Factor XI Antibodies and Methods of Use

Abstract

To provide monoclonal antibodies and antigen binding fragments thereof that bind to human Factor XI and activated Factor XI ("Factor XIa"), as well as to provide pharmaceutical compositions comprising the same and treatment methods.SOLUTION: The antibodies (e.g., human monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies) presented herein bind with similarly high affinity to the catalytic domain (CD) of human FXIa and FXI and induce an inactive protease domain conformation in FXIa.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application is filed on 20 U.S. Provisional Application No. 62 / 184,955, filed June 26, 2015; and Benefit of U.S. Provisional Application No. 62 / 341,568, filed May 25, 2016 claims.

[0002] This application has been filed electronically in ASCII format and is incorporated by reference in its entirety. The AS created on June 23, 2016 contains a sequence listing incorporated herein. The CII copy is named "PAT056955-WO-PCT_SL.txt" and is 45,685 bytes long. It is.

[0003] background Thrombosis is a condition that is caused by inherited risk factors known as thrombophilia or hypercoagulable states, as well as acquired risk factors. It refers to the formation of a blood clot inside a blood vessel following a combination of risk factors. Increased platelet reactivity and activation of clotting factors are some of the fundamental features of thrombosis. Thrombosis can occur in both the venous and arterial circulation and can occur in the deep venous system. This can result in deep vein thrombosis (DVT), pulmonary embolism, and stroke. If a thrombus occurs in the artery, ischemia occurs downstream, leading to acute coronary syndrome (ACS), ischemic stroke, and other conditions. Thrombus formation within the venous system can result in hemorrhagic stroke, acute limb ischemia, and thrombus formation in the deep veins. This typically leads to thrombosis, pulmonary embolism, and chronic thromboembolic pulmonary hypertension. Blood may also form in the left atrial appendage of patients with atrial fibrillation (AF) and become dislodged. The resulting thrombus can lead to potentially serious complications, namely thromboembolic stroke and systemic embolism. Thrombosis may result. Low molecular weight heparin (LMWH), thrombin inhibitor All currently available antithrombotic drugs, including factor Xa (FXa) inhibitors, It is associated with a significant risk of bleeding (Weitz JI (2010) Thromb. Haemost. 103, 62). The development of antithrombotic agents that do not affect the blood flow and therefore do not result in bleeding complications is essential. , is highly desired.

[0004] Current anticoagulants are either injected or taken orally. is widely used and offers a therapeutic profile superior to that of previously applied unfractionated heparin. Over the past few decades, the most commonly used oral anticoagulants have been: Warfarin has a narrow therapeutic window, which means that it is difficult to treat coagulation. They require frequent monitoring and exhibit a variety of drug-drug interactions. Orally available direct FXa and thrombin inhibitors enter the anticoagulant market, Its application is increasing.

[0005] LMWH, FXa inhibitors, and thrombin inhibitors all contribute to postoperative intravenous Prevention of thromboembolic disease, treatment of spontaneous DVT and pulmonary embolism, and treatment of atrial fibrillation However, these anticoagulants also have the potential to reduce the risk of stroke, which is caused by older drugs such as Bleeding complications generally comparable to those observed with flufenaconazole and unfractionated heparin In the ADVANCE-2 clinical trial, the FXa inhibitor apixaban ( Eliquis) has evaluated the efficacy of the LMWH enoxaparin in patients undergoing total knee replacement surgery. Acute apixaban therapy was compared with enoki mushroom in preventing venous thromboembolic disease. Although more effective than saparin, both drugs were associated with a significant risk of bleeding. Patient-related bleeding occurred in 4% of patients receiving apixaban and 4% of patients receiving enoxaparin. It occurred in 5% of patients treated with (Lassen, MR, et al. (2009) N. Engl. J. Med. 361, 594).

[0006] The RE-LY trial evaluated the direct thrombin inhibitor dabigatran (Prada xa) compared with warfarin in patients with atrial fibrillation and risk of stroke. (Connolly, SJ, et al. (2009) N. Engl. J. Med. 361, 1139). Therapy was associated with a significant reduction in the risk of stroke or systemic embolism. This complication occurred in 3.1% of patients receiving 150 mg dabigatran daily and 3.1% of patients receiving 150 mg dabigatran daily. It occurred in 3.4% of patients receiving flufenaconazole (p=0.31).

[0007] Atrial fibrillation (AF) remains the most common cardiac arrhythmia in clinical practice. AF accounts for approximately one-third of all hospitalizations for rhythm disorders. It is estimated to affect over 2.3 million patients in the United States and over 10 million in the United States. Due to the increasing proportion of the aging population, this number continues to grow rapidly. It is estimated that approximately 5% of people and 10% of people over the age of 80 develop AF. However, the prevalence of AF is increasing beyond that explained by age alone. Hypertension, congestive heart failure, left ventricular hypertrophy, coronary artery disease, and diabetes, as well as obstructive sleep AF risk factors such as apnea are also on the rise. The number of individuals affected by AF is increasing. In fact, it is expected that the prevalence of HIV in Western populations will increase two to three times over the next 30 years (Kannel and Benjamin (2008) Med Clin North Am. 2008; 92:17-40; Bunch, et al. (2012) JI nnovations of Card Rhythm Manag 2012; 3: 855-63).

[0008] The major risk of AF is a 4-5 fold increase in embolic stroke. The risk of stroke increases sharply with age, rising to 23.5% for those aged 80-89. AF is associated with a doubling of mortality in both sexes (Kannel and Benjamin 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. al 2012).

[0009] Most patients with AF require approximately 10 years of therapy in their lifetime to prevent cardioembolic stroke and systemic embolism. The CHA2DS2-VASc risk score has been validated. to predict thromboembolic risk in patients with atrial fibrillation and identify patients who will benefit from anticoagulation therapy. It is a widely used stratification tool to identify individuals with pulmonary embolism (LIP 2011; Camm, et al. (2011) 12) Eur Heart J 2012; 33: 2719-2747) Accumulating evidence suggests that CHA2DS2-VASc , and scores such as CHADS2 to identify patients who will develop stroke and thromboembolism. It is at least as accurate, or perhaps even better, than these and involves AF. It is crucial to identify patients who are at "true low risk" It is estimated that 85-90% of patients with AF require anticoagulation therapy.

[0010] The efficacy of vitamin K antagonists (VKAs) in reducing stroke and systemic embolism A meta-analysis of six trials evaluating the effects of cerebrospinal fluid therapy on stroke risk was A highly significant reduction was observed (relative reduction in risk of stroke was 67%). All-cause mortality was significantly reduced with dose-adjusted VKAs compared with controls (26%) (Hart, Pearce, and Aguilar (2007) Ann Intern Med 2007;146:857-867). The INR (international normalized ratio) target value between (Hylek et al (2003) N Engl J Med; 349:1019-1026), international guidelines and national guidelines It has been widely adopted as a guideline.

[0011] Recently, new oral anticoagulants (NOAs), also called direct oral anticoagulants (DOACs), have become available. C) have been approved and introduced into clinical practice. These drugs have been shown to reduce the risk of thromboembolic disease. It is at least as effective as, or even better than, warfarin in reducing (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 reduce the most serious complications of warfarin, namely hemorrhagic stroke and cranial It was also associated with a significant reduction in internal bleeding. Major bleeding events were associated with well-administered warfarin therapy. In addition, NOACs were associated with a smaller reduction in vasopressin than warfarin. are associated with the potential for serious drug-drug interactions and should not be used without routine monitoring. This is expected to facilitate their use in routine medical practice.

[0012] Despite recent improvements, the risk of bleeding associated with anticoagulant use remains high. For example, the annual incidence of major bleeding and clinically relevant non-major bleeding is 14. 9% and in the ROCKET study, the incidence of rivaroxaban-treated patients was significantly higher. The annual incidence of major bleeding events was 3.6% (Patel et al 2011). and major bleeding in patients defined as having a HAS Bled risk score of ≥3. The annual incidence rate was >5% (Gallego, et al. (2012) Carc Arrhythm Electrophysiol. ; 5:312-318). Major bleeding is a particularly significant clinical outcome, e.g., in ROCKE In the T study, when major bleeding occurred, the all-cause mortality rate was 20% in the rivaroxaban group. The incidence of major bleeding events was 26.1% in the thrombolytic group and 0.4% in the thrombolytic group. rivaroxaban and warfarin groups, 4.7% and 5% of patients, respectively. Stroke and systemic embolism occurred in 4% of cases (Piccini, et al. (2014) Eur Heart J; 35 Hospitalization, transfusion of blood products, and utilization of blood resources also contribute to the occurrence of hemorrhage. The risk of bleeding was also significantly affected by the lack of anticoagulation in eligible patients. This is also the main reason. The Euro Heart Survey on Arthritis, including data from hospitalized AF patients In ial Fibrillation, 67% of eligible patients received oral anticoagulant therapy at discharge. They were only given a coagulant (Nieuwlaat, et al (2005) Eur Heart J;26, 2422-243 4).

[0013] Therefore, it is a treatment with efficacy comparable to existing treatments, but with reduced bleeding tendency, stroke, and total stroke. May reduce thromboembolic complications of AF, including systemic embolism, cognitive decline, and mortality Therefore, there is a large unmet medical need for safer treatments.

