode the entire or substantially the entire sequence of the variable region of the heavy chain and/or light chain of the FXIa-binding antibody described above. Due to the degeneracy of the genetic code, different nucleotide sequences will encode each of the amino acid sequences of the immunoglobulin.

Nucleic acid molecules of the invention can encode both the variable region and the constant region of an antibody. Some of the nucleotide sequences of the invention comprise nucleotides encoding a heavy chain sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the heavy chain sequence set forth in SEQ ID NO: 11 or 31. Some other nucleotide sequences comprise nucleotides encoding a light chain sequence that is substantially identical (e.g., at least 80%, 90%, or 99%) to the light chain sequence set forth in SEQ ID NO: 21 or 41.

Polynucleotide sequences can be prepared de novo by solid-phase DNA synthesis or PCR mutagenesis of an existing sequence (e.g., the sequences described in the Examples section below) encoding an FXIa-binding antibody or a binding fragment thereof. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method described in Narang et al. , 1979, Meth. Enzymol. 68:90; the phosphodiester method described in Brown et al. , Meth. Enzymol. 68:109, 1979; the diethyl phosphoramidite method described in Beaucage et al. , Tetra. Lett., 22:1859, 1981; and the solid support method described in U.S. Patent No. 4,458,066. The introduction of mutations into a polynucleotide sequence by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al. , Nucleic Acids Res. 19:967, 1991; and Eckert et al. , PCR Methods and Applications 1:17, 1991.

The invention also relates to expression vectors and host cells for producing the FXI and/or FXIa-binding antibodies described above. Various expression vectors can be used to express polynucleotides encoding FXIa-binding antibody chains or binding fragments. Both viral-based and non-viral expression vectors can be used to produce antibodies in a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for protein or RNA expression, and human artificial chromosomes (see, for example, Harrington et al. , Nat Genet 15:345, 1997). For example, nonviral vectors useful for expressing FXIa-binding polypeptide polynucleotides in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1/His, pEBVHis A, B, and C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other proteins. Useful viral vectors include retrovirus, adenovirus, adeno-associated virus, herpesvirus, SV40, papillomavirus, Epstein-Barr HBP, vaccinia virus, and Semliki Forest virus (SFV) vectors. See Brent et al. , supra; Smith, Annu. Rev. Microbiol. 49: 807, 1995; and Rosenfeld et al. , Cell 68: 143, 1992.

The choice of expression vector depends on the intended host cells in which the vector will be expressed. Expression vectors typically contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to polynucleotides encoding the FXIa-binding antibody chain or fragment. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequences in the absence of inducing conditions. Inducible promoters include, for example, the arabinose, lacZ, metallothionein, or heat shock protein promoters. Cultures of transformed microorganisms can be grown under non-inducing conditions without affecting the population of coding sequences, the expression products of which are better tolerated by host cells. In addition to promoters, other regulatory elements may also be necessary or desirable for efficient expression of the FXIa-binding antibody chain or fragment. These elements typically include an ATG initiation codon and an adjacent ribosome binding site or other sequences. In addition, expression efficiency can be increased by incorporating enhancers appropriate for the cellular system used (see, e.g., Scharf et al. , Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al. , Meth. Enzymol., 153: 516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

Expression vectors can also provide a secretion signal sequence for the formation of a fusion protein with polypeptides encoded by inserted FXIa-binding antibody sequences. More commonly, inserted FXI and/or FXIa-binding antibody sequences are linked to signal sequences prior to incorporation into the vector. Vectors used to receive sequences encoding the variable domains of the light and heavy chains of FXI and/or FXIa-binding antibodies sometimes also encode constant regions or portions thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions, resulting in the production of intact antibodies or fragments thereof. Typically, such constant regions are human.

Host cells for containing and expressing the FXI and/or FXIa binding chain sequences of the antibody may be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention. Other suitable microbial hosts include bacilli such as Bacillus subtilis and other enterobacteria such as Salmonella , Serratia , and various Pseudomonas species. These prokaryotic hosts may also contain expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of different well-known promoters will be contained, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally with an operator sequence, and also include ribosome binding site sequences and the like, for the initiation and termination of transcription and translation. Other microorganisms, such as yeast, can also be used to express the FXIa-binding polypeptides of the invention. Insect cells can also be used in combination with baculovirus vectors.

In some preferred embodiments, mammalian host cells are used to express and produce the FXI and/or FXIa binding polypeptides of the present invention. These include any normal mortal or normal or abnormal immortalized animal or human cells. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells. The use of mammalian cell cultures for the expression of polypeptides is generally described, for example, in Winnacker, FROM GENES TO CLONE, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may contain expression control sequences such as an origin of replication, a promoter, and an enhancer (see, for example, Queen, et al. , Immunol. Rev. 89: 49-68, 1986), as well as necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences.

These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, developmental stage-specific, and/or modulated or regulated. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human CMV immediate early promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the host cell type. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other host cells. (See generally Sambrook et al. , supra.) Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88: 223, 1997), agent-enhanced DNA uptake, and ex vivo transduction. Long-term, high-yield production of recombinant proteins often requires stable expression. For example, cell lines stably expressing FXIa-binding antibody chains or binding fragments can be generated using expression vectors of the invention, which contain a viral origin of replication or endogenous expression elements and a selectable marker gene. Following vector introduction, cells can be allowed to grow for 1-2 days in enriched medium before being transferred to a selective medium. The selectable marker confirms resistance to selection, and its presence allows cells that successfully express the introduced sequences to grow in the selective medium. Stable, stably transfected cells can be expanded using culture methods appropriate for the specific cell type.

Construction of the scaffold or Fc region

Engineered antibodies of the invention include those in which the framework residues in the VH and/or VL have been modified, for example, to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain framework residues that differ from those in the germline sequence from which the antibody was derived. Such residues can be identified by comparing the framework sequences of the antibody with the germline sequences from which the antibody was derived. To return the framework sequences to their germline configuration, somatic mutations can be "back-mutated" to the germline sequence, for example, by site-directed mutagenesis. Such "back-mutated" antibodies are also within the scope of the invention.

Another type of framework modification involves mutating one or more residues in the framework region, or even one or more CDR regions, to remove T-cell epitopes to reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 by Carr et al.

In addition to or as an alternative to modifications made in the framework and CDR regions, antibodies of the invention can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the EU Kabat index.

In one embodiment, the CH1 hinge region is modified such that the number of cysteine residues in the hinge region is altered, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease antibody stability.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. Specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain boundary region of the Fc hinge fragment such that the antibody's binding to staphylococcal protein A (SpA) is impaired compared to SpA binding by the native Fc hinge domain. This approach is described in more detail in U.S. Patent No. 6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. Various approaches can be used. For example, one or more of the mutations described in U.S. Patent No. 6,277,375 to Ward can be used. Alternatively, to increase the biological half-life, the antibody can be altered in the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 to Presta et al.

In other embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered binding affinity for an effector ligand but retains the ability of the original antibody to bind the antigen. The effector ligand whose affinity is altered can be, for example, an Fc receptor or complement component C1. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both issued to Winter et al.

In another embodiment, one or more amino acids selected from the amino acid residues can be replaced with a different amino acid residue such that the antibody's ability to bind C1q is altered and/or complement-dependent cytotoxicity (CDC) is reduced or eliminated. This approach is described in more detail in U.S. Patent No. 6,194,551 to Idusogie et al.

In another embodiment, one or more amino acid residues are altered to alter the antibody's ability to fix complement. This approach is described in more detail in PCT publication WO 94/29351 by Bodmer et al.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody comprising the VL CDR and VH CDR of NOV1401) comprises an Fc region of human IgG (e.g., IgG1) having two amino acid substitutions, D265A and P329A, to reduce the likelihood of ADCC or CDC induced by any surface-bound FXI. Such alanine substitutions have been shown to result in decreased ADCC and CDC (see, e.g., Idosugie et al. , J. Immunol. 164:4178-4184, 2000; Shields et al. , J. Biol. Chem. 276:6591-6604, 2001).

In another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fcγ receptor by changing one or more amino acids. This approach is described in more detail in PCT publication WO 00/42072 by Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped and variants with improved binding parameters have been described (see Shields, RL et al. , 2001 J. Biol. Chen. 276: 6591-6604).

In another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody (i.e., an antibody without glycosylation) can be produced. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can lead to an increase in the affinity of the antibody for the antigen. This approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, an antibody with an altered glycosylation pattern can be produced, for example, a hypofucosylated antibody containing fewer fucosyl residues or an antibody containing more bisected GlcNac structures. Such altered glycosylation patterns have been shown to enhance the ADCC activity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation apparatus. Cells with an altered glycosylation apparatus are described in the art and can be used as host cells for expressing recombinant antibodies of the invention to produce antibodies with an altered glycosylation pattern. For example, EP 1 176 195 by Hang et al. describes a cell line with a functional disruption of the FUT8 gene encoding fucosyltransferase, resulting in antibodies expressed in cells of such a cell line being characterized by hypofucosylation. PCT Publication No. WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also results in hypofucosylation of antibodies expressed in such a host cell (see also Shields, RL et al. , 2002 J. Biol. Chem. 277: 26733–26740). PCT Publication No. WO 99/54342 by Umana et al. Cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) have been described, resulting in antibodies expressed in these cell lines exhibiting increased numbers of bisected GlcNac structures, leading to increased ADCC activity of the antibodies (see also Umana et al. , 1999 Nat. Biotech. 17: 176–180).

Methods for constructing modified antibodies

As described above, FXIa-binding antibodies having the VH and VL sequences or full-length heavy and light chain sequences described herein can be used to generate new FXIa-binding antibodies by altering the full-length heavy chain and/or light chain sequences, the VH and/or VL sequences, or the sequences of the constant region(s) attached thereto. Thus, in another aspect of the invention, the structural features of the FXIa-binding antibody of the invention are used to generate structurally related FXIa-binding antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human FXIa, as well as inhibition of one or more functional properties of FXIa (e.g., inhibition of FXIa binding to the FXIa receptor, inhibition of FXIa-dependent cell proliferation).

For example, one or more CDR regions of the antibodies of the present invention, or mutant forms thereof, can be combined recombinantly with known framework regions and/or other CDR regions to create additional recombinantly engineered FXIa-binding antibodies of the invention, as described above. Other types of modifications include those described in the previous section. The starting material for the engineering is one or more of the VH and/or VL sequences provided herein, or one or more of their CDR regions. To create an engineered antibody, it is not necessary to actually produce (i.e., express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein, or one or more of their CDR regions. Rather, the information contained in the sequence(s) is used as starting material to create “second generation” sequence(s) derived from the original sequence(s), and the “second generation” sequence(s) is then produced and expressed as a protein.

Accordingly, in another embodiment, the invention relates to a method for producing an FXIa-binding antibody, comprising a heavy chain variable region sequence of an antibody comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 3 and 23, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 4 and 24, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 5 and 25; and a light chain variable region sequence of an antibody comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 13 and 33, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 14 and 34, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 15 and 35; altering at least one amino acid residue in the sequence of the variable region of the heavy chain of the antibody and/or the sequence of the variable region of the light chain of the antibody to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention relates to a method for producing an FXIa-binding antibody, comprising a heavy chain variable region sequence of an antibody comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 6 and 26, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 7 and 27, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 8 and 28; and a light chain variable region sequence of an antibody comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 16 and 36, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 17 and 37, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 and 38; altering at least one amino acid residue in the sequence of the variable region of the heavy chain of the antibody and/or the sequence of the variable region of the light chain of the antibody to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention relates to a method for producing an FXIa-binding antibody optimized for expression in a mammalian cell, comprising: a full-length antibody heavy chain sequence having a sequence selected from the group consisting of SEQ ID NOs: 11 or 31; and a full-length antibody light chain sequence having a sequence selected from the group consisting of SEQ ID NOs: 21 or 41; altering at least one amino acid residue in the full-length antibody heavy chain sequence and/or the full-length antibody light chain sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. In one embodiment, the alteration in the heavy or light chain occurs in the framework region of the heavy or light chain.

A modified antibody sequence can also be obtained by screening antibody libraries having fixed CDR3 sequences or minimal key binding determinants, as described in US2005/0255552, and different CDR1 and CDR2 sequences. Screening can be performed using any method suitable for screening antibodies from antibody libraries, such as phage display technology.

Standard molecular biology techniques can be used to produce and express the altered antibody sequence. The antibody encoded by the altered antibody sequence(s) is an antibody that retains one, several, or all of the functional properties of the FXIa-binding antibodies described herein, wherein the functional properties include, but are not limited to, specific binding to human, cynomolgus macaque, rat, and/or mouse FXIa; and inhibition of FXIa-dependent cell proliferation by the antibody in a cell proliferation assay using F36E and/or Ba/F3-FXIaR.

In specific embodiments of the antibody engineering methods of the invention, mutations can be introduced randomly or selectively throughout all or part of the coding sequence of an FXIa-binding antibody, and the resulting modified FXIa-binding antibodies can be screened for binding activity and/or other functional properties as described herein. Methods for making mutations are described in the art. For example, PCT Publication WO 02/092780 by Short describes methods for generating and screening mutant antibodies using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03/074679 by Lazar et al. describes the use of computational screening methods to optimize the physicochemical properties of antibodies.

In specific embodiments of the invention, antibodies were engineered to remove deamidation sites. Deamidation is known to cause structural and functional changes in a peptide or protein. Deamidation can lead to a decrease in biological activity, as well as changes in the pharmacokinetics and antigenicity of a protein pharmaceutical ( Anal Chem . 2005 Mar 1; 77(5): 1432-9).