[0014] overview The present invention relates to human coagulation factor XI and XIa (activated factor XI) (hereinafter referred to as "activated factor XI" in some cases). Thus, monoclonal antibodies that bind to FXI (also referred to as "FXI," "FXIa," and similar terms) ANTIBODY, PHARMACEUTICAL COMPOSITION COMPRISING THE SAME, AND METHOD OF TREATMENT COMPRISING THE SAME - Patent application Thrombotic or thromboembolic diseases / disorders (e.g., thrombotic stroke, atrial fibrillation, atrial fibrillation) Prevention of stroke in fibrillation (SPAF), deep vein thrombosis, venous thromboembolism, pulmonary embolism, Acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension It is effective in preventing and treating pulmonary embolism (pulmonary embolism, systemic embolism) but does not carry the risk of bleeding. The development of antithrombotic agents that pose little or only minimal bleeding risk would be a promising solution. A significant unmet medical need will be met.

[0015] In specific embodiments, the antibodies presented herein (e.g., human monoclonal antibodies, Human FXIa and FXI (humanized monoclonal antibodies) It binds with similar high affinity to the catalytic domain (CD) of FXIa, and inactivates Induce protease domain conformation.

[0016] The isolated anti-FXI and / or anti-FXIa antibodies described herein, e.g., The full-length IgG with two binding sites described herein may bind to FXI and / or FXI a, equilibrium dissociation constant (K D ) are bonded together. For example, The isolated antibody has a specific binding affinity to human FXI and / or FXIa of 100 pM or less, 50 pM or less , 45pM or less, 40pM or less, 35pM or less, 20pM or less, or 10pM or less K D More specifically, the isolated antibodies described herein may also bind to human FXI. and / or FXIa by surface plasmon resonance (SPR), e.g., BIACORE ( or less than 34 pM as measured by the Equilibrium Titration Assay (SE) K below 4 pM as measured by T D It is also possible to combine FXI and / or FXIa in the IL-1440 cells were measured by the BIACORE™ assay. K < 53 pM measured by SET or < 4 pM measured by SET D It is also possible to combine In a specific embodiment, the isolated antibodies described herein (e.g., NOV1401) are capable of inhibiting the growth of the inflammatory bowel disease. ) in human FXI and FXIa, e.g., by equilibrium titration assay (SET). The concentrations measured are approximately 5 pM (e.g., 4.7 pM) and 2 pM (e.g., 1. 3 pM or less apparent K D In a specific embodiment, the nucleotides described herein are linked at Anti-FXI / FXIa antibodies are used to detect FXI / FXIa in cynomolgus monkeys using SET (e.g., about 12.5 (±6.6) pM for FXIa as measured by the ELISA (see Example 2) and an apparent K of approximately 5.0 (±0.7) pM D In a specific embodiment, The anti-FXI / FXIa antibodies described herein are derived from rabbit FXI and / or FXIa antibodies. with a K of approximately 20(±2)nM D In a specific embodiment, the antibodies described herein bind to FXI / FXIa antibodies are antibodies against human, cynomolgus monkey, and rabbit FXI and / or FXIa. It binds to XIa but does not specifically bind to mouse or rat FXI.

[0017] The isolated anti-FXI antigen-binding fragment and / or anti-FXIa antigen-binding fragment described herein Synthetic fragments, such as Fab fragments and other fragments containing a single binding site, can be used to bind FXI. and / or FXIa with an equilibrium dissociation constant (K D ) to join them. For example, The isolated antigen-binding fragments described herein bind to human FXI and / or FXIa. , 10nM or less, 5nM or less, 1nM or less, 500pM or less, 305pM or less, 62pM More specifically, the isolated antigen-binding fragments described herein may bind with a KD of It also binds to human FXI and / or FXIa with a KD of 305 pM or less. is also possible.

[0018] The present invention provides isolated antibodies or antibodies that bind to human, rabbit, and cynomolgus monkey FXIa. The present invention also relates to antigen-binding fragments of the catalytic domain of FXI and / or FXIa. an isolated antibody or antigen-binding fragment thereof that binds to the surface of the antibody, specifically the active site region; Also related.

[0019] The present invention also provides antibodies that bind to FXI and / or FXIa and are listed in Table 1 ( For example, an isolated antibody or its antigen-binding The present invention also relates to the antibodies and / or antigen-binding fragments thereof described herein. "Competition" refers to the ability of both antibodies (or their binding fragments) to bind to the same or overlapping FXI domains. and / or FXIa epitopes (e.g., those well known to those skilled in the art). (as determined in a competitive binding assay by any of the methods described above) As used herein, an antibody or antigen-binding fragment thereof refers to a competing antibody or antigen-binding fragment thereof. The fragment may have the same FXI epitope and / or F domain as the antibody or antigen-binding fragment of the present invention. XIa epitope or overlapping FXI and / or FXIa epitopes FXI and / or FXIa antibodies of the present invention, so long as they do not bind to the target antigen. or do not "compete" with the antigen-binding fragment (e.g., NOV1401 or NOV1090). As used herein, a competing antibody or antigen-binding fragment thereof is (i) an antibody or antigen-binding fragment of the present invention. sterically blocking (e.g., competing) the antigen-binding fragment or antigen-binding fragment from binding to its target. Antibodies bind to nearby, non-overlapping FXI and / or FXIa epitopes and physically prevent the antibody or antigen-binding fragment of the invention from binding to its target. and / or (ii) different, non-overlapping FXI epitopes and / or F XIa epitope and bind to FXI protein and / or FXIa protein. The FXI and / or FXIa antibodies or antigen-binding fragments of the invention are conformationally The conformational change is such that it can no longer bind in the form that occurs in the absence of the conformational change. The antibody does not include a competing antibody or antigen-binding fragment thereof that induces an alteration in the protein.

[0020] In one embodiment, an antibody that binds to FXI and / or FXIa and is described in Table 1 The isolated antibodies or antigen-binding fragments thereof that further compete for binding with the antibodies of Table 1 are In another embodiment, FXI and and / or bind to FXIa and further compete for binding with the antibodies listed in Table 1 The isolated antibody or antigen-binding fragment thereof binds to all of the epitopes to which the antibody in Table 1 binds. Combine.

[0021] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to active FXI (FXIa). When it binds to the active FXI (FXIa) catalytic domain, it binds to FXIa and its cofactors. In another embodiment, the conformation of the The isolated antibody or antigen-binding fragment thereof has four N-terminal amino acids in the inactive conformation. residues, loops 145, 188, and 220, are displaced relative to the active conformation. Further induces shifting and / or irregular changes.

[0022] In one embodiment, the isolated antibody or antigen-binding fragment thereof is an antibody that binds to FXI (e.g., human FXI). ), but upon binding to FXI, the FXI catalytic domain binds to loops 145, 188, and and 220 are regular in the active conformation as well as in the structure of the FXIa catalytic domain. Prevents users from taking actions.

[0023] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to FXI but does not bind to FXI. Upon binding to ATP, the FXI catalytic domain binds to four residues at the N-terminus, loops 145, 188, and and 220 are in the active conformation, which is similarly regular in the structure of the FXIa catalytic domain. Prevents theft of personal information.

[0024] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to FXI but does not bind to FXI. Upon binding to FXIa, it induces a conformational change within the zymogen structure, allowing it to bind to FXIa. The inhibitory FXI conformation closely related to the inhibitory FXI conformation observed when This further induces the FXI catalytic domain to assume an active conformation. Prevents the taking of a position.

[0025] In one embodiment, the isolated antibody or antigen-binding fragment thereof is FXI and / or FXI binds to FXI and / or FXIa but not to FXI and / or FXIa When the antibody:antigen complex is formed, the catalytic domain of a 20, compared to the uncomplexed structure of the catalytic domain of active factor XI (FXIa). This causes a shift and / or loss of orientation relative to the original.

[0026] In one embodiment, the isolated antibody or antigen-binding fragment thereof is FXI and / or FXI binds to FXI and / or FXIa but not to FXI and / or FXIa When the catalytic domain of a forms an antibody:antigen complex, the four N-terminal residues, loop 145 , 188, and 220, the catalytic domain of active factor XI (FXIa) complexed This causes a shift and / or loss of orientation compared to the unmodified structure.

[0027] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to active FXI (FXIa). It binds to the FXI (FXIa) catalytic domain and modifies its conformation by constituting loop 145 , 188, and 220 are shifted and / or disoriented relative to the active conformation. This causes the enzyme to change to an inactive conformation in which tropism is lost.

[0028] In one embodiment, the isolated antibody or antigen-binding fragment thereof binds to FXI but does not bind to thymo Induce a conformational change in the acetylcholinesterase (AH) structure, which results in a conformational change in the acetylcholinesterase (AH) structure upon binding to FXIa. The inhibitory FXI conformation closely related to the observed inhibitory FXI conformation This prevents the catalytic domain from adopting an active conformation. Stop.

[0029] The present invention also provides antibodies that bind to the same epitope as the antibodies listed in Table 1 (e.g., NOV1401). The present invention further relates to an isolated antibody or antigen-binding fragment thereof that binds to the polypeptide.

[0030] The binding affinities of the isolated antibodies and antigen-binding fragments described herein are determined by lysis level. It can be determined by equilibrium titration (SET). The SET method is known in the art. Alternatively, the isolated antibodies or fragments described herein may be used in combination with other antibodies or fragments of the present invention. The binding affinity of the fragments can be determined by, for example, surface protease activity in a BIACORE™ assay. This can be determined by plasmon resonance measurements. ) Methods for kinetic assays are known and are described in more detail below.

[0031] The isolated anti-FXI and / or FXIa antibodies and anti- The original binding fragments were used to identify Factor IX (also known as FIX), Factor X ( FX), and / or thrombin, directly or indirectly, and / or or platelet receptor binding, thereby inhibiting the intrinsic coagulation pathway and / or Alternatively, activation of the common coagulation pathway can be prevented.