In specific embodiments of the invention, the antibodies were engineered to increase their pI value and improve their properties as therapeutics. The pI value of a protein is a key factor determining the overall biophysical properties of a molecule. Antibodies with low pI values are known to be less soluble, less stable, and prone to aggregation. Furthermore, the purification of antibodies with a low pI value is complex and can be problematic, especially during large-scale production for clinical use. Increasing the pI value of the anti-FXI/FXIa antibodies, or Fab fragments, of the invention results in improved solubility, allowing the antibodies to be formulated at higher concentrations (>100 mg/mL). Formulating antibodies at high concentrations (e.g., (>100 mg/mL) has the advantage of allowing higher antibody doses to be administered, which in turn allows for a reduction in dosing frequency, an important advantage in the treatment of chronic diseases, including thrombotic and/or thromboembolic diseases. Higher pI values may also promote increased FcRn-mediated recycling of the IgG antibody variant, thus allowing the drug to remain in the body for a longer period, resulting in fewer injections being required. Finally, the overall stability of the antibody is significantly enhanced by high pI values, leading to increased shelf life and biological activity.in vivoPreferably, the pI value is greater than or equal to 8.2.

The functional properties of the altered antibodies can be assessed using standard assays available in the art and/or described herein, such as those provided in the Examples section (e.g., ELISA).

Prophylactic and therapeutic use

The FXI and/or FXIa binding antibodies described herein (e.g., the antibodies listed in Table 1, such as anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401) can be used at a therapeutically useful concentration to treat a thromboembolic disease or disorder (e.g., thrombotic stroke, atrial fibrillation, stroke prevention in atrial fibrillation (SIP), deep vein thrombosis, venous thromboembolism, pulmonary embolism, acute coronary syndromes (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, or systemic embolism) by administering to a subject in need thereof an effective amount of the antibodies, or antigen-binding fragments, of the invention. The present invention relates to a method for treating a thromboembolic disease (e.g., thrombotic diseases) by administering to a subject in need thereof an effective amount of antibodies of the invention. The present invention relates to a method for treating thromboembolic diseases (e.g., thrombotic stroke, atrial fibrillation, stroke prevention in atrial fibrillation (SIPAF), deep vein thrombosis, venous thromboembolism, pulmonary embolism, acute coronary syndromes (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, or systemic embolism) by administering to a subject in need thereof an effective amount of antibodies of the invention.

The antibodies described herein (e.g., the antibodies listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401) can be used, among other things, to prevent, treat, or ameliorate thromboembolic conditions or diseases, including, but not limited to, the thrombotic diseases described in more detail herein.

The antibodies provided herein (e.g., the antibodies listed in Table 1, such as anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401) can also be used in combination with other agents to prevent, treat, or ameliorate thromboembolic diseases. For example, statin therapy can be used in combination with the anti-FXIa antibodies and antigen-binding fragments of the invention to treat patients with thrombotic and/or thromboembolic diseases.

In a specific embodiment, the present invention provides a method for treating or preventing stroke in a patient with atrial fibrillation, comprising administering to a patient in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for controlling or preventing risks or conditions associated with atrial fibrillation (AF), such as embolic stroke and systemic embolism, in a patient with atrial fibrillation, comprising administering to a patient in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing conditions associated with atrial fibrillation (AF), such as embolic stroke and systemic embolism, in a patient with atrial fibrillation, comprising administering to a patient in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401. In specific embodiments, the patient with AF has a high risk of bleeding.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing deep vein thrombosis or related conditions in a subject (e.g., a subject suffering from or at risk of developing deep vein thrombosis), comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing venous thromboembolism (VTE) or conditions related thereto in a subject (e.g., a subject suffering from or at risk of developing venous thromboembolism), comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401. In specific embodiments, subjects receiving treatment with an anti-FXI/FXIa antibody provided herein have experienced 1) a primary unprovoked VTE with a low risk of bleeding, 2) a recurrent unprovoked VTE, or 3) a VTE associated with thrombophilia, including cancer patients.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing pulmonary embolism or related conditions in a subject (e.g., a subject suffering from, or at risk of, pulmonary embolism), comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing acute coronary syndromes (ACS) or related conditions in a subject, comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing ischemic stroke in a subject (e.g., a subject suffering from or at risk of developing ischemic stroke), comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing acute limb ischemia in a subject, comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing chronic thromboembolic pulmonary hypertension in a subject, comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing systemic embolism in a subject (e.g., a subject suffering from or at risk of developing systemic embolism), comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In a specific embodiment, the present invention provides a method for treating, managing, or preventing thromboembolic conditions associated with catheter insertion (e.g., a Hickman catheter in cancer patients), in which catheters become clogged with clots, and extracorporeal membrane oxygenation (ECMO), in which clots form in the tubes, comprising administering to a subject in need thereof an effective amount of an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401.

In specific embodiments, subjects in need of treatment with an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401, may include:

- subjects with indications for chronic anticoagulant therapy (e.g., in case of AF, thrombus in the left ventricle, previous cardioembolic stroke)

- subjects with moderate or high risk of major bleeding;

- subjects undergoing elective or primary percutaneous coronary intervention (PCI) with stent placement who may require dual antiplatelet therapy (aspirin and P2Y12 receptor antagonists) to prevent stent thrombosis.

In specific embodiments, one of the following conditions can be treated or managed with an anti-FXI/FXIa antibody described herein, such as an anti-FXI/FXIa antibody listed in Table 1, such as NOV1401, or anti-FXI/FXIa antibodies comprising the VL CDR and VH CDR of NOV1401:

- thromboembolism in subjects with suspected or confirmed cardiac arrhythmia such as paroxysmal, persistent or permanent atrial fibrillation or atrial flutter;

- prevention of stroke in atrial fibrillation (PAFI), one of the subgroups of patients are patients with AF undergoing percutaneous coronary intervention (PCI);

- treatment of acute venous thromboembolic events (VTE) and extended secondary prevention of VTE in patients with a high risk of bleeding;

- cerebral and cardiovascular events in secondary prevention after transient ischemic attack (TIA) or non-disabling stroke and prevention of thromboembolic events in heart failure with sinus rhythm;

- left atrial clot formation and thromboembolism in subjects undergoing cardioversion for cardiac arrhythmia;

- thrombosis before, during and after the ablation procedure due to cardiac arrhythmia;

- venous thrombosis, this includes, but is not limited to, treatment and secondary prevention of deep and superficial vein thrombosis in the lower extremities or upper extremities, thrombosis in the abdominal and thoracic veins, sinus thrombosis and jugular vein thrombosis;

- thrombosis on any artificial surface in the veins, such as a catheter or pacemaker wires;

- pulmonary embolism in patients with or without venous thrombosis;

- chronic thromboembolic pulmonary hypertension (CTEPH);

- arterial thrombosis at the site of a ruptured atherosclerotic plaque, thrombosis at an intra-arterial graft or catheter, and thrombosis in apparently normal arteries, including, but not limited to, acute coronary syndromes, ST-segment elevated myocardial infarction, non-ST-segment elevated myocardial infarction, unstable angina, stent thrombosis, thrombosis of any artificial surface in the arterial circulation, and pulmonary artery thrombosis in subjects with and without pulmonary hypertension;

- thrombosis and thromboembolism in patients undergoing percutaneous coronary intervention (PCI);

- cardioembolic and cryptogenic strokes;

- thrombosis in patients with invasive and non-invasive malignant neoplasms;

- thrombosis in the cavitary catheter;

- thrombosis and thromboembolism in patients in serious condition;

- cardiac thrombosis and thromboembolism, including, but not limited to, cardiac thrombosis after myocardial infarction, cardiac thrombosis associated with conditions such as cardiac aneurysm, myocardial fibrosis, cardiac enlargement and heart failure, myocarditis and artificial surfaces in the heart;

- thromboembolism in patients with valvular heart disease, with or without atrial fibrillation;

- thromboembolism in mechanical or biological valve prostheses;

- thromboembolism in patients with natural or artificial heart patches, arterial or venous conduits after heart surgery for simple or complex heart defects;

- venous thrombosis and thromboembolism after knee replacement surgery, hip replacement surgery, as well as orthopedic surgery, thoracic or abdominal surgery;

- arterial or venous thrombosis after neurosurgery, including intracranial surgery and spinal cord surgery;

- congenital or acquired thrombophilia, including, but not limited to, factor V Leiden mutation, prothrombin mutation, antithrombin III deficiency, protein C and protein S deficiency, factor XIII mutation, familial dysfibrinogenemia, congenital plasminogen deficiency, elevated factor XI levels, sickle cell disease, antiphospholipid syndrome, autoimmune disease, chronic bowel disease, nephrotic syndrome, hemolytic uremia, myeloproliferative disease, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria and heparin-induced thrombopenia;

- thrombosis and thromboembolism in chronic renal failure; and

- thrombosis and thromboembolism in patients undergoing hemodialysis and in patients undergoing extracorporeal membrane oxygenation.

In a particular aspect, the present invention provides methods for controlling bleeding in a patient receiving or administered an anti-FXI/FXIa antibody as provided herein (e.g., an antibody as set forth in Table 1, such as an anti-FXI/FXIa antibody comprising the VL CDR and VH CDR of NOV1401), such as bleeding associated with trauma, surgery, menstruation, or childbirth, comprising reversing the anticoagulant effect. FXI deficiency is rarely associated with spontaneous bleeding episodes; in particular aspects, bleeding is most often associated with trauma, surgery, menstruation, or childbirth. Prolonged bleeding may occur following major trauma or following surgery on organs with tissues that have increased fibrinolytic activity, such as the oral mucosa, nasal mucosa, genital tract mucosa, or urinary tract mucosa. Dental extractions, tonsillectomy, and uterine or prostate ablation are examples of surgical procedures that carry a high risk of bleeding. Individuals with this condition also have a strong tendency to nosebleeds and subcutaneous hemorrhages, and, less commonly, uterine or intestinal bleeding. The incidence of spontaneous muscle or joint bleeding and intracranial bleeding is not increased in patients with FXI deficiency. Vein puncture is generally not associated with prolonged bleeding. Other genetic mutations associated with FXI deficiency may contribute to the tendency toward heterogeneous and unpredictable bleeding in patients with severe FXI deficiency. Concomitant use of antiplatelet drugs, other anticoagulants, and fibrinolytic agents may increase the risk of bleeding.

In specific embodiments, the present invention provides a method for controlling bleeding in a patient receiving treatment with an anti-FXI/FXIa antibody as provided herein (e.g., an antibody as listed in Table 1, such as an anti-FXI/FXIa antibody comprising the VL CDR and VH CDR of NOV1401), comprising temporarily reversing the anticoagulant effect for a time sufficient to stop the bleeding. In specific embodiments, the step of reversing the anticoagulant effect comprises (i) infusion therapy using colloids, crystalloids, human plasma, or plasma proteins such as albumin; or (ii) transfusion of packed red blood cells or whole blood. In a particular embodiment, therapeutic agents for reversing the effect of anticoagulants, such as in an emergency situation, include, but are not limited to, prohemostatic blood components such as fresh frozen plasma (FFP), prothrombin complex concentrates (PCCs) and activated PCCs [(aPCCs); e.g., factor VIII bypass inhibitor (FVII)], as well as recombinant activated factor VII (rFVIIa). In one embodiment, a regimen comprising rFVIIa at 30 mcg/kg, followed by rFVIIa at 15-30 mcg/kg every 2-4 hours for 24-48 hours in addition to 1 g tranexamic acid every 6 hours for 5-7 days, has the potential to restore hemostasis and stop bleeding in subjects receiving treatment with an anti-FXI/FXIa antibody provided herein (e.g., NOV1401 or an antibody comprising the VL CDR and VH CDR of NOV1401) undergoing major surgery and in patients with an inaccessible area of active bleeding. For example, Riddell et al. reported four patients with severe FXI deficiency without an inhibitor undergoing surgery (Riddell et al. , 2011, Thromb. Haemost.; 106: 521–527); patients were given rFVIIa 30 μg/kg and 1 g tranexamic acid IV at induction of anesthesia. Subsequent bolus doses of 15–30 μg/kg rFVIIa were administered at 2–4-hour intervals, guided by rotational thromboelastometry (ROTEM) results. Patients received rFVIIa at the above doses for 24–48 hours. Administration of 1 g tranexamic acid every six hours was continued for five days. In this small group of patients, rFVIIa administered at doses as low as 15-30 mcg/kg in combination with tranexamic acid was safe and effective in correcting the hemostatic defect in patients with severe FXI deficiency in this study. In another study of 4 patients with severe FXI deficiency with an inhibitor (autologous neutralizing anti-FXI antibodies, typically acquired after transfusion or administration of blood products to patients with severe FXI deficiency) undergoing 5 surgeries, the authors (Livnat et al. , 2009, Thromb. Haemost.; 102: 487-492) used the following protocol: 1 g tranexamic acid orally two hours before surgery, then patients received an additional 1 g tranexamic acid IV immediately before surgery. Recombinant FVIIa, at doses ranging from 15 to 30 mcg/kg, was administered by infusion at the completion of surgery. Patients then took 1 g of tranexamic acid orally every 6 hours for at least 7 days. Fibrin glue was sprayed onto the removed gallbladder bed in one patient. This protocol ensured normal hemostasis in patients with severe FXI deficiency and an inhibitor.

In one aspect, fibrin glue can be used to restore local hemostasis during dental surgery in patients with FXI deficiency (Bolton-Maggs (2000) Haemophilia; 6 (S1): 100-9). In a specific embodiment, relating to methods for controlling bleeding in patients receiving treatment with an anti-FXI/FXIa antibody provided herein (e.g., NOV1401), a regimen comprising taking 1 g of tranexamic acid every 6 hours for 5-7 days in combination with the use of fibrin glue can be used to create local hemostasis in subjects undergoing minor surgery and in subjects with an accessible bleeding area, including bleeding in the mouth and nose.

Pharmaceutical compositions

The invention relates to pharmaceutical compositions comprising FXIa-binding antibodies (intact antibodies or binding fragments) formulated with a pharmaceutically acceptable carrier. The compositions may also contain one or more other therapeutic agents suitable for the treatment or prevention of, for example, thromboembolic diseases (e.g., thrombotic diseases). Pharmaceutically acceptable carriers enhance the effect or stabilize the composition, or can be used to facilitate the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-retarding agents, and the like, which are physiologically compatible.

The pharmaceutical composition of the present invention can be administered by various routes known in the art. The route and/or method of administration varies depending on the desired results. Intravenous (IV), intramuscular (IM), intraperitoneal (IP), or subcutaneous (SC) administration, or administration near the target area, is preferred. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody, bispecific, or multispecific molecule, can be coated with a material to protect the compound from the action of acids and other environmental conditions that can inactivate the compound.

In certain aspects, the anti-FXI/FXIa antibodies described herein (e.g., the antibodies listed in Table 1, such as NOV1401, or antibodies comprising the LCDR and HCDR regions of NOV1401) are formulated at a concentration of from about 75 mg/1 ml to about 200 mg/1 ml, in vials of liquid for subcutaneous injection. In certain embodiments, the pharmaceutical composition comprises a pharmaceutical carrier or excipient, such as sucrose and polysorbate 20. In certain embodiments, the pharmaceutical composition comprises L-histidine and/or histidine HCl monohydrate. In certain embodiments, the pharmaceutical composition has a pH of about 4-7 or 5-6.

In certain aspects, the anti-FXI/FXIa antibodies described herein (e.g., the antibodies listed in Table 1, such as NOV1401, or antibodies comprising the LCDR and HCDR regions of NOV1401) are formulated at a concentration of 150 mg/1 mL in vials of liquid for subcutaneous injection. In one embodiment, the 150 mg/mL liquid preparation comprises 150 mg of an anti-FXI/FXIa antibody, L-histidine, histidine HCl monohydrate, sucrose, and polysorbate 20, and has a pH of 5.5 ± 0.5. The composition should be sterile and liquid. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride. Long-term absorption of injectable compositions can be achieved by including an absorption delaying agent, such as aluminum monostearate or gelatin.

The pharmaceutical compositions of the invention can be prepared by methods well known and commonly practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th edition, 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured in accordance with GMP requirements. Typically, a therapeutically effective dose or an effective dose of an FXIa-binding antibody is used in the pharmaceutical compositions of the invention. FXIa-binding antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Dosage regimens are adjusted to ensure the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased depending on the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of the active compound calculated to achieve the desired therapeutic effect, in association with the required pharmaceutical carrier.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied to achieve an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, without toxicity to the patient. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular compositions of the present invention used, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, other drugs, compounds, and/or materials used in combination with the particular compositions used, the age, gender, body weight, pathological condition, general health, and previous medical history of the patient receiving treatment, and similar factors.

A physician may begin administering doses of the antibodies of the invention used in the pharmaceutical composition at levels lower than those necessary to achieve the desired therapeutic effect and gradually increase the doses until the desired effect is achieved. In general, effective doses of the compositions of the present invention for the treatment of thrombotic and/or thromboembolic diseases described herein vary depending on a variety of factors, including the route of administration, the target site, the physiological condition of the patient, other drugs administered, and whether the use is prophylactic or therapeutic. Therapeutic doses must be titrated to optimize safety and efficacy. For systemic administration of the antibody, doses range from about 0.01 to 15 mg/kg of subject body weight. For intravenous administration (e.g., subcutaneous administration) of the antibody, the dose may range from 0.1 mg to 5 mg or from 1 mg to 600 mg. For example, the anti-FXI/FXIa antibody described herein can be administered at a dose of 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, or 5.0 mg/kg. An illustrative treatment regimen includes systemic administration once every two weeks or once monthly, or once every 3 to 6 months. An illustrative treatment regimen includes systemic administration once weekly, once every two weeks, once every three weeks, once monthly, or once every 3-6 months, or as needed (PRN).

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose of 3 mg/kg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose of 10 mg/kg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose of 30 mg/kg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose of 50 mg/kg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose of 100 mg/kg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose in the range of 5 mg to 600 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, e.g., intravenously or subcutaneously at a dose of about 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 90 mg, 100 mg, 120 mg, 150 mg, 180 mg, 200 mg, 210 mg, 240 mg, 250 mg, 270 mg, 300 mg, 330 mg, 350 mg, 360 mg, 390 mg, 400 mg, 420 mg, 450 mg, 480 mg, 500 mg, 510 mg, 540 mg, 550 mg, 570 mg or 600 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 5 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 15 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 50 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 150 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 300 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, subcutaneously at a dose of 600 mg.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, e.g., intravenously or subcutaneously, at a dose sufficient to achieve a prolongation of the mean aPTT duration by 2-fold or more for a period of no more than 30 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, or 50 days.

In a specific embodiment, an anti-FXI/FXIa antibody described herein (e.g., an antibody listed in Table 1, such as NOV1401, or an antibody comprising the VL CDR and VH CDR of NOV1401) is administered, for example, intravenously or subcutaneously at a dose sufficient to achieve a prolongation of the mean aPTT duration by 2-fold or more for a period of no more than 42 days.

The antibody is typically administered multiple times. Intervals between doses may be weekly, biweekly, monthly, or yearly. Intervals may also be irregular, determined by measuring the patient's blood levels of FXI-binding antibody and/or FXIa. Alternative dosing intervals may be determined by the physician, with doses administered monthly or as needed to ensure efficacy. With some systemic administration methods, doses are adjusted to achieve plasma antibody concentrations of 1-1000 mcg/mL or 1-1200 mcg/mL, and with some methods, 25-500 mcg/mL. Alternatively, the antibody can be administered as a sustained-release preparation, requiring less frequent administration. Doses and frequency of administration vary depending on the half-life of the antibody and its target in the patient's body. Generally, human and humanized antibodies have a longer half-life in humans than chimeric and non-human antibodies. Doses and frequency of administration may vary depending on whether the use is prophylactic or therapeutic. For prophylactic use, a relatively low dose is administered infrequently over a long period of time. Some patients continue to receive the drug for life. For therapeutic use, a relatively high dose may need to be administered at relatively short intervals until disease progression slows or stops, and preferably until the patient experiences a partial or complete amelioration of disease symptoms. The drug can then be administered prophylactically.

EXAMPLES

The following examples are provided to further illustrate the invention, but not to limit its scope. Other embodiments of the invention will be obvious to those skilled in the art and are within the scope of the appended claims.

Example 1

Panning of the human Fab phage library

Several panning strategies were used to select antibodies recognizing human factor XI. Therapeutic antibodies against different variants of the human factor XI catalytic domain protein and rabbit factor XIa were generated by selecting clones of factor XI-binding antibodies using a commercially available phage display library, the Morphosys HuCAL PLATINUM ^® library, as a source of antibodies. The phagemid library is based on the HuCAL ^® concept (Knappike et al., 2000, J Mol Biol 296:57-86) and utilizes CysDisplay™ technology to display Fab fragments on the phage surface (WO01/05950). Liquid-phase panning strategies were used to isolate anti-factor XI antibodies.

Cross-reactivity analysis

Purified Fab fragments were tested in an ELISA assay for binding to different biotinylated variants of the human factor XI catalytic domain proteins (factor XI catalytic domain, factor XIa, and factor XIa) and rabbit factor XIa. For this purpose, 384-well Maxisorp™ plates (Nunc) were coated with neutravidin at a concentration of 10 μg/ml in PBS overnight at 4°C. Antigen binding to neutravidin via biotin was performed at room temperature (RT) for 30 minutes. Binding of Fab fragments at different concentrations was detected using F(ab) _2- specific goat anti-human IgG antibodies conjugated to alkaline phosphatase (diluted 1:5000) using the fluorescent substrate Attophos (Roche, catalog no. 11681982001). Fluorescence emission at 535 nm was recorded with excitation at 430 nm.

IgG modification and IgG expression

To express full-length IgG in CAP-T cells, fragments of the variable domains of the heavy (VH) and light (VL) chains were subcloned from Fab expression vectors into the corresponding ^pMorph®_hIg vectors for human IgG1. Two amino acid substitutions (D265A and P329A) were made in the Fc region to reduce the likelihood of ADCC or CDC caused by any surface-bound FXI. These alanine substitutions have been shown to reduce the likelihood of ADCC and CDC (see, e.g., Idosugie et al. , J. Immunol. 164: 4178–4184, 2000; Shields et al., J. Biol. Chem. 276: 6591–6604, 2001). Cell culture supernatant was collected 7 days after transfection. After filter sterilization, the solution was subjected to protein A affinity chromatography using an automated liquid handling workstation. Samples were eluted in 50 nM citrate, 140 nM NaOH, adjusted to neutral pH with 1 M Tris buffer, and filter sterilization (0.2 μm pore size) was performed. Protein concentrations were determined by UV spectrophotometry at 280 nm, and IgG purity was analyzed by SDS-PAGE under denaturing reducing conditions.

Example 2

Linking data

Surface plasmon resonance (SPR) analysis of the FXI catalytic domain.

SPR measurements were performed using a BIACORE™ T200 surface plasmon resonance optical biosensor (BIACORE™, GE Healthcare, Uppsala). S-series (CM5) sensor chips, immobilization kits, and regeneration buffer were purchased from GE Healthcare (Uppsala). Two different assay variants were performed depending on the ligand format, IgG or Fab. First, the surface was activated with N- hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N- ethylcarbodiimide hydrochloride (EDC). NOV1401-Fab was covalently attached to the activated dextran matrix on the CM5 chip using a standard amine coupling method (GE Healthcare, Uppsala). For NOV1401-IgG, a capture assay was performed, and goat anti-human IgG-Fc antibody (JIR) was immobilized on the chip at 14,000 EO. Remaining active surface groups were inactivated with ethanolamine (EA). A reference well without immobilized ligand was prepared, and the system was equilibrated with 1x HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P2O, pH 7.4, Teknova H8022).

All binding experiments were performed at 25°C with a flow rate of 50 μl/min using HBS-EP+ buffer. For the capture assay, NOV1401-IgG was captured until an EO of 80 was reached. For kinetic studies, a dilution series of the FXI catalytic domain with a concentration range of 0-200 nM in HBS-EP+ buffer was used. The association time was 120 s, and the dissociation time was 180 s. The surface was regenerated by a single injection of 10 mM glycine, pH 1.5 (contact time 60 s, stabilization time 120 s). Data processing, as well as determination ofk _on ,k _off And K _D was performed using the T200 BiaEvaluation software version 1.0. Double control (subtraction of the reference and blank samples) was used to correct for volumetric effects and other system artifacts. Fitting of sensorgrams was performed using the 1:1 binding model (R_maxset to "global").

Equilibrium solution titration (ESS) for FXI and FXIa

Twenty-two serial 1.6-fold dilutions of antigens were prepared in sample buffer (PBS, pH 7.4, containing 0.5% BSA and 0.02% Tween 20), and NOV1401-Fab (200 pM for huFXI and 500 pM for huFXIa) or NOV1401 (10 pM for huFXI and huFXIa) antibody at a constant concentration were added to the antigen at each concentration. Starting concentrations for the antigen dilution series were 100 nM for huFXIa and 20 nM for huFXI (Fab assay) or 1 nM (IgG assay). 60 µl/well of each dilution mixture were added in duplicate to the wells of a 384-well polypropylene microtiter plate (MTP). Sample buffer served as a negative control, and a sample containing no antigen served as a positive control (Bmax). The plates were sealed and incubated overnight at RT on a shaker. A standard 384-well MTP (MSD) was coated with 30 µl/well of huFXIa at a concentration of 0.1 µg/ml (for huFXIa and huFXI) diluted in PBS, sealed, and incubated overnight at 4°C.

After incubation and three washes with TBST (TBS containing 0.05% Tween 20), the MSD antigen-coated plate was blocked by adding 50 μl/well of blocking buffer (PBS containing 5% BSA) and incubated for 1 h at RT on a shaker. The washing step was repeated, and 30 μl/well of the Fab/IgG antigen preparation was transferred from the polypropylene MTP to the MSD antigen-coated plate and incubated for 20 min at RT on a shaker. After an additional washing step, 30 μl of ECL-labeled goat anti-human IgG/Fab (MSD) detection antibody at a concentration of 0.5 μg/ml diluted in sample buffer was added to each well and incubated for 30 min at RT with shaking. After washing the plate three times, 35 µl of MSD reading buffer was added to each well. Electrochemiluminescence (ECL) signals were acquired and read using an MSD Sector Imager 6000.

The mean ECL signal was calculated from duplicate wells in each assay. Data were adjusted for baseline values by subtracting the lowest value from all data points and plotted against the corresponding antigen concentrations. K _D values were determined by plotting the signal according to a 1:1 (for Fab) or 1:2 (for IgG) binding model (according to Piehler et al., 1997).

Results

The results are summarized in Tables 3 and 4. For both NOV1401 formats, Fab and IgG, K _D values of approximately 20 nM were obtained for the FXI catalytic domain when determined using BIACORE™. Fab affinities for both activated and zymogen FXI were in the pM range and were 66- and 300-fold higher than the affinity for the catalytic domain, respectively. Given their high affinity, these interactions were measured in TPP assays. NOV1401-Fab demonstrated fivefold higher affinity for zymogen FXI (62 pM) than for activated FXI (305 pM). NOV1401-IgG affinity values for both dimeric zymogen and activated FXI are labeled as apparent K _D values because the interaction could be influenced by avidity effects.

To confirm that NOV2401 also binds cynomolgus macaque FXI, TPP experiments were performed with activated cynomolgus macaque FXI and cynomolgus macaque FXI zymogen, yielding apparent K _D values of 12.5 ± 6.6 pM (N = 2) and 5.0 ± 0.7 pM (N = 2), respectively. Thus, the affinity values of NOV1401 for cynomolgus macaque FXI proteins (active form and zymogen) were comparable to those for binding to human FXI (Table 3).

Table 2. K values _D and kinetic parameters of NOV1401-Fab/IgG binding to the human FXI catalytic domain as determined using BIACORE™. Catalytic domain k _a (1/Ms) k _d (1/s) K _D (nM) n NOV1401-Fab 3.2 ± 0.5 E+4 6.1 ± 1.8 E-4 19 ± 6 3 NOV1401-IgG 4.2 ± 1.6 E+4 8.8 ± 2.2 E-4 21 ± 3 2

Table 3. K _D values of NOV1401-Fab/IgG for activated and zymogen FXI determined by the equilibrium solution titration (ESS) method. *Only apparent K _D values are shown, as avidity effects could influence the interaction. NOV1401-Fab K _D [pm] n Activated human FXI 305 ± 8 3 Human zymogen FXI 62 ± 18 3 NOV1401-IgG Activated human FXI 4.7 ± 2.1* 3 Human zymogen FXI 1.3 ± 0.3* 3

Literature: Piehler et al.Assessment of affinity constants by rapid solid phase detection of equilibrium binding in a flow system, J. Immunol. Meth. 1997, 189-206.