[0032] The isolated anti-FXI and / or FXIa antibodies and anti- The original binding fragments were used to identify Factor IX (also known as FIX), Factor X ( FX), and / or thrombin, directly or indirectly, at concentrations of 100 nM or less. , 50nM or less, 35nM or less, 25nM or less, 10nM or less, or 5.2nM or less I C 50 More specifically, the isolated antibodies or The antigen-binding fragments thereof are those of Factor IX (also known as FIX), Factor X ( FX), and / or thrombin, directly or indirectly, at concentrations of 100 nM or less. , 50nM or less, 35nM or less, 25nM or less, 10nM or less, or 5.2nM or less I C 50 More specifically, the isolated antibodies or antigens thereof described herein may be inhibited by Binding fragments include Factor IX (also known as FIX), Factor X (FX), and and / or thrombin, direct or indirect activation, IC<=35nM, <=25nM, <=20nM, or <=18nM 50 Blocked by More specifically, the isolated antibodies or antigen-binding fragments thereof described herein may , Factor IX (also known as FIX), Factor X (FX), and / or Direct or indirect activation of thrombin is controlled by 100 nM or less, 50 nM or less, 35 nM or less IC< M, < 25 nM, < 10 nM, or < 5 nM 50 It can be hindered by In certain embodiments, the anti-FXI / FXIa antibody or its antigen-binding fragment thereof described herein is The fragment inhibits FXIa-mediated activation of its natural substrate, FIX, at concentrations below 2 nM. For example, an IC of 1.8 nM 50 and inhibits.

[0033] Isolated anti-FXI and / or anti-FXIa antibodies, or antigen-binding fragments thereof Inhibition of FXI- and / or FXIa-mediated FIX activation, for example, using fragments inhibits the intrinsic and / or common coagulation pathways through Therefore, isolated anti-FXI / FXIa antibodies, or These antigen-binding fragments can be used to prevent blood clotting or the spread of blood clotting. The antibody or its antigen-binding fragment inhibits FXI-mediated activation of FIX, thereby , preventing and treating coagulation disorders such as deep vein thrombosis and stroke (e.g., ischemic stroke); or can be used to improve

[0034] In specific embodiments, anti-FXI and / or anti-FXIa antibodies, or antibodies thereof The antigen-binding fragment of is determined, for example, by the aPTT assay described in the Examples section. It increases the clotting time (i.e., the time it takes for a clot to form) of human plasma in a concentration-dependent manner. In a specific embodiment, the aPTT can be increased. in the range of 10 nM to 20 nM, e.g., about 14 nM or At a total anti-FXI antibody (e.g., NOV1401) concentration of 15 nM, compared to baseline In certain embodiments, the antibody against FXI and / or FXIa is or These antigen-binding fragments can be determined, for example, by the aPTT assay described in the Examples section. As shown, the IC50 ranges from 5 nM to 20 nM, e.g., about 13 nM, in a concentration-dependent manner. , it is possible to prolong the clotting time of human plasma.

[0035] In specific embodiments, the anti-FXI antibodies and / or anti-FXI antibodies described herein The aPTT antibodies, or antigen-binding fragments thereof, can be used, for example, in the aPTT assay described in the Examples section. For example, at least 1.1-fold, 1.2-fold, 1.0-fold, 1.2-fold, 1.0-fold, 1.1 ... 0.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, or 2x, It is possible to extend the clotting time of human plasma (e.g., the time it takes for a clot to begin to form). In specific embodiments, the anti-FXI and / or anti-F antibodies described herein are XIa antibodies, or antigen-binding fragments thereof, can be used in, for example, the aPTT assay described in the Examples section. at least 1.4-fold, 1.5-fold, 1.6-fold, or 1.0-fold as determined by assay. It can extend the clotting time (i.e., the time it takes for a clot to start forming) of human plasma by 7 times. It is possible.

[0036] In specific embodiments, the anti-FXI and / or anti-FXIa antibodies described herein The antibodies, or their antigen-binding fragments, bind to the IL-1 receptor in the presence of very low concentrations of tissue factor (TF). We investigated the effect of FXIa inhibition on the thrombin → FXIa feed-forward loop. The amount of thrombin in human plasma is measured by the thrombin generation assay (TGA). In certain embodiments, the antibodies described herein can reduce the vasoconstriction in a concentration-dependent manner. Anti-FXI and / or anti-FXIa antibodies, or antigen-binding fragments thereof, are isolated from human blood. In plasma thrombin generation assay (TGA), the range of 10 nM to 30 nM, e.g. , IC of approximately 20 nM or 24 nM 50 value and a residual thrombin concentration of about 159 nM. The amount of thrombin can be reduced.

[0037] In a specific embodiment herein, the catalytic domain of human FXI and / or FXIa The terminal elimination half-life (t 1 / 2 ) for about 1 Incubate for 4 to 15 days with the antibody (e.g., NOV1401 or the HCDR of NOV1401). 1-3 and LCDR1-3), or antigen-binding In a specific embodiment, such an anti-FXI / FXIa antibody exhibits a specific fragment of about It exhibits an absolute subcutaneous (sc) bioavailability of 61-66%.

[0038] In a specific embodiment, the present invention provides a method for the preparation of a human FXI antibody that specifically binds to human FXI and / or FXIa. The antibodies or antigen-binding fragments thereof provided herein (e.g., NOV1401, Table 1 The antibodies described in have the following characteristics: (i) For example, apparent K of approximately 1–2 pM and 4–5 pM, respectively. D So, human F specifically binds to the catalytic domains (CDs) of XI and FXIa; (ii) Coagulation assessed by activated partial thromboplastin time (aPTT) assay Prolonging blood time; (iii) F by activated factor XII (FXIIa) and thrombin, respectively Inhibiting thrombin generation in human plasma through inhibition of XI activation; (iv) In FXI- / - mice reconstituted with human FXI, antithrombotic and anticoagulant activities were observed. exhibiting coagulation activity; (v) For example, in cynomolgus monkeys, free FXI (FXI f ) levels or or prolonging the reduction; (vi) For example, the terminal elimination half-life of the whole antibody in cynomolgus monkeys is about 14 to 15 days. Between; (vii) specifically binds to human and monkey FXI and / or FXIa; does not specifically bind to mouse or rat FXI and / or FXIa; and (viii) The following residues of human FXI (numbered according to Swissprot): Pro 410, Arg413, Leu415, Cys416, His431, Cys432, T yr434, Gly435, Glu437, Tyr472~Glu476, Tyr521 ~Lys527, Arg548, His552, Ser575, Ser594~Glu5 97, and one or more (e.g., two, three or more) of Arg602 to Arg604 , 4, 5, 6, or 7 or more), or in part or in whole to come into contact with One or more of (e.g., two, or three, or four, or five, or six, or seven), or all of the above.

[0039] The isolated anti-FXI antibody and / or isolated FXIa antibody described herein, or The antigen-binding fragments of the present invention may be monoclonal antibodies, human or humanized antibodies, chimeric antibodies, single-chain antibodies, or the like. an antibody, a Fab fragment, an Fv fragment, an F(ab')2 fragment, or an scFv fragment, and / or It may be of an IgG isotype (e.g., IgG1, such as human IgG1). In embodiments, the anti-FXI and / or anti-FXIa antibodies described herein are recombinant. In specific embodiments, the anti-FXI antibodies and / or antibodies described herein are human antibodies. Alternatively, the anti-FXIa antibody is a human IgG1 / lambda (λ) antibody. The anti-FXI and / or anti-FXIa antibodies described herein inhibit effector F that have been engineered to reduce the potential for function (e.g., ADCC and / or CDC) a human Fc domain, e.g., a human Fc domain containing a D265A substitution and / or a P329A substitution It is a human IgG1 / lambda (λ) antibody containing IgG1.

[0040] An isolated anti-FXI antibody and / or an isolated FXIa antibody or antibody thereof as described herein. The antigen-binding fragments of these also include those in which the amino acid sequence is the same as the human VH germline sequence or the human VL germline sequence. The frameworks were also replaced with antibody frameworks derived from each of the cell lineage sequences. It may include.

[0041] Another embodiment of the invention is a Fab having the complete heavy and light chain sequences of the Fabs set forth in Table 1. More specifically, the present invention relates to an isolated antibody or antigen-binding fragment thereof. The combined fragment can have the heavy and light chain sequences of NOV1090 and NOV1401.

[0042] A further aspect of the invention is the heavy and light chain variable domains of the Fabs listed in Table 1. More specifically, the present invention includes an isolated antibody or antigen-binding fragment thereof having the sequence The antigen-binding fragments are derived from the heavy and light chain variable domains of NOV1090 and NOV1401. It may have a main sequence.

[0043] Further aspects of the present invention include the Kabat CDR, the IMGT CDR, the Chothia CDR, Heavy chain variable domain CDRs of the antibodies listed in Table 1, including CDRs or combined CDRs. R (i.e., HCDR1, HCDR2, and HCDR3) sequences and the light chain variable domain and an isolated CDR (i.e., LCDR1, LCDR2, and LCDR3) sequence. More specifically, an isolated antibody or antigen-binding fragment thereof The fragment may be a Kabat CDR, an IMGT CDR, a Chothia CDR, or a combination For example, the CDRs of NOV1090 and NOV1401 are listed in Table 1. CDR1 sequence, HCDR2 sequence, HCDR3 sequence, LCDR1 sequence, LCDR2 sequence, and and LCDR3 sequences.

[0044] The present invention also provides a method for the production of a nucleic acid molecule comprising: a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 3 and 23; heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 4 and 24, and SEQ ID NOs: 5 and 25 and a heavy chain CDR3 selected from the group consisting of human FXI and / or human FXI In another aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that binds to a. The isolated antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 13 and 33. a light chain CDR1 selected from the group consisting of SEQ ID NOs: 14 and 34; and 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.

[0045] The present invention also provides a light chain CDR1, sequence 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 CDR2 selected from the group consisting of SEQ ID NOs: 15 and 36; and 35, comprising a light chain CDR3 selected from the group consisting of human FXI and / or human Also disclosed is an isolated antibody or antigen-binding fragment thereof that binds to FXIa.

[0046] The present invention also provides isolated antibodies or antigen binding sites thereof that bind to FXI and / or FXIa. a synthetic fragment comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2 and LCDR3, and HCDR1, HCDR2, and HCDR3 are SEQ ID NOs: 3, 4, and 5, and LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 1 3, 14, and 15, or HCDR1, HCDR2, and HCDR3 are SEQ ID NOs: 23, 24, and 25, and LCDR1, LCDR2, and LCDR3 are selected from the group consisting of the sequences Also relates to an isolated antibody or antigen-binding fragment thereof comprising numbers 33, 34, and 35.

[0047] The present invention also provides an isolated antibody or antigen-binding fragment thereof that binds to FXI and / or FXIa. a functional fragment comprising HCDR1, HCDR2, and HCDR3, and LCDR1, L CDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3 are LCDR1, LCDR2, LCDR3 contain SEQ ID NOs: 43, 44, and 45, respectively. an isolated antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 47, 37, and 15, respectively; It also concerns one side.

[0048] The present invention also provides isolated antibodies or antigen-binding antibodies that bind to FXI and / or FXIa. a functional fragment comprising HCDR1, HCDR2, and HCDR3, and LCDR1, L CDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3 are and LCDR1, LCDR2, and LCDR3 are selected from the group consisting of SEQ ID NOs: 46, 4, and 5, respectively. isolated antibodies or antigen-binding fragments thereof comprising SEQ ID NOs: 33, 14, and 15, respectively; Also related.

[0049] The present invention also provides the variable heavy chain sequences of SEQ ID NOs: 9 and 29 as defined by Chothia. HCDR1, HCDR2, and HCDR3, and the variable light sequences of SEQ ID NOs: 19 and 39 The present invention also relates to an antibody or antigen-binding fragment having the LCDR1, LCDR2, and LCDR3 of the LCDR1, LCDR2, and LCDR3 chains. In another embodiment of the invention, the antibody or antigen-binding fragment is , HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain sequences of SEQ ID NOs: 9 and 29 DR3, and LCDR1, LCDR2, and LCDR3 of the light chain variable domain sequences of SEQ ID NOs: 19 and 39 R2, and LCDR3.

[0050] The present invention also relates to the HCD of the variable heavy chain of SEQ ID NOs: 9 and 29 as defined by IMGT. R1, HCDR2, and HCDR3, and the L of the variable light chain of SEQ ID NOs: 19 and 39 Also relates to an antibody or antigen-binding fragment having CDR1, LCDR2, and LCDR3. In another embodiment of the invention, the antibody or antigen-binding fragment is HCDR1, HCDR2, and HCDR3, and LCDR1, L of the light chain variable domain sequences of SEQ ID NOs: 19 and 39 It may have a CDR2, and an LCDR3.

[0051] In one embodiment of the invention, the isolated antibody or antigen-binding fragment thereof is selected from SEQ ID NOs: 9 and 29. The isolated antibody or antigen-binding fragment comprises a heavy chain variable domain sequence selected from the group consisting of: , and may further comprise a light chain variable domain sequence, in which case the heavy chain variable domain and In combination with the light chain variable domain, it forms the antigen-binding site for FXIa. In particular, the light chain variable domain sequence can be selected from SEQ ID NOs: 19 and 39, wherein the isolated antibody or antigen-binding fragment thereof binds to FXI and / or FXIa. Combine.

[0052] The present invention also provides a light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 19 and 39. An isolated antibody or antigen thereof comprising a sequence and binding to human FXI and / or human FXIa. The invention also relates to isolated antibodies or antigen-binding fragments, which further comprise a heavy chain variable domain sequence. In this case, the light chain variable domain and the heavy chain variable domain may be combined. In addition, it forms an antigen-binding site for FXI and / or FXIa.

[0053] Isolated antibodies or antigen-binding fragments thereof that specifically bind to FXI and / or FXIa heavy and light chains comprising the sequences of SEQ ID NOs: 9 and 19; or 19 and 39, respectively; The antibody may have a chain variable domain.

[0054] The present invention further provides a method for detecting a sequence selected from the group consisting of SEQ ID NOs: 9 and 29 and at least 8 Overlapping sequences with 0%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity The present invention also relates to an isolated antibody or antigen-binding fragment thereof comprising a chain variable domain, wherein the antibody In one embodiment, the isolated antibody or its antigen binds to FXI and / or FXIa. The binding fragment also has at least one sequence selected from the group consisting of SEQ ID NOs: 19 and 39. having 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity In a further aspect of the invention, the isolated antibody or antigen-binding fragment comprises , HCDR1, HCDR2, HCD, as defined by Kabat and listed in Table 1 In a specific embodiment, a single The isolated antibody or antigen-binding fragment may be defined by Chothia, IMGT, or a combination. HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2 are listed in Table 1. R2, and LCDR3.

[0055] The present invention also relates to a method for detecting a nucleic acid sequence comprising at least 8 sequences selected from the group consisting of SEQ ID NOs: 19 and 39. 0%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity The present invention also relates to an isolated antibody or antigen-binding fragment thereof having a chain variable domain, wherein the antibody The body binds to FXI and / or FXIa.

[0056] In another aspect of the invention, an isolated antibody or antibody thereof that binds to FXI and / or FXIa. The antigen-binding fragment of may have a heavy chain comprising the sequence of SEQ ID NO: 11 or 31. The isolated antibody may In addition, in combination with the heavy chain, it binds to human FXI and / or human FXIa. In particular, the light chain may comprise a sequence comprising SEQ ID NO: 21 or 41. In particular, isolated antibodies or antibodies thereof that bind to FXI and / or FXIa. The original binding fragments include the sequences of SEQ ID NOs: 11 and 21; or 31 and 41, respectively. The antibody may have heavy and light chains comprising:

[0057] The present invention still further provides a method for the preparation of a nucleic acid molecule comprising administering to a subject a nucleic acid molecule having at least one sequence selected from the group consisting of SEQ ID NO: 11 or 31. The present invention also relates to an isolated antibody or antigen-binding fragment thereof comprising a heavy chain having 90% sequence identity with the In one embodiment, the antibody binds to FXI and / or FXIa. The isolated antibody or antigen-binding fragment thereof may also be selected from the group consisting of SEQ ID NO: 21 or 41. at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence It also includes a light chain having sequence identity of:

[0058] The present invention still further provides a method for the preparation of a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 21 or 41. All of the sequences share 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity. The present invention also relates to an isolated antibody or antigen-binding fragment thereof comprising a light chain comprising F Binds to XI and / or FXIa.

[0059] The present invention also provides a composition comprising an isolated antibody or antigen-binding fragment thereof described herein. The present invention also relates to an antibody composition in combination with a pharmaceutically acceptable carrier. In particular, the present invention provides antibodies of Table 1, such as antibodies NOV1090 and NOV1401. The present invention further includes pharmaceutical compositions comprising the isolated antibodies of Table 1 or antigen-binding fragments thereof. The present invention also relates to pharmaceutical compositions comprising a combination of two or more of the antibodies or antigen-binding fragments thereof. .

[0060] The present invention also provides a method for the production of a human ovarian tumor suppressor comprising administering to a mammalian subject the present invention, ... a mammalian subject the present invention, and a human subject the present invention. In particular, the nucleic acid is selected from the group consisting of SEQ ID NOs: 10 and 30. At least 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the sequence In a further aspect of the invention, the sequence has a sequence identity of SEQ ID NO: 10 or 30. is.

[0061] The present invention also provides a nucleic acid encoding a variable light chain having a sequence selected from SEQ ID NOs: 20 and 40. In particular, the nucleic acid is selected from the group consisting of SEQ ID NOs: 20 and 40. The sequence selected is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence selected. In a further aspect of the invention, the sequences have 9% sequence identity with SEQ ID NOs: 20 and 4. It is 0.

[0062] The present invention also relates to a method for detecting a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 20 and 40, and at least 9 0% sequence identity It also relates to isolated nucleic acids.

[0063] The present invention also provides vectors comprising one or more of the nucleic acid molecules described herein. Also relates to.

[0064] The present invention also relates to a recombinant DNA sequence encoding the heavy chain of the antibody described above, and 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, In this case, the DNA sequence is operably linked to a promoter and is expressed in the host cell. It is contemplated that the antibody may be a human monoclonal antibody. It is also contemplated that the host cell is a non-human mammalian cell.

[0065] The present invention also relates to a method for detecting FXI and / or FXIa expression and / or intrinsic coagulation pathway expression. A method for reducing activation of the coagulation pathway and / or common coagulation pathway, comprising administering to cells an effective amount of a subject is contacted with a composition comprising an isolated antibody or antigen-binding fragment thereof described herein. The present invention also relates to a method including the steps of:

[0066] The present invention also relates to a method for inhibiting the binding of FXI and / or FXIa to FIX. wherein the cells are treated with an effective amount of an isolated antibody or antigen-binding fragment thereof described herein. The present invention also relates to a method comprising contacting a subject with a composition comprising:

[0067] It is contemplated that the cell is a human cell. It is further contemplated that the cell is in a subject. In one embodiment, it is contemplated that the cells are platelets. The subject may be a human. It is still further contemplated that:

[0068] The present invention also relates to a method of treating, ameliorating, or preventing a thromboembolic disorder in a subject. and administering to the subject an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein. In one aspect, the thromboembolic disease is a thrombus. Prevention of sexual disorders (e.g., thrombosis, thrombotic stroke, atrial fibrillation, stroke in atrial fibrillation (S PAF, deep vein thrombosis, venous thromboembolism, and pulmonary embolism). It is also contemplated that

[0069] Any of the foregoing isolated antibodies or antigen-binding fragments thereof may be referred to as monoclonal antibodies. or an antigen-binding fragment thereof.