Example 3

Biochemical assay: FXIa inhibition in an activity assay using a fluorescent peptide as a substrate

The activity of human FXIa (Kordia Life Science NL, catalog number HFXIa 1111a) was determined by monitoring the cleavage of a fluorescently labeled peptide with the sequence D-Leu-Pro-Arg*Rh110-D-Pro (catalog number BS-2494; Biosyntan GmbH, Berlin, Germany). In the substrate sequence above, * indicates a labile bond, D-Leu: D-leucine, Pro: proline, Arg: arginine, Rh110: rhodamine 110, D-Pro: D-proline). FXIa-mediated cleavage of the labile bond of the peptide substrate results in an increase in the fluorescence intensity of rhodamine 110 using excitation and emission wavelengths of 485 nm and 535 nm, respectively. Fluorescence intensity was measured continuously on a Safire2 plate reader (TECAN, Maennedorf, Switzerland) at room temperature (RT). The assay buffer contained 50 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM CaCl2 _, and 0.05% (w/v) CHAPS. For the final activity assay, human FXIa and the BS-2494 substrate were used at assay concentrations of 0.1 nM and 0.5 μM, respectively. Under these conditions, the increase in fluorescence intensity versus time was linear for at least 60 minutes.

To test the inhibitory activity of antibodies, serial dilutions of antibodies were prepared in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ ) containing 0.05% (w/v) CHAPS. Two μl of the antibody solution were preincubated with 10 μl of FXIa solution (in assay buffer) for 60 min at RT. After the preincubation step, 10 μl of BS-2494 substrate (diluted in assay buffer) were added and the enzymatic reaction was allowed to proceed for 60 min, after which the fluorescence intensity was measured. Fluorescence intensity values were converted to percent inhibition using control reactions (signal from reactions without inhibition is equivalent to 0% inhibition and signal in the reaction mixture without enzyme is equivalent to 100% inhibition) and the following formula for converting values:

y=100% - [FI(x)-FI(min)]/[FI(max)-FI(min)],

where y represents the % inhibition at antibody concentration x, FI(x) represents the fluorescence intensity measured at antibody concentration x, FI(min) represents the fluorescence intensity measured in the control reaction in the absence of antibody, and FI(max) represents the fluorescence intensity measured in the control reaction without inhibition. Data were analyzed using Origin 7.5SR6 software (OriginLab Corporation, USA). IC ₅₀ values from averaged data were calculated using the logistic function:

y=A2+(A1-A2)/(1+(x/IC ₅₀ )^p),

where y represents the % inhibition at antibody concentration x, A1 represents the minimum inhibition value, and A2 represents the maximum inhibition value. The exponent, p, is the Hill coefficient.

Figure 6A shows a representative concentration-response curve for the inhibition of full-length human FXIa enzymatic activity by the NOV1401 antibody. The results demonstrate that NOV1401 inhibits full-length human FXIa enzymatic activity in a concentration-dependent manner (Figure 6A). A logistic curve fit yielded an _IC50 value of approximately 160 pM.

Example 4

Anticoagulant activity of anti-FXIa antibodies

The antithrombotic activity of NOV1401 and NOV1090 antibodies was tested in the activated partial thromboplastin time (aPTT) assay and the thrombin generation assay (TGA).

aPTT analysis:

Lyophilized normal human plasma "Coagulation Control N" (catalog no. 5020050) was purchased from Technoclone GmbH (Vienna, Austria). It consisted of pooled plasma from selected healthy donors with added sodium citrate. The clotting time determined for this normal plasma reflects normal concentrations of coagulation factors involved in clot formation. Lyophilized plasma was stored at 4°C. Before use, the plasma was resuspended in 1 ml of distilled water by gently inverting the tube and then incubated for 10 minutes at RT.

The intrinsic pathway activating reagent "aPTT-s" (catalog no. TE0350) was purchased from SYCOmed (Lemgo, Germany) and contained a phospholipid and silicate (colloidal) in a buffer solution (sodium chloride, polyethyleneglycol 20000; sucrose, sodium azide). The solution was stored at 4°C.

Calcium chloride (catalog no. C1016-500G; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was prepared in bidistilled water at a stock concentration of 25 mM.

Tris/HCl buffer pH 7.5, UltraPure (catalog no. 15567-027; Life Technologies Corporation, NY, USA) and phosphate-buffered saline (PBS, catalog no. P4417-100TAB; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were used to solubilize the compounds.

3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate hydrate (CHAPS, catalog no. C3023-25G) and anhydrous dimethyl sulfoxide (DMSO, catalog no. 276855-100 mL) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).

Blood clotting time measurements were performed using an Amelung KC4A bead coagulometer (purchased from SYCOmed, Lemgo, Germany), a semiautomated mechanical clot detection system. The system uses a special cuvette (catalog no. AI4000; SYCOmed) containing a stainless steel bead (catalog no. AI5000; SYCOmed).

The sample is added to the cuvette. After the appropriate incubation period, the cuvette is placed in the measuring cell of the coagulometer. The measuring cell rotates slowly, causing the cuvette to rotate along its longitudinal axis. Since the cuvette is positioned at a slight angle, gravity and inertia ensure that the ball always remains at the lowest point of the cuvette. A magnetic sensor is located directly opposite the ball's position. Adding the initiating reagent starts the timer. As blood clots, fibrin threads form in the reaction mixture. The fibrin threads pull the ball away from its inertial position, causing a pulse in the magnetic sensor. This pulse electronically stops the timer. The sequence of events was as follows (Table 4a):

Table 4a Analysis stage Solution aPTT analysis
volume [µl] 1 Diluted compound or diluent 50 2 Human blood plasma 50 3 Reagent aPTT-s 50 4 Incubation for 3 minutes at 37°C with rotation 5 25 mM calcium chloride 50 6 Instant start timer 7 The timer stops when a clot forms.

Samples were measured in duplicate at 37°C in an Amelung bead coagulometer.

Figure 6B shows a representative concentration-response curve for the NOV1401 antibody, which resulted in a concentration-dependent prolongation of the clotting time in the aPTT assay. The results indicate that NOV1401 results in a concentration-dependent prolongation of the clotting time in the aPTT assay of human plasma. The clotting time in the aPTT assay was doubled compared to the baseline value at a NOV1401 concentration of approximately 14 nM. The calculated _IC50 value was approximately 13 nM.

Thrombin generation assay (TGA) :

For AOT analysis, lyophilized normal human plasma (Coagulation Control N) was purchased from Technoclone GmbH (catalog number 5020040, lot no. 1P37B00) and reconstituted in distilled water at the volume suggested by the manufacturer.

The substrate solution was prepared using the fluorogenic substrate Z-Gly-Gly-Arg-AMC from Technoclone GmbH (catalog number 5006230, lot no. 8F41B00). Aliquots of the lyophilized substrate were stored at 4°C. The substrate was redissolved in the volume of distilled water specified on the vial 20 minutes before use in the assay. The reconstituted substrate solution contained the fluorogenic peptide at a concentration of 1 mM and _CaCl2 at a concentration of 15 mM.

Two different reagents, TGA RD (catalog no. 500622) and TGA RC low (catalog no. 5006213), for initiating the intrinsic and extrinsic pathways of coagulation activation, respectively, were purchased from Technoclone GmbH (Vienna, Austria). The initiating reagent, platelet poor plasma (PPP)-reagent low, was purchased from Thrombinoscope (TS31.00, lot no. PPL1409/01) and reconstituted with the volume of distilled water indicated on the bottle. PPP-reagent low contains a mixture of phospholipids and tissue factor at a very low concentration. The reagent was diluted 8-fold in 80 mM Tris/HCl, pH 7.4, 0.05% (w/v) CHAPS immediately before use.

Samples were aliquoted and measured in 96-well black, clear-bottom plates purchased from Costar (catalog number 3603). For automated transfer, samples were placed in a 96-well V-bottom plate (Costar, 3894) and transferred using the CyBio automated system (Analytik Jena US, Woburn, MA, USA).

Recovered human plasma, PPP-reagent low, and substrate were prewarmed for 10 min in a 37°C water bath. Serial 1:3 dilutions of antibody in PBS were prepared in a 96-well plate, starting with a NOV1401 concentration of 5 μM (5x highest final concentration from 1 μM), for a total of 8 dilutions. 222 μL of the primer reagent were mixed with 1108 μL of the substrate solution to obtain a 10+50 primer/substrate mixture. 80 μL were added to each well of a 96-well V-bottom plate for subsequent transfer using an automated system. The plate was stored at 37°C. Reagents were added according to the schedule given in Table 4b.

Table 4b Analysis stage Solution Volume [µl] 1 Antibody solutions (8 dilutions) 20 2 Plasma mother solution 20 5 minutes of incubation at 37°C in a thermomixer at 300 rpm 3 Initiator/substrate mixture 10+50

Initiator/substrate mixtures were transferred using an automated system. After addition of the mixtures, measurements were immediately recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively, using a Synergy Neo instrument (BioTek Instrument Inc., Winooski, VT, USA). Samples were measured in duplicate at 37°C in a plate reader for 90 minutes at 55-second intervals.

To obtain peak thrombin concentrations, the data were processed using the AOT estimator file provided by Technoclone. Data were fit using GraphPad to plot peak thrombin concentration versus antibody concentration. The data were fitted to a nonlinear regression model using GraphPad Prism5 (GraphPad Software Inc., La Jolla, CA, USA). The IC ₅₀ was determined using the built-in four-parameter dose-response equation (variable slope): y = trough + (high - trough) / (1 + 10^((LogIC ₅₀ - x) * slope)), where y is the maximum thrombin concentration generated at inhibitor concentration x; and high and trough are the thrombin concentrations without inhibitor and at the highest inhibitor concentration, respectively.

Figure 6C shows a representative concentration-response curve for the NOV1401 antibody, demonstrating concentration-dependent inhibition of thrombin generation in the AOT assay. An _IC50 value of 24 nM and a residual thrombin concentration of 159 nM (dashed line) were calculated from this concentration-response curve.

Example 5

Protein expression, complex formation, crystallization, and structure determination of the NOV1401 (Fab)-FXI (catalytic domain) complex

The structure of the Fab fragment of the NOV1401 antibody, obtained by papain cleavage, in complex with the catalytic domain of factor XI was determined by co-crystallization at a resolution of 2.04 Å.

Protein expression :

The expression construct for the FXI catalytic domain consisted of amino acid residues 388–625 (Swissprot P03951) with an unpaired cysteine residue, C500, mutated to cysteine, an N-terminal extension consisting of the amino acids MGSS (SEQ ID NO: 49), an octahistidine tag (SEQ ID NO: 50), a PreScission™ cleavage site, and an enterokinase cleavage site. The construct was assembled by gene synthesis, cloned into the pET24a expression vector, and expressed as inclusion bodies in E. coli strain BL21 (DE3) cells grown in LB medium. Inclusion bodies were solubilized in a mixture of 50 mM Tris/HCl, pH 8.0, 6.0 M guanidine chloride, and 50 mM DTT for 2 h, completely denaturing the recombinant protein. Refolding buffer (0.5 M Tris, pH 8.0, 0.9 M arginine HCl, 5 mM GSH, 0.5 mM GSSG, 1 mM EDTA) was quickly added in large excess to solution IB to give a final protein concentration of 50 μg/ml and incubated at 4°C for 5 days. Refolding and disulfide bridge formation were achieved by dialysis with buffer A (50 mM Tris, pH 8.0) for 3 days.

The protein in the reconstituted conformation was loaded onto an anion exchange chromatography column containing Q ^- Sepharose® FF (GE Healthcare), equilibrated and washed with buffer A. Unbound recombinant protein was collected in the column runoff and wash fractions. The pH was adjusted to 7.4 by dialysis with 50 mM Tris buffer, pH 7.4, before removal of the N-terminal tag sequence using enterokinase (enterokinase:recombinant protein ratio 1:100, incubation time 2.5 h). The digestion reaction was stopped by loading the sample onto a benzamidine-Sepharose 4 FF (high sub) benzamidine affinity chromatography column (GE Healthcare) equilibrated and washed with buffer B (50 mM Tris, pH 7.4, 0.5 M NaCl) and eluted with buffer C (buffer B containing 50 mM benzamidine). The active catalytic domain of FXI was loaded onto an XK 26/600 Superdex 75 size-exclusion chromatography column (GE Healthcare) equilibrated with 20 mM Na-acetate buffer, pH 5.3, containing 75 mM NaCl. The final protein concentration was 1.07 mg/mL.

The NOV1401 Fab fragment was generated by IgG digestion with papain. The final Fab concentration in PBS was 11 mg/ml. Digestion was performed overnight at 37°C using papain (Roche Diagnostics 108 014; 10 mg/ml) added to the antibody at a ratio of 1:100 (by weight) in the presence of 1 mM cysteine (added to the IgG stock solution). The cleavage was stopped by the addition of 50 μM of the papain-specific inhibitor E64, (N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]agmatine), and the cleavage products were passed through a small protein A column (5 ml) to remove the Fc fragment. The Fab fragments were collected in the column runoff buffer, dialyzed against PBS, concentrated to their final concentration by ultrafiltration, and sterilized by filtration (0.22 μm).

Complex formation, crystallization and structure determination :

The FXI catalytic domain and Fab were mixed in an equimolar ratio and concentrated to a final concentration of 9 mg/mL.

Crystals used for data collection were prepared at 277K using sessile drop vapor diffusion by mixing 0.3 µL of reservoir solution (0.2 M ammonium chloride, 20% PEG 3350), 0.2 µL of Fab-FXI complex, and 0.1 µL of seed crystal from crystals obtained in the first round of crystal screening.