[0070] Non-limiting embodiments of the present disclosure are described in the following aspects: 1. An isolated anti-FXI antibody that binds within the catalytic domain of FXI and / or FXIa; and and / or an isolated anti-FXIa antibody or fragment thereof. 2. Isolated antibodies that bind to one or more epitopes of anti-FXI and / or FXIa or a fragment thereof, wherein the epitope is Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu43 7, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr 521, Arg522, Lys523, Leu524, Arg525, Asp526, L ys527, Arg548, His552, Ser575, Ser594, Trp595 , Gly596, Glu597, Arg602, Glu603, and Arg604 An isolated antibody or fragment thereof comprising two or more amino acid residues thereof. 3. The epitope is Pro410, Arg413, Leu415, Cys416, His 431, Cys432, Tyr434, Gly435, Glu437, Tyr472, L ys473, Met474, Ala475, Glu476, Tyr521, Arg522 , Lys523, Leu524, Arg525, Asp526, Lys527, Arg5 48, His552, Ser575, Ser594, Trp595, Gly596, Gl four or more of the following amino acids: u597, Arg602, Glu603, and Arg604 The isolated antibody or fragment of embodiment 2, comprising an acid residue. 4. The epitope is Pro410, Arg413, Leu415, Cys416, His 431, Cys432, Tyr434, Gly435, Glu437, Tyr472, L ys473, Met474, Ala475, Glu476, Tyr521, Arg522 , Lys523, Leu524, Arg525, Asp526, Lys527, Arg5 48, His552, Ser575, Ser594, Trp595, Gly596, Gl six or more of the following amino acids: u597, Arg602, Glu603, and Arg604 The isolated antibody or fragment of embodiment 2, comprising an acid residue. 5. The epitope is Pro410, Arg413, Leu415, Cys416, His 431, Cys432, Tyr434, Gly435, Glu437, Tyr472, L ys473, Met474, Ala475, Glu476, Tyr521, Arg522 , Lys523, Leu524, Arg525, Asp526, Lys527, Arg5 48, His552, Ser575, Ser594, Trp595, Gly596, Gl Eight or more of the following amino acids: u597, Arg602, Glu603, and Arg604 The isolated antibody or fragment of embodiment 2, comprising an acid residue. 6. The epitope is Pro410, Arg413, Leu415, Cys416, His 431, Cys432, Tyr434, Gly435, Glu437, Tyr472, L ys473, Met474, Ala475, Glu476, Tyr521, Arg522 , Lys523, Leu524, Arg525, Asp526, Lys527, Arg5 48, His552, Ser575, Ser594, Trp595, Gly596, Gl The monoclonal antibody of embodiment 2, comprising residues u597, Arg602, Glu603, and Arg604. Isolated antibodies or fragments. 7. The epitope is composed of the amino acid residues Pro410, Arg413, and Lys527, and the amino acid residues Le u415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu47 6, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp 526, Arg548, His552, Ser575, Ser594, Trp595, G of ly596, Glu597, Arg602, Glu603, and Arg604 and one or more amino acid residues. 8. The epitope is composed of the amino acid residues Pro410, Arg413, and Lys527, and the amino acid residues Le u415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu47 6, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp 526, Arg548, His552, Ser575, Ser594, Trp595, G of ly596, Glu597, Arg602, Glu603, and Arg604 and four or more amino acid residues. 9. The epitope is composed of the amino acid residues Pro410, Arg413, and Lys527, and the amino acid residues Le u415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu47 6, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp 526, Arg548, His552, Ser575, Ser594, Trp595, G of ly596, Glu597, Arg602, Glu603, and Arg604 and 6 or more amino acid residues. 10. Isolated anti-FXI antibodies that bind within the catalytic domain of FXI and / or FXIa and / or isolated anti-FXIa antibodies or fragments thereof, XIa to one or more of factor IX, factor XIIa, and thrombin An isolated antibody or fragment that blocks binding. 11. FXI and / or FXIa, factor IX, factor XIIa, or thrombin 11. The method of claim 10, wherein the antibody blocks binding to one or more of the clotting pathway components, Isolated antibody or fragment. 12. Binding of one or more of FIX, FXI, and FXIa to platelet receptors The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment blocks binding of 13. An isolated antibody or antibody of embodiment 1 that prevents activation of the intrinsic coagulation pathway or the common coagulation pathway. piece. 14. Human FXI protein and / or human FXIa protein, BIACORE ≤ 34 nM as measured by the ® assay or by the equilibrium titration assay in solution (S K below 4 pM measured by ET) D An isolated antibody or fragment thereof that binds at 15. At least 90% identity to at least one of the CDRs listed in Table 1 2. The isolated antibody or fragment of embodiment 1, comprising at least one complementarity determining region having the same identity as the antibody or fragment of embodiment 1. 16. The isolated antibody or fragment of embodiment 1, comprising CDR1, CDR2, and CDR3 of Table 1. . 17. An isolated variant of the antibody or fragment of embodiment 1, wherein the antibody or fragment is selected from the group consisting of C, D, E, F, G, H, I, H, I, M ... and a variant of CDR1, CDR2, or CDR3. Isolated mutants with at least 1-4 amino acid changes in one of R3 . 18. The monoclonal antibody of embodiment 1, comprising a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 5 and 25. Isolated antibodies or fragments. 20. A VH selected from the group consisting of SEQ ID NOs: 9 and 29, or 90% thereof an amino acid sequence having an identity of SEQ ID NO: 19; and an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 39; 2. The method of claim 1, comprising administering to a mammalian subject the present invention, a human or animal subject to the present invention ... Isolated antibodies or fragments. 21. A VH selected from the group consisting of SEQ ID NOs: 9 and 29, or 95% thereof an amino acid sequence having an identity of SEQ ID NO: 19; and an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 39; 2. The method of claim 1, comprising administering to a mammalian subject the present invention, a human or animal subject to the present invention ... Isolated antibodies or fragments. 22. A VH selected from the group consisting of SEQ ID NOs: 9 and 29, or 97% thereof an amino acid sequence having an identity of SEQ ID NO: 19; and an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and 39; 2. The method of claim 1, comprising administering to a mammalian subject the VL of Isolated antibodies or fragments. 23. The monoclonal antibody of embodiment 1, comprising a variable heavy chain sequence selected from the group consisting of SEQ ID NOs: 9 and 29. Isolated antibodies or fragments. 24. The antibody of embodiment 1, comprising a variable light chain sequence selected from the group consisting of SEQ ID NOs: 19 and 39. Isolated antibody or fragment. 25. A variable heavy chain selected from the group consisting of SEQ ID NO: 9 and 29; and SEQ ID NO: 19 and 39. The isolated antibody or fragment of embodiment 1, comprising a variable light chain sequence selected from the group consisting of: . 26. An antibody or fragment comprising the variable heavy chain sequence of SEQ ID NO: 9 and the variable light chain sequence of SEQ ID NO: 19. and an antibody or fragment comprising the variable heavy chain sequence of SEQ ID NO: 29 and the variable light chain sequence of SEQ ID NO: 39. 2. The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment is selected from the group consisting of: 27. CDR1 of the heavy chain variable region selected from the group consisting of SEQ ID NO: 46; CDR2 selected from the group consisting of: CDR3 selected from the group consisting of SEQ ID NO: 33; CDR1 of the light chain variable region selected from the group consisting of: and a CDR3 selected from the group consisting of SEQ ID NO: 15. Isolated antibodies or fragments. 28. CDR1 of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 3 and 23; SEQ ID NOs: CDR2 selected from the group consisting of CDRs 4 and 24; CDR3 selected from the group consisting of CDRs 5 and 25 CDR3 of the light chain variable region selected from the group consisting of SEQ ID NOs: 13 and 33 1; CDR2 selected from the group consisting of SEQ ID NOs: 14 and 34; and SEQ ID NO: 15 and 35. The isolated antibody or fragment of embodiment 1, comprising a CDR3 selected from the group consisting of: 29. CDR1 of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 6 and 26; SEQ ID NOs: CDR2 selected from the group consisting of CDRs 7 and 27; CDR3 selected from the group consisting of CDRs 8 and 28 CDR3 of the light chain variable region selected from the group consisting of SEQ ID NOs: 16 and 36 1; CDR2 selected from the group consisting of SEQ ID NOs: 17 and 37; and SEQ ID NO: 18 and 38. The isolated antibody or fragment of embodiment 1, comprising a CDR3 selected from the group consisting of: 30. CDR1 of the heavy chain variable region of SEQ ID NO: 3; CDR2 of the heavy chain variable region of SEQ ID NO: 4; CDR3 of the heavy chain variable region of sequence number 5; CDR1 of the light chain variable region of sequence number 13; 14; and CDR3 of the light chain variable region of SEQ ID NO: 15. The isolated antibody or fragment of embodiment 1. 31. CDR1 of the heavy chain variable region of SEQ ID NO: 23; CDR2 of the heavy chain variable region of SEQ ID NO: 24 CDR3 of the heavy chain variable region of SEQ ID NO: 25; CDR1 of the light chain variable region of SEQ ID NO: 33; CDR2 of the light chain variable region of SEQ ID NO: 34; and CDR3 of the light chain variable region of SEQ ID NO: 35 The isolated antibody or fragment of embodiment 1, comprising 32. CDR1 of the heavy chain variable region of SEQ ID NO: 6; CDR2 of the heavy chain variable region of SEQ ID NO: 7; CDR3 of the heavy chain variable region of sequence number 8; CDR1 of the light chain variable region of sequence number 16; 17; and CDR3 of the light chain variable region of SEQ ID NO: 18. The isolated antibody or fragment of embodiment 1. 33. CDR1 of the heavy chain variable region of SEQ ID NO: 26; CDR2 of the heavy chain variable region of SEQ ID NO: 27 CDR3 of the heavy chain variable region of SEQ ID NO: 28; CDR1 of the light chain variable region of SEQ ID NO: 36; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 38 The isolated antibody or fragment of embodiment 1, comprising 34. A pharmaceutical composition comprising an antibody or fragment thereof according to one of the above embodiments and a pharmaceutically acceptable carrier. Finished product. 35. An embodiment that binds to the same epitope as the isolated antibody or fragment of any of the preceding embodiments. An isolated antibody or fragment of IgG1-like. 36. For binding to human FXI protein and / or human FXIa protein, 10. The isolated antibody or fragment of embodiment 1, which competes with the isolated antibody or fragment of any preceding embodiment. Piece. 37. The isolated antibody of embodiment 1, selected from the group consisting of NOV1090 and NOV1401. Body or fragment. 38. A method of treating a thromboembolic disorder, comprising administering an effective amount of a compound according to any of the preceding embodiments. A pharmaceutical composition comprising the antibody or fragment is administered to a subject suffering from a thromboembolic disorder. A method including steps. 39. Subject has one or more of the following: ischemic stroke and deep vein thrombosis associated with atrial fibrillation The method of embodiment 38, wherein the patient is suffering from multiple 40. The method of embodiment 38, wherein the subject is suffering from an ischemic stroke associated with atrial fibrillation. 41. A method of treating a thromboembolic disorder, comprising administering an effective amount of a compound according to any of the preceding embodiments. A pharmaceutical composition comprising the antibody or fragment is administered in combination with statin therapy to treat patients with thromboembolic disorders. to an affected subject. 42. A medicament comprising the antibody of any of the preceding aspects. 43. A nucleic acid encoding one or more of the antibodies of any of the preceding embodiments. 44. A vector comprising the nucleic acid of embodiment 43. 45. A host cell comprising the vector of embodiment 44. 46. ​​Upon binding to the active FXI (FXIa) catalytic domain, FXIa is The four residues at the N-terminus, loops 145, 188, and 220, are responsible for the active conformation. inactive conformation that is shifted and / or disordered compared to the The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment is modified to a polypeptide. Upon binding to FXI, the FXI catalytic domain binds to loops 145, 188, and 22. 0 adopts an active conformation that is as regular as in the structure of the FXIa catalytic domain The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment prevents 48. Upon binding to FXI, the FXI catalytic domain binds to four residues at the N-terminus, loop 145. , 188, and 220 are regular active components, similar to those in the structure of the FXIa catalytic domain. 2. The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment is prevented from assuming a conformation. 49. Upon binding to FXI, it induces a conformational change within the zymogen structure, resulting in the formation of FX Inhibition closely correlates with the inhibitory FXI conformation observed when bound to Ia By further conferring the active FXI conformation, the FXI catalytic domain The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment is prevented from adopting a conformation. 50. Binds to FXI and / or FXIa and acts as a catalytic receptor for FXI and / or FXIa Upon forming the antibody:antigen complex, the active sites of loops 145, 188, and 220 are activated. compared to the uncomplexed structure of the catalytic domain of human factor XI (FXIa) The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment causes a shift in and / or loss of orientation of the target molecule. 51. Binds to FXI and / or FXIa and inhibits the catalytic activity of FXI and / or FXIa When the antibody:antigen complex is formed, the four N-terminal residues, the loop 145, and the loop 188 and 220 uncomplexed portions of the catalytic domain of active factor XI (FXIa). Isolation of embodiment 1, causing a shift and / or loss of orientation compared to the original structure. Antibodies or fragments. 52. Binds to active FXI (FXIa) and mediates its coactivation with the FXI (FXIa) catalytic domain. The conformation of the ATP is such that loops 145, 188, and 220 are in the active conformation. to an inactive conformation that is shifted and / or disoriented relative to the The isolated antibody or fragment of embodiment 1, wherein the antibody or fragment is fused to a polypeptide.

[0071] definition Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains.

[0072] The terms "FXI protein," "FXI antigen," and "FXI" are used interchangeably. It is used to refer to the factor XI protein in different species. Factor XI is a limited protein. When converted to an active serine protease by protein degradation, it activates the intrinsic pathway of blood coagulation. A glycoprotein present in human plasma at a concentration of 25-30 nM is a zymogen that participates in the Mammalian plasma coagulation factor XI is a protein.

[0073] The terms "FXIa protein," "FXIa antigen," and "FXIa" are used interchangeably. It is commonly used to refer to the activated form of FXI protein in different species. Factor XI mediates thrombin through the contact phase of blood coagulation, or on the platelet surface Upon activation, it is converted to its active form, coagulation factor Xla (FXIa). During this activation of factor XI, an internal peptide bond is broken in each of the two chains, forming disulfide bonds. Serine protein consisting of two heavy chains and two light chains held together by amino acid bonds This serine protease results in activated factor Xla. The enzyme FXIa converts coagulation factor IX to IXa, which then converts coagulation factor X to IXa. Activates factor Xa (Xa), which then activates factor II / thrombin For example, human FXI has the sequence shown in Table 1 (SEQ ID NO: 1) and can mediate and literature (Mandle RJ Jr, et al. (1979) Blood;54(4):850; NCBI Reference Sequence :AAA51985).

[0074] In the context of the present invention, the terms "FXI" and "FXIa" (and the like) refer to naturally occurring FXI. Mutants and variants of the FXIa protein and the FXIa protein, respectively, The amino acid sequence of the natural primary structure (amino acid sequence) described in the above report and Includes mutants and variants having substantially the same amino acid sequence.

[0075] The terms "catalytic domain," "serine protease catalytic domain," and the terms used herein Similar terms used in [the text] refer to the amino acid sequence of the amino acid sequence, counted from Glu1 at the N-terminus of the mature protein in circulation. This also refers to the amino acids Ile370 to Val607 at the C-terminus of FXI. It can also be described as groups 388-625. As used herein, the term "active site" refers to a The term refers to a catalytically active amino acid sequence containing the amino acids His413, Asp462, and Se557. This refers to a triad of catalytic residues (Bane and Gailani (2014) Drug Disc. 19(9)).

[0076] The term "about" in reference to a number x means, for example, x±10%.

[0077] As used herein, the term "antibody" refers to whole antibodies and any antigen-binding fragments (including The term "antigen-binding portion" refers to a single chain of an antibody or its derivatives. Contains at least two heavy (H) chains and two light (L) chains interconnected by bond bonds Each heavy chain is a glycoprotein. The heavy chain constant region comprises three domains: CH1, CH2, and C Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (H3). The light chain constant region contains one domain, CL. The VH and VL regions The complementarity-determining regions are interspersed with more conserved regions called framework regions (FR). Each VH and VH can be further subdivided into regions of hypervariability called CDRs. Each VL has three CDRs and CDRs arranged in the following order from amino terminus to carboxy terminus: and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of immunoglobulins are responsible for the binding of various cells of the host tissue or immune system (e.g., Fc. It mediates binding to factors including the first component of the classical complement system (Clq) and the first component of the classical complement system (C1q). Ugh.

[0078] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody The term refers to a compound that retains the ability to specifically bind to a given antigen (e.g., Factor XIa (FXIa)). The term refers to one or more fragments of an intact antibody that retain the antigen-binding function of an antibody. This function can be fulfilled by an antigen-binding portion or antigen-binding fragment of an antibody. Examples of binding fragments encompassed within the term include Fab fragments, VL domains, VH domains, C Monovalent fragment consisting of the L domain and CH1 domain; disulfide in the hinge region F(ab)2 fragment, a bivalent fragment containing two Fab fragments linked by a bridge; an Fd fragment consisting of the VL domain and CH1 domain of a single arm of an antibody; Fv fragments consisting of a VH domain and a VL domain; single domain fragments consisting of a VH domain or a VL domain dAb fragments (Ward et al., 1989 Nature 341:544-546); and isolated complementarity determinations Contains the CDR region.

[0079] Furthermore, the two domains of the Fv fragment, the VL domain and the VH domain, are separated by individual They are encoded by genes, but can be extracted using recombinant methods to produce them as a single protein chain. They can be attached by artificial peptide linkers that allow for the creation of The VL and VH regions are paired to form a monovalent molecule (known as a single-chain Fv (scFv)). See, for example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are These antibody fragments contain one or more antigen-binding portions or fragments. Fragments are obtained using conventional techniques known to those skilled in the art, and the fragments have the same utility as intact antibodies. are screened for.

[0080] Antigen-binding fragments also include single domain antibodies, maxibodies, minibodies, intrabodies, antibody, diabody, triabody, tetrabody, v-NAR, and bis-scFv (See, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136) The antigen-binding portion of an antibody can also be incorporated into a type III fibroblast. Polypeptides such as fibronectin (Fn3) (fibronectin polypeptide monobody) (See U.S. Patent No. 6,703,199, which describes a scaffold based on It can be grafted.