For data collection, crystals were snap-frozen in liquid nitrogen. Data were acquired on a Swiss Light Source X10SA synchronous beamline at 1.00002 Å using a Pilatus pixel detector (Dectris) at 100K. Data processing and scaling were performed using XDS and XSCALE (Kabsch, W. (2010) Acta Cryst. D66, 125-132). The crystal diffracted to a resolution of 2.04 Å with unit cell dimensions a=191.27, b=53.22, c=65.164, alpha=90.0, beta=94.56, gamma=90.0 (space group C2) with one copy of the complex per asymmetric unit.

The structure of the complex was solved by molecular replacement using the structures of the FXI catalytic domain and the truncated Fab fragment, previously determined in our own laboratory, as search models using PHASER (McCoy, AJ et al. (2007) J. Appl. Cryst. 40, 658–674). Alternating rounds of refinement and reconstruction were performed using buster and coot rsp. (Bricogne, G. et al. (2010) BUSTER version 2.9. Cambridge, United Kingdom: Global Phasing Ltd.; Emsley, P. and Cowtan, K. (2004). Acta Crystallogr. D60, 2126–2132). The data obtained and refinement statistics are presented in Table 5.

Table 5. Data set and refinement statistics Data set Space group C2 Cell dimensions a , b , c (Å) 191.27; 53.22, 65.16 α, β, γ (°) 90; 94.56; 90 Resolution (Å) 64.96-2.04 (2.09-2.04) R _sym 0.099 (0.571) I /σ I 10.8 (2.7) Completion (%) 95.9 (98.4) Redundancy 3.2 Clarification Resolution (Å) 64.96-2.03 Number of reflections 40088 R _slave / R _free 0.217/0.282 Number of atoms Protein 5071 Water 413 Standard deviations Bond lengths (Å) 0.01 Bond angles (°) 1.19

*Values in brackets correspond to the highest resolution region.

Description of the structure:

Structural data showed that the binding epitope of the NOV1401 antibody binds to the active site surface, with the heavy chain CDR3 loop covering portions of the S3, S2, S1-beta, and S1' subsites. Adjacent heavy chain CDR1 and CDR2 loops induce conformational changes in loops 145 and 220 of FXI (chymotrypsin numbering). Additionally, the four N-terminal residues of FXI, as well as residues surrounding Asp189, become disordered; both of these regions are key for FXI catalytic activity. The conformational change in loop 145 results in closure of the S1 pocket by Arg144 and of the S2' subsite by Tyr143. Thus, antibody binding induces the adoption of the inhibited conformation by FXI through multiple mechanisms.

The observed inhibited form has features similar to those described for the full-length FXI zymogen (PDB 2F83). Portions of the FXI catalytic domain that changed conformation and became disordered due to antibody binding are also disordered in the zymogen. This explains the strong binding of NOV1401 to FXI in its zymogen form.

The inventors' data indicating that NOV1401 does not inhibit FXI zymogen activation are explained by the distance between the binding epitope and the FXIa zymogen activation cleavage site. NOV1401 binds both FXI and FXIa. X-ray crystallographic analysis of the Fab-FXI CD complex revealed a unique binding mode and mechanism of FXIa inhibition. NOV1401 binds to the active site of FXIa (Figure 4) and induces conformational changes in the four N-terminal residues and loops of the catalytic domain, resulting in an inactive conformation. This inactive conformation has similar features to the structure of the inactive catalytic domain in zymogen (Figure 5), explaining how both FXI and FXIa can be bound with high affinity by the NOV1401 antibody. For example, three loops of the catalytic center (e.g., loop 220, loop 188, and loop 145), which are disordered in the zymogen structure, are also disordered or misaligned in the NOV1401 Fab-FXI CD complex structure, and the N-terminal salt bridge observed in the active conformation is absent in both the zymogen and NOV1401 Fab-FXI CD complex structures (Table 6). Thus, NOV1401 appears to induce conformational changes in the CD that result in an inactive, zymogen-like conformation.

Table 6: Structural characteristics of FXIa KD, FXIa KD in complex with NOV1401 and FXI zymogen (KD): FXIa KD Complex NOV1401-FXI CD Zymogen FXI Salt bridge Ile16-Asp194 + - - Loop 145 ordered displaced disordered Loop 188 ordered disordered disordered Loop 220 ordered displaced disordered

Example 6

Epitope mapping based on X-ray structural analysis

The FXI residues in contact with Fab, analyzed using AREAIMOL (Briggs, P.J. (2000) CCP4 Newsletter No. 38) by determining the differences in surface area of residues when calculated without bound Fab and in complex with Fab, are listed in Table 7 and Table 8a (Swissprot numbering):

Table 7: FXI Epitope
Epitope (underlined: contacts of light chain and heavy chain): Light chain contacts
Pro410
Arg413
His431
Tyr434
Gly435
Glu437
Tyr472
Lys473
Met474
Glu476
Tyr521
Leu524
Arg525
Asp526
His552 Heavy chain contacts
Leu415
Cys416
His431
Cys432
Tyr434
Tyr472
Met474
Ala475
Glu476
Tyr521
Arg522
Lys523
Leu524
Arg525
Asp526
Lys527
Arg548
Ser575
Ser594
Trp595
Gly596
Glu597
Arg602
Glu603
Arg604

The FXI epitope is formed by the following residues:

Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472-Glu476, Tyr521-Lys527, Arg548, His552, Ser575, Ser594-Glu597, Arg602-Arg604.

Table 8a: FXI residues in contact with NOV1401 (epitope) Remainder Difference in area PRO A 410 -11.0 ARG A 413 -36.3 LEU A 415 -3.6 CYS A 416 -2.1 HIS A 431 -36.5 CYS A 432 -0.6 TYR A 434 -108.1 GLY A 435 -31.6 GLU A 437 -3.7 TYR A 472 -13.0 LYS A 473 -40.1 MET A 474 -73.1 ALA A 475 -12.0 GLU A 476 -40.4 TYR A 521 -18.5 ARG A 522 -46.6 LYS A 523 -74.7 LEU A 524 -147.1 ARG A 525 -212.6 ASP A 526 -17.7 LYS A 527 -0.2 ARG A 548 -11.6 HIS A 552 -4.0 SER A 575 -7.7 SER A 594 -8.7 TRP A 595 -20.9 GLY A 596 -17.5 GLU A 597 -49.0 ARG A 602 -18.5 GLU A 603 -2.0 ARG A 604 -41.0

The epitope mapped by X-ray crystallography to the catalytic domain sequence (residues that form the epitope are shown in bold and underlined):

Table 8b lists the antibody residues that contact FXI (paratope).

Table 8b: NOV1401 residues in contact with FXI (paratope).
L, light chain; H, heavy chain Differences in residues Square SER L 27 -1.80 GLY L 30 -5.00 SER L 31 -52.60 ASN L 32 -21.00 ASP L 33 -22.00 TYR L 50 -36.00 LYS L 51 -54.20 TYR L 53 -41.40 ASN L 54 -25.50 LYS L 67 -6.90 TRP L 92 -44.30 GLN L 94 -72.00 ARG L 95 -5.70 PHE L 97 -54.70 ASP L 98 -2.70 VAL L 99 -0.10 PHE H 27 -2.00 THR H 28 -20.50 SER H 30 -13.80 THR H 31 -78.90 ALA H 33 -10.80 TRP H 47 -12.70 SER H 52 -2.20 TYR H 59 -62.00 TYR H 60 -0.80 GLU H 99 -1.70 SER H 101 -51.30 TYR H 102 -116.60 LEU H 103 -175.00 TYR H 104 -140.20 SER H 105 -1.30

Example 7

The effect of anti-FXI antibody on FeCl-induced ₃ thrombosis in mice

FXI-deficient mice (FXI ^-/- mice) on a C57Bl background were obtained from Novartis (E. Hanover, NJ) and used to evaluate the antithrombotic efficacy of NOV1401. Following intravenous administration of human FXI (hFXI), these mice acquire a wild-type thrombophilic phenotype upon exposure to a thrombogenic stimulus. In the studies described herein, thrombosis was induced in the carotid artery by applying ferric chloride (FeCl ₃ ) to the arterial surface.

NOV1401 was administered by bolus injection via the jugular vein to anesthetized mice 15 minutes before thrombosis induction. Antibody doses ranged from 0.24 mg/kg to 0.47 mg/kg. FXI ^-/- mice were induced to restore deficiency with human FXI by injecting 0.47 mg/kg human FXI via the jugular vein 10 minutes before _FeCl3 stimulation. Two 1 mm x 1.5 mm filter papers soaked in 3.5% _FeCl3 were applied to opposite sides of the carotid artery, in contact with its adventitial surface, and removed after 3 minutes, followed by thorough irrigation with saline. Carotid artery blood flow was measured using a Transonic flow sensor. Baseline blood flow was determined for 5 minutes before FeCl ₃ application and then for 30 minutes after FeCl ₃ application (i.e., during the thrombogenic period). At the end of the experiment, a blood sample was collected from the vena cava into a syringe containing 3.8% sodium citrate solution, plasma was obtained, and aPTT analysis was performed.

Figure 1A shows the effect of NOV1401 on FeCl3 _- induced thrombosis in FXI ^-/- mice administered human FXI (a mouse model with humanized FXI). Figure 1B shows the effect of NOV1401 on aPTT in the same mouse model. Figure 1C shows prolongation of aPTT in FXI ^-/- mice compared to wild-type mice.

The NOV1401 antibody completely inhibited _FeCl3- induced thrombus formation in hFXI-treated FXI ^-/- mice (Figure 1A) starting at a concentration of 0.24 mg/kg. A very robust dose response was observed, likely reflecting an all-or-none stoichiometric antithrombotic response. The aPTT duration was prolonged by 1.6-fold in the high-dose group relative to vehicle controls (Figure 1B), which corresponded to the same level of prolongation as observed in the case of genetic FXI deficiency (Figure 1C), indicating maximal effect. These results demonstrate that NOV1401 has antithrombotic activity in a mouse model of FeCl3 _- induced thrombosis.

Example 8

Effects of anti-FXI antibody on free FXI levels and aPTT in cynomolgus monkeys

To evaluate the pharmacokinetic (PK) profile and pharmacological effects of an anti-FXI/FXIa antibody such as NOV1401, the antibody was administered by subcutaneous (SC) or intravenous (IV) injections to cynomolgus monkeys in a dose-escalation study.

The anticoagulant effect of NOV1401 was studied in cynomolgus macaques, testing the antibody’s ability to prolong aPTT and reduce free FXI (FXI _c ) levels following a single intravenous (N=2) or subcutaneous (N=2) injection of 3 mg/kg. A second dose of 10 mg/kg was administered to all animals, followed by a third dose of 30 mg/kg to determine whether the effects observed at the 3 mg/kg dose could be potentiated at higher doses. These results demonstrate that NOV1401 has robust anticoagulant activity in cynomolgus macaques. The pharmacodynamics (PD) of NOV1401, characterized by its anticoagulant effect as determined by aPTT and FXI _c levels, were then compared with the PK profile. The comparison revealed a good PK/PD correlation.

Animals were administered either intravenous (N=2) or subcutaneous (N=2) injection of NOV1401 on study day 1 at 3 mg/kg, day 85 at 10 mg/kg, and day 114 at 30 mg/kg. Blood samples were collected into sodium citrate tubes at 15 minutes and 2 hours post-dose for IV-dosed animals and for all animals at baseline, 6, 24, 48, 72, and 96 hours post-dose (for Days 1, 85, and 114) and 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50, 53, 57, 60, 64, 66, 71, 75, and 78 days post-dose (for Day 1 dosing only). Blood was also collected on days 92, 95, 99, 102, 107, 110, 121, 124, 128, and before dosing on day 114. All blood samples were centrifuged, plasma samples were obtained, and frozen at approximately -70 C or below.

Total plasma concentrations of NOV1401 were measured in a standard ELISA assay for the detection of human IgG using a sandwich enzyme-linked immunosorbent assay with mouse anti-human IgG monoclonal antibodies as the capture antibody and HRP-labeled goat anti-human IgG antibodies as the detection antibody.

To measure free FXI in plasma samples containing both FXI and NOV1401, unbound FXI was captured with immobilized NOV1401, and FXI already complexed with NOV1401 was washed away. Plate-bound FXI was then detected with the Fc-containing murine antibody 14E11, a monoclonal antibody that binds the A2 domain of FXI and has been described in the literature (Cheng, et al. Blood, 116: 3981–3989, 2010). The very high affinity of NOV1401 for both FXI and FXIa, as well as the different binding sites for NOV1401 and the detection antibody 14E11, allowed for accurate measurements of free FXI.

ELISA plates (384-well LUMITRAC™ 600 HB) were coated with NOV1401 (5 μg/mL in PBS) to bind free FXI. After blocking (milk blocking solution: KPL #50-82-01, 1:20 dilution) and washing the plates with wash buffer (PBS; 0.05% Tween 20), plasma samples diluted 1:40 in assay buffer (50 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM _CaCl2 , 5 mM EDTA, and 0.05% (w/v) CHAPS) were incubated at RT for 30 min and washed 3 times with wash buffer. The detection antibody 14E11 was added at a concentration of 1 μg/mL in dilution buffer (1.7 mM sodium phosphate monobasic, 8.1 mM sodium phosphate dibasic heptahydrate, 0.15 M sodium chloride, 0.7% Triton X-100, and 0.1% sodium azide, pH 7) containing 0.7% casein. After washing the plates with wash buffer, the secondary detection antibody, peroxidase-labeled anti-mouse IgG (Sigma #A5278), was added at a concentration of 0.5 μg/mL in dilution buffer containing 0.4% casein. After washing the plates with wash buffer, 50 μl of chemiluminescent peroxidase substrate solution (LumiGLO, KPL #54-61-01) was added, and the luminescent signal was immediately read on a multi-mode plate reader (SPECTRAMAX M5E). The concentration of free FXI in each sample was determined using a standard curve constructed with human FXI (zymogen) from Enzyme Research Laboratories (catalog #HFXI 1111), starting with 100 nM FXI with a dilution factor of 2 and 22 dilution steps. The lower limit of quantification (LLOQ) was 0.24 nM, taking into account a 1:40 dilution before measurement.

Plasma samples collected at all time points were analyzed for aPTT, and aPTT results were compared with total plasma NOV1401 concentrations and free FXI levels. Figures 2A and 2B show changes in clotting time in the aPTT assay relative to total plasma NOV1401 levels for animals receiving both i.v. and s.c. doses. 3A and 3B Changes in clotting time in the aPTT assay relative to free FXI levels are shown for animals administered intravenous and subcutaneous doses.

For intravenously administered NOV1401, peak total plasma levels of NOV1401 were observed 15 minutes after dosing (Figure 2A). At this time, aPTT was approximately double that of baseline in both animals and remained at this level for an average of 5-6 weeks. The mean prolongation of aPTT from 15 minutes postdose throughout the measurement period before declining to baseline was 2.0 ± 0.02-fold and 1.9 ± 0.03-fold for each animal.

By day 85, prior to the second dose, aPTT had reached baseline levels and NOV1401 plasma concentrations had fallen below 10 nM. A second 10 mg/kg dose was administered on day 85, resulting in at least approximately a 3-fold increase in total NOV1401 plasma concentrations and a prolongation of aPTT similar to that observed after the first dose. A third 30 mg/kg dose was administered on day 114, while aPTT was still prolonged, and did not result in any significant further prolongation of aPTT, despite another at least 3-fold increase in total NOV1401 plasma concentrations (Figure 2A). Thus, doses of NOV1401 greater than 3 mg/kg resulted in aPTT prolongation comparable to that seen at the 3 mg/kg dose and did not appear to increase the degree of aPTT prolongation. As expected, SC administration of NOV1401 resulted in a slower increase in aPTT than IV administration, but the degree of prolongation was comparable to that seen in the IV group (Figure 2B). In two animals, aPTT was prolonged from baseline for an average of 5–6 weeks. The mean fold of aPTT prolongation was similar to that seen in animals receiving the IV dose: 2.0 ± 0.03 and 1.8 ± 0.02 from the 6-hour post-dose time point throughout the measurement period before decreasing to baseline. As with IV administration, administration of higher doses did not result in higher aPTT values despite higher plasma exposure of NOV1401.

The results in Figures 2A-2B show that NOV1401 results in prolongation of aPTT in cynomolgus macaques.

The mean baseline plasma FXI _c concentration was 10.9 ± 0.3 nM in the IV group and declined rapidly (within 15 min) after NOV1401 administration (Figure 3A). Plasma FXI _c levels remained low before total NOV1401 plasma levels declined to 15 nM - 25 nM (Figure 2A, Figure 3A). In the SC group, the mean baseline FXI _c concentration was 14.3 ± 1.0 nM. FXI _c concentrations were sharply reduced from baseline concentrations 6 hours post-dose (Figure 3B) and remained low before NOV1401 plasma levels declined to 15 nM - 25 nM (Figure 2B, Figure 3B). FXI levels _again dropped sharply after the second 10 mg/kg dose in all animals and remained low until the end of the study. Two higher doses did not result in further reductions in FXI levels _relative to baseline.

In all treated animals, the decline and recovery of FXI _c levels was transient and inversely proportional to the NOV1401-induced prolongation of aPTT, confirming that NOV1401 inhibits the function of the intrinsic coagulation pathway (prolongs aPTT) by reducing FXI _{c levels.}

These results (e.g., Figures 3A and 3B) demonstrate that NOV1401 reduces plasma FXI levels _in cynomolgus macaques. No signs of excessive bleeding were observed at venipuncture sites or during postmortem examination in cynomolgus macaques. Furthermore, no occult blood was detected in the stool during the study.

A robust anticoagulant effect of NOV1401 was also observed in cynomolgus macaques in a repeat-dose toxicity study lasting 13 weeks for subcutaneous administration or 4 weeks for intravenous administration. In this study, NOV1401 was administered weekly at doses of 10 mg/kg (N=3, males and females combined) and 100 mg/kg (N=5, males and females combined) subcutaneously for 13 weeks (14 doses) or at a dose of 50 mg/kg (N=3, males and females combined) intravenously for 4 weeks (5 doses). The control group (N=5, males and females combined) received vehicle for 13 and 4 weeks subcutaneously and intravenously, respectively. FXI:C was assessed by measuring clotting times in cynomolgus monkey plasma samples in the presence of FXI-deficient human plasma (single-stage aPTT). aPTT and FXI:C were measured on study days 2, 23, and 79 in the s.c. groups and on days 2 and 23 in the i.v. group. A 2.1- to 3-fold prolongation of aPTT was observed in all animals and all administration groups (Figure 7A). The effect was maintained throughout the dosing phase of the study, and no dose dependence was observed, similar to the previous dose-escalation study. FXI:C decreased by 88-95% in animals in all administration groups and remained at these levels throughout the dosing phase of the study (Figure 7B). The effect on FXI:C was independent of dose across this dose range.

No macroscopic or microscopic signs of bleeding, including excessive bleeding, were observed at venous puncture sites (including subcutaneous and intravenous injection sites, as well as blood sampling sites) or during postmortem examination. Furthermore, no occult blood was detected in the stool at the end of the study. Furthermore, there were no deaths of animals and no test substance-related effects on clinical signs, body weight, food intake, ophthalmological and electrocardiographic parameters, hematology parameters, clinical chemistry parameters, or urinalysis results. No target organ toxicities were identified.

Increased thyroid weight was observed in males in the 100 mg/kg subcutaneous dose group. However, the toxicological significance of these findings is inconclusive, as there were no histological correlations. Thyroid weights varied widely among animals, and abnormal values were observed only in animals of the same sex. Microscopic examination revealed dose-dependent fibrosis at subcutaneous injection sites in both males and females in the 10 and 100 mg/kg/week subcutaneous dose groups. These findings were not considered unfavorable.

No significant signs of toxicity were observed in a single ascending dose study or a general toxicity study with repeated dosing in cynomolgus macaques for up to 13 weeks. Therefore, the highest subcutaneous dose level administered in the 13-week GLP toxicity study (100 mg/kg/week) was defined as the no-absorbable object area (NOAEL).

Example 9

Pharmacokinetics in cynomolgus macaques - single dose

Cynomolgus macaques (female, N=2) were given a single dose of 3 mg/kg NOV1401 either IV or SC and observed until plasma _FXIc and aPTT concentrations returned to pre-dose levels. Animals were then given a single dose of 10 mg/kg NOV1401 either IV or SC, followed 2 weeks later by a 30 mg/kg dose either IV or SC, followed by an additional 2-week observation period. The PK of NOV1401 was assessed by measuring total NOV1401 levels. Total NOV1401 exposure, determined by either the maximum observed total NOV1401 concentration ( _Cmax ) or the area under the total NOV1401 concentration-time curve ( _AUC0-14d ), were comparable among individual animals within each group. Exposure (C _max or AUC _0-14d ) was approximately dose-dependent with each route of administration (Table 9). C _max was approximately 3-fold higher in the IV group than in the SC group. However, total plasma concentrations of NOV1401 were similar in both groups after the initial distribution phase. The terminal half-life (t _1/2 ) was estimated for each animal using a two-compartment model after a dose of 3 mg/kg. The t _1/2 period was 14-15 days (N = 2). Absolute bioavailability after SC injection was 61-66% (3 dose levels). Anti-NOV1401 antibodies were not detected in any animal after either IV or SC administration.

Table 9 : Mean pharmacokinetic parameters after administration of a single (escalating) dose in female cynomolgus macaques Dose (mg/kg) Route of administration t _max (hour) ^* C _max (mcg/ml) AUC _0-14d (mcg d/ml) 3 intravenously 0.25 96.0 544 3 PC 168 36.0 360 10 intravenously 0.25 325 1810 10 PC 132 101 1160 30 intravenously 1.08 1,170 6770 30 PC 132 344 4140

* t _max is presented as a mean value.

Example 10

Toxicokinetics in cynomolgus macaques - repeated dosing

Cynomolgus macaques were administered weekly doses of 10 or 100 mg/kg NOV1401 SC for 13 weeks (14 doses) or 50 mg/kg NOV1401 IV for 4 weeks (5 doses). NOV1401-treated animals were exposed to NOV1401 during the dosing phase of the study; control animals were unexposed. No sex-related differences in plasma exposure to total NOV1401 were observed. The increase in exposure (both C _max and AUC _0-7d ) was dose-dependent in both males and females (Table 10). Anti-NOV1401 antibodies were detected in 5 of 6 animals in the 10 mg/kg/week SC group, 1 of 10 animals in the 100 mg/kg/week SC group, and 1 of 6 animals in the 50 mg/kg/week IV group. Total NOV1401 exposure was not abnormal in any of the SC groups. Only one anti-drug antibody (ADA)-positive animal had an AUC _0-7d value on study day 22, which was lower than that of other animals in the same group (50 mg/kg/week IV). This animal had no effect on aPTT prolongation and no toxicity was observed.

Table 10 : Mean toxicokinetic parameters for the penultimate dose (study day 85 for the s.c. groups, study day 22 for the i.v. group) in the 13-week/4-week GLP toxicity study in cynomolgus monkeys (males and females combined) Dose (mg/kg/week) Route of administration t _max (hour) ^* C _max (mcg/ml) AUC _0-14d (mcg d/ml) 10 PC 24-120 719 3100 100 PC 72-120 5630 23400 50 intravenously 0.25-96 1990 10700

* t _max is presented as the range of observed values.

Example 11

Study of the dose escalation effect in humans

Human studies are being conducted to evaluate the safety and tolerability of anti-FXI/FXIa antibodies, such as NOV1401, following a single dose in healthy subjects. A total of approximately 60 healthy men and postmenopausal/surgically sterilized women, aged 18-55 years, are participating in this study. Good health is determined based on medical history, physical examination, neurological examination, vital signs, electrocardiogram (ECG), and laboratory test results at screening. Eligible subjects have a body weight of at least 50 kg and a body mass index (BMI) of 18-35 kg/ ^m2 . BMI = body weight (kg)/[height (m)] ² .

Six subcutaneous dose levels of 5, 15, 50, 150, 300, and 600 mg will be tested in a human study, provided that the predicted mean duration of aPTT prolongation ≥ 2-fold is not maintained for ≥ 42 days at any dose tested. Two interim analyses (IAs) will be conducted to confirm dose selection at the two highest dose levels. If the model-predicted mean duration of aPTT prolongation is ≥ 2-fold for more than 42 days at the 300 mg or 600 mg dose, the dose may be reduced, for example, based on a simulation model, to ensure that the mean duration of aPTT prolongation does not exceed 2-fold for ≥ 42 days. Non-limiting illustrative dose adjustments may include dose reductions using dose reduction steps of 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg.

The first three dose escalation steps are performed in increments of ≈1/2 log. The last two dose escalation steps are ≤2-fold increments to reduce the risk of prolonged target saturation and increase aPTT prolongation.

The maximum duration of a 2-fold prolongation of aPTT for a given number of days (e.g., 30 days, 35 days, 40 days, 42 days, etc.) with FXI-targeted therapy can be estimated based on genetic data demonstrating a moderate bleeding phenotype in patients with severe FXI deficiency, data from patients with FXI deficiency with an acquired inhibitor, and results from human studies such as FXI-ASO (see, e.g., Liu et al. , (2011) “ISIS-FXIRx, a novel and specific antisense inhibitor of factor XI, caused significant reduction in FXI antigen and activity and increased aPTT without causing bleeding in healthy volunteers.” Presented at the 53rd Annual Meeting and Exhibition of the American Society of Hematology, San Diego, California. Blood; 118: Abstract 209) when multiple doses of FXI-ASO are administered over 6 weeks resulted in a strong and sustained depletion of FXI for >6 weeks (42 days) without bleeding episodes. In specific embodiments, model analysis predicts that a maximum aPTT prolongation of ≈2.7-fold (relative to the pre-dose state) can be transiently achieved with a 50 mg subcutaneous dose of NOV1401 (in a 60 kg subject). In specific embodiments, higher doses are predicted to result in a 2.7-fold increase in the duration of this maximum aPTT prolongation.

Subjects will be monitored throughout the study for safety parameters and/or study endpoints, such as physical examination, neurological examination, vital signs, electrocardiographic examination (ECG), laboratory tests, and adverse events (AEs), including serious AEs (SAEs), up to and including day 106 post-dose.

The effect of anti-FXI/FXIa antibodies (e.g., NOV1401) on aPTT is assessed based on the relative change from baseline. Total plasma concentrations of anti-FXI/FXIa antibodies (e.g., NOV1401) are measured to assess single-dose PK in these subjects.

To assess the immunogenicity (IG) of anti-FXI/FXIa antibodies (eg, NOV1401), screening and confirmation for ADA are performed.

Free and total FXI levels, as well as FXI coagulation activity (FXI:C), are measured to assess the effects of anti-FXI/FXIa antibodies (e.g., NOV1401) on target binding and target-related PD parameters.

D-dimer, prothrombin fragments 1,2 (F1,2), and prothrombin-antithrombin complex (TAT) levels are assessed to determine the effects of anti-FXI/FXIa antibodies (eg, NOV1401) on thrombogenesis parameters.

To study the effects of anti-FXI/FXIa antibodies (e.g., NOV1401) on other coagulation parameters in subjects, the following parameters can be assessed: prothrombin time (PT), thrombin time (TT), and diagnostic laboratory parameters of coagulation such as thrombin-activated fibrinolysis inhibitor, fibrinogen, tissue plasminogen activator (tPA), and AOT.

Biomarkers studied may include, but are not limited to: D-dimer, FXI activity, PT/INR, TB, F1,2, fibrinogen, AOT, ATIF activity, TAT, PAI-1 antigen, IPTF activity, tPA activity, and ffW activity.

Inclusion by reference

All literature cited in this document, including patents, patent applications, articles, publications, manuals, etc., and references cited therein, even if not specifically mentioned, are hereby incorporated by reference in their entirety.

Equivalents

The foregoing written specification is deemed sufficient to enable one skilled in the art to practice the present invention. The foregoing specification and examples describe in detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. However, it should be understood that, no matter how thoroughly described, the invention may be practiced in a variety of ways, and the invention should be construed in accordance with the appended claims and any equivalents thereto.