[0081] The antigen-binding fragment forms a pair of antigen-binding regions in combination with a complementary light chain polypeptide. , and assembled into a single chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1). (Zapata et al., 1995 Protein Eng. 8(10):1057-1062; and U.S. Pat. No. 5,641,870).

[0082] As used herein, the term "affinity" refers to the affinity of an antibody to a single antigenic site. It refers to the strength of the interaction between the antibody and the antigen. Within each antigenic site, the variable regions of the antibody "arms" The region interacts with the antigen at multiple sites through weak non-covalent forces, and the interaction The larger the affinity, the stronger the affinity. The term "high affinity" as used herein for the b fragments generally refers to -9 K below M D (e.g., 10 -10 K below M D , 10 -11 K below M D , 10 - 12 K below MD , 10 -13 K below M D , 10 -14 K below M D etc.) The term "antigen-binding fragment" refers to a human antibody or antigen-binding fragment thereof.

[0083] The term "amino acid" includes naturally occurring and synthetic amino acids, as well as naturally occurring It also refers to amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. The amino acids in the saccharin are those encoded by the genetic code, as well as those that are later modified. O-phosphoric acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoryl Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e. hydrogen, carboxyl group, amino group, and R group, e.g., homoserine, norleucine methionine sulfoxide, methionine methyl sulfonium, and methionine methyl sulfonium. Such analogs refer to compounds that have modified R groups (e.g., norleucine) or modified They have a peptide backbone but retain the same basic chemical structure as naturally occurring amino acids. Acid mimetics are compounds that have a structure that is different from the general chemical structure of an amino acid, but which mimic the structure of a naturally occurring amino acid. Refers to chemical compounds that function in a similar manner to acids.

[0084] As used herein, the term "binding specificity" refers to a molecule that reacts with only one antigenic determinant. Refers to the capacity of an individual antigen-binding site.

[0085] "specifically" ( The phrase "bind (or selectively)" refers to a heterogeneous population of proteins and other biological materials. cognate antigens in the IL-1 domain (e.g., human FXI and / or FXIa or cynomolgus monkeys) The term "binding reaction" refers to a binding reaction that determines the presence of FXI and / or FXIa in a protein. The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably with "antibody specific for an antigen." The term "antibody that binds to" is used interchangeably with the term "antibody that binds to."

[0086] The term "FXI and / or FXIa mediated" refers to FXI and / or FXIa mediated XIa is a cytoplasmic factor that is involved in the synthesis of factor IX (also known as FIX), factor X (FX), and and / or by directly or indirectly activating thrombin, and / or By binding to platelet receptors, β-glucan mediates the intrinsic and / or common coagulation pathways. This refers to the fact that it is mediated through

[0087] The term "hemostasis" refers to the primary mechanism for stopping blood flow at the site of injury and wound healing. Each represents a major mechanism for restoring vascular patency during healing. In physical thrombosis, there are three mechanisms: primary arrest, which refers to the interaction of activated platelets with the vessel wall; Blood, fibrin formation, and a process called fibrinolysis are simultaneously activated.

[0088] Terms such as "clotting and the coagulation cascade" and "cascade model of coagulation" are used to describe wound healing. Protein-based systems used to stabilize blood clots that form to seal the The coagulation pathway is a proteolytic cascade. Each enzyme in the pathway is present in plasma. Upon activation, it undergoes proteolytic cleavage to release the active factor from the precursor molecule , exists as a zymogen (in an inactive form). The coagulation cascade regulates the activation process. The pathway functions as a series of positive and negative feedback loops that control the Thrombin, which can convert soluble fibrinogen into fibrin, which forms blood clots. The purpose is to produce

[0089] The process of thrombin generation involves three phases: the intrinsic pathway, and the activated clotting factor FXa (activated clotting factor). The extrinsic pathway, an alternative pathway for generating activated factor X, and thrombin can be divided into a final common pathway resulting in the formation of nd Monroe DM (2005) Curr Hematol Rep. 4:391 -396; Johne J, et al. (2006) Biol Chem. 387:173-178).

[0090] Platelet aggregation occurs when a blood vessel breaks and substances that normally do not come into direct contact with the bloodstream aggregate. These substances (mainly collagen and von Willebrand factor) The thrombus allows platelets to adhere to the fractured surface. When platelets are stimulated, they release chemicals that attract more platelets to the damaged area, a process known as platelet aggregation. These two processes are the first response to stop bleeding.

[0091] As used herein, the term "thromboembolic disorder" or similar terms refers to a disorder characterized by an intrinsic coagulation pathway. pathway and / or the common coagulation pathway are abnormally activated or not naturally inactivated. refers to any number of conditions or diseases that are not cured (e.g., without treatment). , thrombotic stroke, atrial fibrillation, prevention of stroke in atrial fibrillation (SPAF), deep vein thrombosis These include, but are not limited to, coronary heart disease, venous thromboembolism, and pulmonary embolism. Catheter-related conditions in which the catheter becomes thrombosed (e.g., Hickman catheterization in cancer patients) This includes extracorporeal membrane oxygenation (ECMO) where the tubing can cause blood clots. Ugh.

[0092] As used herein, the term "thromboembolic" or similar terms also refers to the anti- Anti-FXI Ab and / or anti-FXIa Ab or antigen-binding fragments thereof are used can prevent or treat the following: Paroxysmal atrial fibrillation or paroxysmal atrial flutter, persistent atrial fibrillation or persistent atrial flutter, or suspected or confirmed cardiac arrhythmias such as permanent atrial fibrillation or permanent atrial flutter Thromboembolism in subjects who have been Subjects with Stroke Prevention in Atrial Fibrillation (SPAF) subpopulations Subjects who are AF patients undergoing percutaneous coronary intervention (PCI); Treatment of acute venous thromboembolic events (VTE) in patients at high risk for bleeding, and and long-term prevention of secondary VTE; In secondary prevention after transient ischemic attack (TIA) or non-disability-inducing stroke For the prevention of cerebral and cardiovascular events, as well as thromboembolic events in heart failure with sinus rhythm cerebral and cardiovascular events in; Left atrium clot formation and thrombosis in subjects undergoing cardiac defibrillation for cardiac arrhythmias thrombosis; Thrombosis before, during, and after ablation procedures for cardiac arrhythmias; Venous thrombosis, which can be deep vein thrombosis or superficial vein thrombosis in the lower or upper extremities. thrombosis of the abdominal and thoracic veins, sinus thrombosis and jugular vein thrombosis including but not limited to treatment and secondary prevention; Thrombosis on any artificial surface within a vein, such as a catheter or pacemaker lead; Pulmonary embolism in patients with or without venous thrombosis; · Chronic thromboembolic pulmonary hypertension (CTEPH); Arterial thrombosis over ruptured atherosclerotic plaques, over intra-arterial prostheses or catheters thrombosis in the arteries of the thorax and in apparently normal arteries, which may be associated with acute coronary syndromes. group, ST-segment elevation myocardial infarction, non-ST-segment elevation myocardial infarction, unstable angina, stent thrombosis, arterial Thrombosis of any artificial surface in the system, and in subjects with or without pulmonary hypertension including but not limited to pulmonary artery thrombosis; Thrombosis and thrombosis in patients undergoing percutaneous coronary intervention (PCI) embolism; Cardioembolic and cryptogenic stroke; Thrombosis in patients with invasive and non-invasive cancer malignancies; Thrombosis across the indwelling catheter; · Thrombosis and thromboembolism in critically ill patients; Cardiac thrombosis and thromboembolism, which are cardiac thrombosis after myocardial infarction, cardiac artery conditions such as aneurysms, myocardial fibrosis, cardiac hypertrophy and dysfunction, myocarditis, and artificial surfaces within the heart. including, but not limited to, cardiothrombosis associated with pulmonary embolism; · Thromboembolism in patients with valvular heart disease, with or without atrial fibrillation; · Thromboembolism across mechanical or biological heart valve prostheses; - After cardiac repair surgery for simple or complex cardiac anomalies, natural or artificial cardiac pads thromboembolism in patients with arterial or venous conduits; Knee replacement, hip replacement, and orthopedic, thoracic, or abdominal surgery venous thrombosis and thromboembolism after After neurosurgery, including intracranial and spinal interventions arterial or venous thrombosis; Factor V Leiden, prothrombin mutations, antithrombin III, prothrombin Protein C deficiency and protein S deficiency, factor XIII mutations, familial fibrinogen deficiency Plasminogenemia, congenital plasminogen deficiency, elevated factor XI levels, sickle cell disease, Antiphospholipid syndrome, autoimmune diseases, chronic intestinal diseases, nephrotic syndrome, hemolytic uremia, bone marrow Proliferative disorders, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and heparin-induced hematoma congenital or acquired thrombophilia, including but not limited to thrombocytopenia; Thrombosis and thromboembolism in chronic kidney disease; and - Thrombosis and thrombosis in patients undergoing hemodialysis and extracorporeal membrane oxygenation Thromboembolism It may also refer to any number of

[0093] The term "chimeric antibody" refers to an antibody that (a) contains an antigen-binding site (variable region) of a different class, effector, or antibody variant. different classes, effector functions, and / or species, or different classes, effector functions, and and / or species-altered constant regions, or entirely different constant regions that confer new properties to the chimeric antibody. The nucleotides can be defined to link to various molecules, such as enzymes, toxins, hormones, growth factors, drugs, etc. antibody molecules in which the constant region or a portion thereof has been modified, replaced, or exchanged; or (b) a variable region or a portion thereof, which has a different or altered antigen specificity; The term "antibody" refers to an antibody molecule in which the variable region has been modified, replaced, or exchanged with a modified variable region. For example, mouse antibodies have their constant regions replaced with constant regions derived from human immunoglobulins. The modification can be achieved by replacing the human constant region. The Ra antibody has reduced antigenicity in humans compared to the original mouse antibody, while still retaining the ability to recognize the antigen. It can retain its specificity in

[0094] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants are those that have identical amino acids. a nucleic acid encoding an amino acid sequence or an essentially identical amino acid sequence, or When a sequence is not coded for, it refers to essentially identical sequences. Due to the degeneracy of the genetic code, A large number of functionally identical nucleic acids encode any given protein. For example, GCA, GCC, GCG, and GCU all contain the amino acid alanine. Thus, alanine is encoded at every position specified by a codon. A codon can be replaced with any of the corresponding codons listed without modifying the polypeptide. Such nucleic acid variations are one type of conservatively modified variation. Any nucleic acid sequence herein that encodes a polypeptide is a "silent mutation." The sequence also describes every possible silent variation of the nucleic acid. Each codon (AUG, which is usually a codon for methionine only, and It was recognized that the codons (except the codon TGG) could be modified to result in a functionally identical molecule. Thus, within each sequence described, the nucleic acid encoding the polypeptide Each silent mutation is implied.