--->

SEQUENCE LIST

<110> NOVARTIS AG

<120> ANTIBODIES TO FACTOR XI AND METHODS OF THEIR USE

<130> PAT056955-WO-PCT

<140>

<141>

<150> 62/341,568

<151> 2016-05-25

<150> 62/184,955

<151> 2015-06-26

<160> 51

<170> PatentIn version 3.5

<210> 1

<211> 625

<212> PROTEIN

<213> Homo sapiens

<400> 1

Met Ile Phe Leu Tyr Gln Val Val His Phe Ile Leu Phe Thr Ser Val

1 5 10 15

Ser Gly Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly

20 25 30

Gly Asp Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val

35 40 45

Val Cys Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu

50 55 60

Ser Pro Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp

65 70 75 80

Ser Val Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser

85 90 95

Gly Tyr Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys Asn Lys

100 105 110

Asp Ile Tyr Val Asp Leu Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser

115 120 125

Val Ala Lys Ser Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val

130 135 140

His Cys His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu

145 150 155 160

His Arg Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr

165 170 175

Arg Ile Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser

180 185 190

Cys Ala Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr

195 200 205

Val Phe Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe

210 215 220

Val Ser Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe Phe Thr

225 230 235 240

Phe Phe Ser Gln Glu Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu

245 250 255

Leu Lys Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg Ile Lys Lys Ser

260 265 270

Lys Ala Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro

275 280 285

Val Phe Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu

290 295 300

Glu Leu Asp Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu

305 310 315 320

Cys Thr Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln

325 330 335

Ala Ser Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser

340 345 350

Asn Gly Ser Pro Thr Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly

355 360 365

Tyr Thr Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile

370 375 380

Lys Pro Arg Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro

385 390 395 400

Trp Gln Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys

405 410 415

Gly Gly Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys

420 425 430

Phe Tyr Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile

435 440 445

Leu Asn Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln

450 455 460

Glu Ile Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp

465 470 475 480

Ile Ala Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln

485 490 495

Arg Pro Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr

500 505 510

Asp Cys Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile

515 520 525

Gln Asn Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu

530 535 540

Cys Gln Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys

545 550 555 560

Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly

565 570 575

Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile

580 585 590

Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr

595 600 605

Thr Asn Val Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala

610 615 620

Val

625

<210> 2

<211> 3278

<212> DNA

<213> Homo sapiens

<400> 2

aggcacacag gcaaaatcaa gttctacatc tgtccctgtg tatgtcactt gtttgaatac 60

gaaataaaat taaaaaaata aattcagtgt attgagaaag caagcaattc tctcaaggta 120

tatttctgac atactaagat tttaacgact ttcacaaata tgctgtactg agagagaatg 180

ttacataaca ttgagaacta gtacaagtaa atattaaagt gaagtgacca tttcctacac 240

aagctcattc agaggaggat gaagaccatt ttggaggaag aaaagcaccc ttattaagaa 300

ttgcagcaag taagccaaca aggtcttttc aggatgattt tcttatatca agtggtacat 360

ttcattttat ttacttcagt ttctggtgaa tgtgtgactc agttgttgaa ggacacctgc 420

tttgaaggag gggacattac tacggtcttc acaccaagcg ccaagtactg ccaggtagtc 480

tgcacttacc acccaagatg tttactcttc actttcacgg cggaatcacc atctgaggat 540

cccacccgat ggtttacttg tgtcctgaaa gacagtgtta cagaaacact gccaagagtg 600

aataggacag cagcgatttc tgggtattct ttcaagcaat gctcacacca aataagcgct 660

tgcaacaaag acatttatgt ggacctagac atgaagggca taaactataa cagctcagtt 720

gccaagagtg ctcaagaatg ccaagaaaga tgcacggatg acgtccactg ccactttttc 780

acgtacgcca caaggcagtt tcccagcctg gagcatcgta acatttgtct actgaagcac 840

acccaaacag ggacaccaac cagaataacg aagctcgata aagtggtgtc tggattttca 900

ctgaaatcct gtgcactttc taatctggct tgtattaggg acattttccc taatacggtg 960

tttgcagaca gcaacatcga cagtgtcatg gctcccgatg cttttgtctg tggccgaatc 1020

tgcactcatc atcccggttg cttgtttttt accttctttt cccaggaatg gcccaaagaa 1080

tctcaaagaa atctttgtct ccttaaaaca tctgagagtg gattgcccag tacacgcatt 1140

aaaaagagca aagctctttc tggtttcagt ctacaaagct gcaggcacag catcccagtg 1200

ttctgccatt cttcatttta ccatgacact gatttcttgg gagaagaact ggatattgtt 1260

gctgcaaaaa gtcacgaggc ctgccagaaa ctgtgcacca atgccgtccg ctgccagttt 1320

tttacctata ccccagccca agcatcctgc aacgaaggga agggcaagtg ttacttaaag 1380

ctttcttcaa acggatctcc aactaaaata cttcacggga gaggaggcat ctctggatac 1440

acattaaggt tgtgtaaaat ggataatgag tgtaccacca aaatcaagcc caggatcgtt 1500

ggaggaactg cgtctgttcg tggtgagtgg ccgtggcagg tgaccctgca cacaacctca 1560

cccactcaga gacacctgtg tggaggctcc atcattggaa accagtggat attaacagcc 1620

gctcactgtt tctatggggt agagtcacct aagattttgc gtgtctacag tggcatttta 1680

aatcaatctg aaataaaaga ggacacatct ttctttgggg ttcaagaaat aataatccat 1740

gatcagtata aaatggcaga aagcgggtat gatattgcct tgttgaaact ggaaaccaca 1800

gtgaattaca cagattctca acgacccata tgcctgcctt ccaaaggaga tagaaatgta 1860

atatacactg attgctgggt gactggatgg gggtacagaa aactaagaga caaaatacaa 1920

aatactctcc agaaagccaa gataccctta gtgaccaacg aagagtgcca gaagagatac 1980

agaggacata aaataaccca taagatgatc tgtgccggct acagggaagg agggaaggac 2040

gcttgcaagg gagattcggg aggccctctg tcctgcaaac acaatgaggt ctggcatctg 2100

gtaggcatca cgagctgggg cgaaggctgt gctcaaaggg agcggccagg tgtttacacc 2160

aacgtggtcg agtacgtgga ctggattctg gagaaaactc aagcagtgtg aatgggttcc 2220

caggggccat tggagtccct gaaggaccca ggatttgctg ggagaggggtg ttgagttcac 2280

tgtgccagca tgcttcctcc acagtaacac gctgaagggg cttggtgttt gtaagaaaat 2340

gctagaagaa aacaaactgt cacaagttgt tatgtccaaa actcccgttc tatgatcgtt 2400

gtagtttgtt tgagcattca gtctctttgt ttttgatcac gcttctatgg agtccaagaa 2460

ttaccataag gcaatatttc tgaagattac tatataggca gatatagcag aaaataacca 2520

agtagtggca gtggggatca ggcagaagaa ctggtaaaag aagccaccat aaatagattt 2580

gttcgatgaa agatgaaaac tggaagaaag gagaacaaag acagtcttca ccattttgca 2640

ggaatctaca ctctgcctat gtgaacacat ttcttttgta aagaaagaaa ttgattgcat 2700

ttaatggcag attttcagaa tagtcaggaa ttcttgtcat ttccatttta aaatatatat 2760

taaaaaaaat cagttcgagt agacacgagc taagagtgaa tgtgaagata acagaatttc 2820

tgtgtggaag aggattacaa gcagcaattt acctggaagt gataccttag gggcaatctt 2880

gaagatacac tttcctgaaa aatgatttgt gatggattgt atatttattt aaaatatctt 2940

gggaggggag gctgatggag atagggagca tgctcaaacc tccctaagac aagctgctgc 3000

tgtgactatg ggctcccaaa gagctagatc gtatatttat ttgacaaaaa tcaccataga 3060

ctgcatccat actacagaga aaaaacaatt agggcgcaaa tggatagtta cagtaaagtc 3120

ttcagcaagc agctgcctgt attctaagca ctgggatttt ctgtttcgtg caaatattta 3180

tctcattatt gttgtgatct agttcaataa cctagaattt gaattgtcac cacatagctt 3240

tcaatctgtg ccaacaacta tacaattcat caagtgtg 3278

<210> 3

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 3

Thr Ala Ala Met Ser

1 5

<210> 4

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 4

Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 5

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 5

Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 6

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 6

Gly Phe Thr Phe Ser Thr Ala

1 5

<210> 7

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 7

Ser Gly Ser Gly Ser Ser

1 5

<210> 8

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 8

Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 9

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 9

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 10

caggtgcaat tgctggaaag cggcggtggc ctggtgcagc cgggtggcag cctgcgtctg 60

agctgcgcgg cgtccggatt caccttttct actgctgcta tgtcttgggt gcgccaggcc 120

ccgggcaaag gtctcgagtg ggtttccggt atctctggtt ctggttcttc tacctactat 180

gcggatagcg tgaaaggccg ctttaccatc agccgcgata attcgaaaaa caccctgtat 240

ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtgaactg 300

tcttacctgt actctggtta ctacttcgat tactggggcc aaggcaccct ggtgactgtt 360

agctca 366

<210> 11

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 11

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 12

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 12

caggtgcaat tgctggaaag cggcggtggc ctggtgcagc cgggtggcag cctgcgtctg 60

agctgcgcgg cgtccggatt caccttttct actgctgcta tgtcttgggt gcgccaggcc 120

ccgggcaaag gtctcgagtg ggtttccggt atctctggtt ctggttcttc tacctactat 180

gcggatagcg tgaaaggccg ctttaccatc agccgcgata attcgaaaaa caccctgtat 240

ctgcaaatga acagcctgcg tgcggaagat acggccgtgt attattgcgc gcgtgaactg 300

tcttacctgt actctggtta ctacttcgat tactggggcc aaggcaccct ggtgactgtt 360

agctcagcct ccaccaaggg tccatcggtc ttccccctgg caccctcctc caagagcacc 420

tctggggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 480

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 540

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 600

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt 660

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaagcagcg 720

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 780

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 840

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggagggagcag 900

tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 960

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1020

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1080

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1140

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1200

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1260

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320

tacacgcaga agagcctctc cctgtctccg ggtaaa 1356

<210> 13

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 13

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Ser

1 5 10

<210> 14

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 14

Lys Asn Tyr Asn Arg Pro Ser

1 5

<210> 15

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 15

Ser Ala Trp Asp Gln Arg Gln Phe Asp Val Val

1 5 10

<210> 16

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 16

Ser Ser Ser Asn Ile Gly Ser Asn Asp

1 5

<210> 17

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 17

Lys Asn Tyr

1

<210> 18

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 18

Trp Asp Gln Arg Gln Phe Asp Val

1 5

<210> 19

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 19

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Lys Asn Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Gln Arg Gln

85 90 95

Phe Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 20

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 20

gatatcgtgc tgacccagcc gccgagcgtg agcggtgcac cgggccagcg cgtgaccatt 60

agctgtagcg gcagcagcag caacattggt tctaacgacg tgtcttggta ccagcagctg 120

ccgggcacgg cgccgaaact gctgatctac aaaaactaca accgcccgag cggcgtgccg 180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa 240

gcagaagacg aagcggatta ttactgctct gcttgggacc agcgtcagtt cgacgttgtg 300

tttggcggcg gcacgaagtt aaccgtccta 330

<210> 21

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 21

Asp Ile Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Lys Asn Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Gln Arg Gln

85 90 95

Phe Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 22

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 22

gatatcgtgc tgacccagcc gccgagcgtg agcggtgcac cgggccagcg cgtgaccatt 60

agctgtagcg gcagcagcag caacattggt tctaacgacg tgtcttggta ccagcagctg 120

ccgggcacgg cgccgaaact gctgatctac aaaaactaca accgcccgag cggcgtgccg 180

gatcgcttta gcggatccaa aagcggcacc agcgccagcc tggcgattac cggcctgcaa 240

gcagaagacg aagcggatta ttactgctct gcttgggacc agcgtcagtt cgacgttgtg 300

tttggcggcg gcacgaagtt aaccgtccta ggtcagccca aggctgcccc ctcggtcact 360

ctgttcccgc cctcctctga ggagcttcaa gccaacaagg cccacactggt gtgtctcata 420

agtgacttct acccgggagc cgtgacagtg gcctggaagg cagatagcag ccccgtcaag 480

gcgggagtgg agaccaccac accctccaaa caaagcaaca acaagtacgc ggccagcagc 540

tatctgagcc tgacgcctga gcagtggaag tcccacagaa gctacagctg ccaggtcacg 600

catgaaggga gcaccgtgga gaagacagtg gcccctacag aatgttca 648

<210> 23

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 23

Thr Ala Ala Met Ser

1 5

<210> 24

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 24

Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 25

Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 26

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 26

Gly Phe Thr Phe Ser Thr Ala

1 5

<210> 27

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 27

Ser Gly Ser Gly Ser Ser

1 5

<210> 28

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 28

Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr

1 5 10

<210> 29

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 29

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 30

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 30

caggtgcagc tgctggaatc aggcggcgga ctggtgcagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt cacctttagc accgccgcta tgagctgggt tcgacaggcc 120

ccagggaaag gcctcgagtg ggtctcaggg attagcggta gcggctctag cacctactac 180

gccgatagcg tgaagggccg gttcactatc tctagggata actctaagaa caccctgtac 240

ctgcagatga atagcctgag agccgaggac accgccgtct actactgcgc tagagagctg 300

agctacctgt atagcggcta ctacttcgac tactggggtc aaggcaccct ggtcaccgtg 360

tctagc 366

<210> 31

<211> 452

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 31

Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Ala

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Gly Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Ala Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 32

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 32

caggtgcagc tgctggaatc aggcggcgga ctggtgcagc ctggcggtag cctgagactg 60

agctgcgctg ctagtggctt cacctttagc accgccgcta tgagctgggt tcgacaggcc 120

ccagggaaag gcctcgagtg ggtctcaggg attagcggta gcggctctag cacctactac 180

gccgatagcg tgaagggccg gttcactatc tctagggata actctaagaa caccctgtac 240

ctgcagatga atagcctgag agccgaggac accgccgtct actactgcgc tagagagctg 300

agctacctgt atagcggcta ctacttcgac tactggggtc aaggcaccct ggtcaccgtg 360

tctagcgcta gcactaaggg cccctccgtg ttccctctgg ccccttccag caagtctacc 420

tccggcggca cagctgctct gggctgcctg gtcaaggact acttccctga gcctgtgaca 480

gtgtcctgga actctggcgc cctgacctct ggcgtgcaca ccttccctgc cgtgctgcag 540

tcctccggcc tgtactccct gtcctccgtg gtcacagtgc cttcaagcag cctgggcacc 600

cagacctata tctgcaacgt gaaccacaag ccttccaaca ccaaggtgga caagcgggtg 660

gagcctaagt cctgcgacaa gacccacacc tgtcctccct gccctgctcc tgaactgctg 720

ggcggccctt ctgtgttcct gttccctcca aagcccaagg acaccctgat gatctcccgg 780

acccctgaag tgacctgcgt ggtggtggcc gtgtcccacg aggatcctga agtgaagttc 840

aattggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcctcg ggaggaacag 900

tacaactcca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960

ggcaaagagt acaagtgcaa agtctccaac aaggccctgg ccgcccctat cgaaaagaca 1020

atctccaagg ccaagggcca gcctagggaa ccccaggtgt acaccctgcc acccagccgg 1080

gaggaaatga ccaagaacca ggtgtccctg acctgtctgg tcaagggctt ctacccttcc 1140

gatatcgccg tggagtggga gtctaacggc cagcctgaga acaactacaa gaccacccct 1200

cctgtgctgg actccgacgg ctccttcttc ctgtactcca aactgaccgt ggacaagtcc 1260

cggtggcagc agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaaccac 1320

tacacccaga agtccctgtc cctgtctccc ggcaag 1356

<210> 33

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 33

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Asp Val Ser

1 5 10

<210> 34

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 34

Lys Asn Tyr Asn Arg Pro Ser

1 5

<210> 35

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 35

Ser Ala Trp Asp Gln Arg Gln Phe Asp Val Val

1 5 10

<210> 36

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 36

Ser Ser Ser Asn Ile Gly Ser Asn Asp

1 5

<210> 37

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 37

Lys Asn Tyr

1

<210> 38

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 38

Trp Asp Gln Arg Gln Phe Asp Val

1 5

<210> 39

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 39

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Lys Asn Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Gln Arg Gln

85 90 95

Phe Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 40

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 40

cagtcagtcc tgactcagcc ccctagcgct agtggcaccc ctggtcaaag agtgactatt 60

agctgtagcg gctctagctc taatatcggc tctaacgacg tcagctggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat aagaactata ataggcctag cggcgtgccc 180

gataggttta gcggatctaa atcagggact tctgctagtc tggctattag cggcctgcag 240

tcagaggacg aggccgacta ctactgtagc gcctgggatc agcgtcagtt cgacgtggtg 300

ttcggcggag gcactaagct gaccgtgctg 330

<210> 41

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 41

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Lys Asn Tyr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Gln Arg Gln

85 90 95

Phe Asp Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 42

<211> 648

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polynucleotide"

<400> 42

cagtcagtcc tgactcagcc ccctagcgct agtggcaccc ctggtcaaag agtgactatt 60

agctgtagcg gctctagctc taatatcggc tctaacgacg tcagctggta tcagcagctg 120

cccggcaccg cccctaagct gctgatctat aagaactata ataggcctag cggcgtgccc 180

gataggttta gcggatctaa atcagggact tctgctagtc tggctattag cggcctgcag 240

tcagaggacg aggccgacta ctactgtagc gcctgggatc agcgtcagtt cgacgtggtg 300

ttcggcggag gcactaagct gaccgtgctg ggtcaaccta aggctgcccc cagcgtgacc 360

ctgttccccc ccagcagcga ggagctgcag gccaacaagg ccaccctggt gtgcctgatc 420

agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcag ccccgtgaag 480

gccggcgtgg agaccaccac ccccagcaag cagagcaaca acaagtacgc cgccagcagc 540

tacctgagcc tgacccccga gcagtggaag agccacaggt cctacagctg ccaggtgacc 600

cacgaggca gcaccgtgga aaagaccgtg gccccaaccg agtgcagc 648

<210> 43

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 43

Gly Phe Thr Phe Ser Thr Ala Ala

1 5

<210> 44

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 44

Ile Ser Gly Ser Gly Ser Ser Thr

1 5

<210> 45

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 45

Ala Arg Glu Leu Ser Tyr Leu Tyr Ser Gly Tyr Tyr Phe Asp Tyr

1 5 10 15

<210> 46

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 46

Gly Phe Thr Phe Ser Thr Ala Ala Met Ser

1 5 10

<210> 47

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 47

Ser Ser Asn Ile Gly Ser Asn Asp

1 5

<210> 48

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

6xHis tag"

<400> 48

His His His His His

1 5

<210> 49

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

peptide"

<400> 49

Met Gly Ser Ser

1

<210> 50

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

8xHis tag"

<400> 50

His His His His His His His

1 5

<210> 51

<211> 238

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence: Synthetic

polypeptide"

<400> 51

Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro Trp Gln Val

1 5 10 15

Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys Gly Gly Ser

20 25 30

Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys Phe Tyr Gly

35 40 45

Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile Leu Asn Gln

50 55 60

Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln Glu Ile Ile

65 70 75 80

Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp Ile Ala Leu

85 90 95

Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln Arg Pro Ile

100 105 110

Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr Asp Cys Trp

115 120 125

Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile Gln Asn Thr

130 135 140

Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu Cys Gln Lys

145 150 155 160

Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys Ala Gly Tyr

165 170 175

Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu

180 185 190

Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile Thr Ser Trp

195 200 205

Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr Thr Asn Val

210 215 220

Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala Val

225 230 235

2

1

<---

Claims (37)

1. A method for treating a thromboembolic disease, comprising administering to a subject suffering from a thromboembolic disease or at risk of developing it, an effective amount of a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment comprises (i) a light chain variable region (VL) comprising the complementarity-determining regions LCDR1, LCDR2, LCDR3 of SEQ ID NO: 39, and (ii) a heavy chain variable region (VH) comprising the complementarity-determining regions HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 29, wherein the antibody or antigen-binding fragment thereof is administered to the subject at a dose in the range of 5 to 600 mg. 2. The method according to claim 1, wherein the antibody or antigen-binding fragment comprises: i. CDR1 of the variable heavy chain SEQ ID NO: 23; CDR2 of the variable heavy chain SEQ ID NO: 24; CDR3 of the variable heavy chain SEQ ID NO: 25; CDR1 of the variable light chain SEQ ID NO: 33; CDR2 of the variable light chain SEQ ID NO: 34; and CDR3 of the variable light chain SEQ ID NO: 35; ii. CDR1 of the variable heavy chain SEQ ID NO: 26; CDR2 of the variable heavy chain SEQ ID NO: 27; CDR3 of the variable heavy chain SEQ ID NO: 28; CDR1 of the variable light chain SEQ ID NO: 36; CDR2 of the variable light chain SEQ ID NO: 37; and CDR3 of the variable light chain SEQ ID NO: 38; iii. The variable heavy chain CDR1 of SEQ ID NO: 43; the variable heavy chain CDR2 of SEQ ID NO: 44; the variable heavy chain CDR3 of SEQ ID NO: 45; the variable light chain CDR1 of SEQ ID NO: 47; the variable light chain CDR2 of SEQ ID NO: 37; and the variable light chain CDR3 of SEQ ID NO: 15; or iv. CDR1 of the variable heavy chain SEQ ID NO: 46; CDR2 of the variable heavy chain SEQ ID NO: 4; CDR3 of the variable heavy chain SEQ ID NO: 5; CDR1 of the variable light chain SEQ ID NO: 33; CDR2 of the variable light chain SEQ ID NO: 14; and CDR3 of the variable light chain SEQ ID NO: 15. 3. The method according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises: (i) the heavy chain variable region sequence of SEQ ID NO: 9 and the light chain variable region sequence of SEQ ID NO: 19; or (ii) the heavy chain variable region sequence of SEQ ID NO: 29 and the light chain variable region sequence of SEQ ID NO: 39; or wherein the antibody comprises: (iii) the heavy chain variable region sequence of SEQ ID NO: 31 and the light chain variable region sequence of SEQ ID NO: 41; or (iv) the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 21. 4. The method according to claim 3, wherein the antibody comprises the substitutions D265A and P329A in the Fc domain. 5. The method of any one of claims 1-4, wherein the subject suffers from or is at risk of developing a thromboembolic disease selected from the group consisting of: venous thromboembolism, paroxysmal atrial fibrillation or atrial flutter, stroke associated with atrial fibrillation or atrial flutter, deep vein thrombosis (DVT), cancer, transient ischemic attack, severe protein S deficiency, thromboembolic stroke, ischemic stroke, systemic embolism, and myocardial infarction. 6. The method of any one of claims 1-5, wherein the subject suffers from one or more of stroke associated with atrial fibrillation and deep vein thrombosis. 7. The method according to any one of claims 1-6, wherein the subject suffers from a stroke associated with atrial fibrillation or atrial flutter. 8. The method according to claim 7, wherein the atrial fibrillation or atrial flutter is paroxysmal atrial fibrillation (PAF). 9. The method according to any one of claims 1-5, wherein the subject suffers from venous thromboembolism or is at risk of developing it. 10. The method according to any one of claims 1-6, wherein the subject suffers from deep vein thrombosis (DVT) or is at risk of developing it. 11. The method of claim 9 or 10, wherein the method further comprises measuring the effectiveness based on the incidence of DVT. 12. The method according to any one of paragraphs 1-11, wherein the subject has a high risk of bleeding. 13. The method according to any one of paragraphs 1-5 or 9-12, wherein the subject undergoes a medical procedure selected from the group consisting of knee replacement surgery, hip replacement surgery, orthopedic surgery, pacemaker placement, catheter insertion, thoracic surgery, and abdominal surgery. 14. The method of claim 13, wherein the medical procedure is knee replacement surgery. 15. The method of any one of claims 1-8, wherein the subject at risk has one or more of hypertension, congestive heart failure, left ventricular hypertrophy, and diabetes mellitus. 16. The method according to any one of claims 1-15, wherein the method further comprises assessing the efficacy of the pharmaceutical composition based on one or more biomarkers selected from the group consisting of free factor XI, total factor XI, factor XI coagulation activity, activated partial thromboplastin time, and D-dimer. 17. The method of any one of claims 1-16, further comprising assessing adverse events with respect to the pharmaceutical composition by measuring the incidence of bleeding and/or the presence of anti-drug antibodies. 18. The method of claim 17, further comprising administering one or more of the following to a patient experiencing an adverse event, wherein the adverse event is a bleeding event: (i) infusion therapy using colloids, crystalloids, human plasma, or plasma proteins such as albumin; (ii) transfusion of packed red blood cells or whole blood; or (iii) administration of fresh frozen plasma (FFP), prothrombin complex concentrates (PCC), activated PCCs (aPCC) such as a factor VIII inhibitor and/or recombinant activated factor VII. 19. The method of any one of claims 1-18, wherein the method further comprises administering to a subject suffering from or at risk of developing a thromboembolic disease an effective amount of statin therapy. 20. The method according to any one of claims 1-19, wherein the subject is administered a dose of about 150 mg of the antibody or antigen-binding fragment thereof. 21. The method according to any one of claims 1-20, wherein the antibody or antigen-binding fragment thereof is administered to the subject subcutaneously or intravenously. 22. The method according to any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof is administered to the subject once a month. 23. The method according to any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof is administered to the subject in a pharmaceutical composition further comprising sucrose, polysorbate, L-histidine and histidine HCl monohydrate. 24. The method of claim 23, wherein the pharmaceutical composition has a pH of about 5.5. 25. The method according to any one of claims 1-24, wherein the antibody is an anti-factor XI and/or anti-activated factor XI (factor XIa) antibody. 26. Use of an antibody or antigen-binding fragment thereof comprising (i) a light chain variable region (VL) comprising the complementarity determining regions LCDR1, LCDR2, LCDR3 of SEQ ID NO: 39, and (ii) a heavy chain variable region (VH) comprising the complementarity determining regions HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 29, for the manufacture of a medicament for the treatment of a thromboembolic disease in a subject in need thereof, wherein the antibody or antigen-binding fragment thereof is administered to the subject at a dose in the range of 5 to 600 mg. 27. The use according to claim 26, wherein the antibody or antigen-binding fragment thereof comprises: i. CDR1 of the variable heavy chain SEQ ID NO: 23; CDR2 of the variable heavy chain SEQ ID NO: 24; CDR3 of the variable heavy chain SEQ ID NO: 25; CDR1 of the variable light chain SEQ ID NO: 33; CDR2 of the variable light chain SEQ ID NO: 34; and CDR3 of the variable light chain SEQ ID NO: 35; ii. CDR1 of the variable heavy chain SEQ ID NO: 26; CDR2 of the variable heavy chain SEQ ID NO: 27; CDR3 of the variable heavy chain SEQ ID NO: 28; CDR1 of the variable light chain SEQ ID NO: 36; CDR2 of the variable light chain SEQ ID NO: 37; and CDR3 of the variable light chain SEQ ID NO: 38; iii. The variable heavy chain CDR1 of SEQ ID NO: 43; the variable heavy chain CDR2 of SEQ ID NO: 44; the variable heavy chain CDR3 of SEQ ID NO: 45; the variable light chain CDR1 of SEQ ID NO: 47; the variable light chain CDR2 of SEQ ID NO: 37; and the variable light chain CDR3 of SEQ ID NO: 15; or iv. CDR1 of the variable heavy chain SEQ ID NO: 46; CDR2 of the variable heavy chain SEQ ID NO: 4; CDR3 of the variable heavy chain SEQ ID NO: 5; CDR1 of the variable light chain SEQ ID NO: 33; CDR2 of the variable light chain SEQ ID NO: 14; and CDR3 of the variable light chain SEQ ID NO: 15. 28. The use according to claim 26 or 27, wherein the antibody is an anti-factor XI and/or anti-activated factor XI (factor XIa) antibody.