[0095] In polypeptide sequences, "conservatively modified variants" refer to chemically similar amino acids. Individual substitutions, deletions, or deletions in a polypeptide sequence resulting in the substitution of corresponding amino acids. Conservative substitutions, which represent functionally similar amino acids, are well known in the art. Such conservatively modified variants are not intended to be limiting unless otherwise specified. This is in addition to, and does not exclude, homologs and alleles. Eight groups: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (G); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (L) isoleucine (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) are mutually Containing amino acids that are conservative substitutions for (see, e.g., Creighton, Proteins (1984) In some embodiments, the term "conservative sequence modification" refers to the modification of an amino acid It does not significantly affect or alter the binding characteristics of the antibody containing the sequence. It is used to refer to an amino acid modification.

[0096] The term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. They usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes and nonconformational epitopes are distinguished by the fact that binding to the former is mediated by denaturing solvents. Competitive binding assays are distinguished by the fact that binding to the ATP is lost in the presence of the ATP but not to the ATP. In the method, one antibody is fused to a second antibody by any of the methods well known to those skilled in the art. Two antibodies are said to "compete" if they are shown to bind to the same epitope.

[0097] As used herein, the term "human antibody" refers to a human antibody that is humanized in the framework and CDR regions. Any of the above ranges is intended to include antibodies having variable regions derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region may also be composed of such human sequences, e.g. For example, derived from human germline sequences or mutated versions of human germline sequences. The human antibodies of the invention may contain amino acid residues that are not encoded by human sequences (e.g., In vitro mutagenesis by random or site-directed mutagenesis mutations introduced into the genome or by somatic mutation in vivo mutations).

[0098] The term "human monoclonal antibody" refers to an antibody displaying a single binding specificity. Both the framework and CDR regions have variable regions derived from human sequences. In one embodiment, a human monoclonal antibody refers to an antibody that is derived from a human immunoglobulin gene. Prepared using phage display technology for screening libraries of vectors do.

[0099] A "humanized" antibody is an antibody that is less immunogenic in humans but retains the reactivity of a non-human antibody. This means that, for example, the non-human CDR regions are retained and the remainder of the antibody is The constant regions and framework portions of the variable regions are replaced with their human counterparts. This can be achieved by, for example, Morrison et al., Proc. Natl. Acad. Sci. U SA, 81:6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44:65-92, 1988; Verho eyen et al., Science, 239:1534~1536, 1988; Padlan, Molec. Immun., 28:489~498, 1991 and Padlan, Molec. Immun., 31:169-217, 1994. Examples include the Xoma technology disclosed in US 5,766,886. Not limited.

[0100] "Identical" or "identical" in the context of two or more nucleic acid sequences or polypeptide sequences The term "percent" refers to two or more sequences or subsequences that are the same. using one of the sequence comparison algorithms below, or by manual alignment and compared for maximum correspondence over a visually measured comparison zone or designated area; When sequenced, the specified percentage of amino acid residues or nucleotides of the two sequences If the tid is the same (i.e., spans the specified range, or if not specified, , 60% identity across the entire sequence, optionally 65%, 70%, 75%, 80%, Two sequences are considered "substantially identical" if they share 85%, 90%, 95%, or 99% identity. Optionally, identity is at least about 50 nucleotides (or 10 amino acids). or more preferably, 100 to 500, or 1000 spanning a region of at least 50,000 nucleotides (or at least 20, 50, or 200 amino acids) in length It exists.

[0101] For sequence comparison, one sequence acts as a reference sequence, against which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are typically Enter the sequence into the computer, specify the subsequence coordinates if necessary, and run the sequence algorithm program. Specify the program parameters. You can also use the default program parameters. Alternatively, alternative parameters can be specified. The sequence comparison algorithm then: The percent sequence identity for the test sequence compared to the reference sequence is determined based on the program parameters. Calculate the points.

[0102] As used herein, a "comparison window" refers to a window in which, after optimally aligning two sequences, the sequences are compared to one another. A reference sequence can be compared with the same number of consecutive positions, typically between 20 and 600, typically between about 50 and about 20 0, more usually a number of consecutive positions selected from the group consisting of about 100 to about 150 This includes reference to a segment by any one of the following: Sequence alignment methods are well known. The optimal sequence alignment for comparison is, for example, Sm The local homology algorithm by Ith and Waterman (1970) Adv. Appl. Math. 2:482c or by homology analysis according to Needleman and Wunsch, J. Mol. Biol. 48:443, 1970. This can also be done by the gender alignment algorithm, see Pearson and Lipman, Proc. Nat'l Acad. Sci. USA 85:2444, 1988, and these algorithms can be used for similarity searches. A computerized implementation of the algorithm (Wisconsin Genetics Software) tware Package, Genetics Computer Group, 57 GAP, BESTFIT, at 5 Science Dr., Madison, WI FASTA, and TFASTA), manual alignment and Visual inspection (e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons , Inc. (see Ringbou, ed., 2003).

[0103] Algorithms Suitable for Determining Percent Sequence Identity and Percent Sequence Similarity Two examples are, respectively, Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402; and and Altschul et al. (1990) J. Mol. Biol. 215:403-410. The BLAST analysis was performed using the ST algorithm and the BLAST 2.0 algorithm. The software for this is provided by the National Center for Biotech This algorithm is published by the Department of When matching with words of the same length in the database sequence, a threshold score T by identifying a short word length W in the query sequence that matches or satisfies This involves first identifying high-scoring sequence pairs (HSPs). Let T be the neighborhood word score threshold. These initial neighborhood word hits are then used to find the most relevant hits for the search. It serves as a seed to start searches to find longer HSPs. The word hits are extended in either direction along each sequence for as far as the core can grow. The cumulative score is calculated based on the parameter M (the number of matching residues per pair) for nucleotide sequences. reward score; always >0) and N (penalty for mismatched residues) For amino acid sequences, the scoring matrix is The cumulative alignment score is calculated using the for the accumulation of one or more negative-scoring residue alignments; , if the cumulative score falls below zero; or if either end of the sequence is reached stops the extension of the word hit in each direction. The W, T, and X used determine the sensitivity and speed of the alignment. The program (for nucleotide sequences) uses a word length (W) of 11 and an expectation (E) of 10. ), M=5, N=-4 as defaults, and a comparison of both strands is performed. The BLASTP program for and 50 BLOSUM62 scoring matrices (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) Alignment (B), 10 periods Use the default values ​​of E, M=5, and N=-4, and compare both strands.

[0104] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., See, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993. One measure of similarity provided by the BLAST algorithm is , an index of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, the minimum total probability (P(N)) of a test nucleic acid being compared to a reference nucleic acid is The minimum sum probability when compared is less than about 0.2, and more preferably less than about 0.01. and most preferably, a nucleic acid is considered similar to a reference sequence if it has a sequence identity of less than about 0.001. can be.

[0105] The percent identity between two amino acid sequences can also be calculated using the PAM120 weighted residue table, ALIGN process using a gap length penalty of 2 and a gap penalty of 4. The program (version 2.0) was developed by E. Meyers and W. Miller (Comput. It can also be determined using the algorithm by Appl. Biosci., 4:11-17, 1988. In addition, the percent identity between the two amino acid sequences is calculated using the Blossom 62 matrix. Ricks or PAM250 matrix and 16, 14, 12, 10, 8, 6 or Uses a gap weighting of 1, 2, 3, 4, 5, or 6 and a length weighting of 1, 2, 3, 4, 5, or 6 the GCG software package (available on the internet at gcg.com) The GAP program was incorporated into the Needleman and Wunsch (J. Mol. Biol. 48:44 4-453, 1970).

[0106] Other than the percentage sequence identity mentioned above, two nucleic acid sequences or polypeptides may have substantial Another indication that the sequence is identical to the sequence encoded by the first nucleic acid is The polypeptide reacts immunologically with an antibody against a polypeptide encoded by a second nucleic acid. Thus, for example, if two peptides have only conservative substitutions, Thus, if different, the polypeptide is typically substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is the The ability of two molecules or their complements to hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences are identical to the sequences of the same primers. The advantage is that sequences can be amplified using

[0107] The term "isolated antibody" refers to 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 referred to as an F (substantially free of antibodies that specifically bind to antigens other than XI and / or FXIa) However, isolated antibodies that specifically bind to FXI and / or FXIa may not bind to other antigens. Additionally, isolated antibodies may have cross-reactivity to other intracellular substances and / or chemicals. It may be substantially free of substances.

[0108] The term "isotype" refers to the antibody class ( For example, IgM, IgE, IgG such as IgG1 or IgG4. Also included are modified versions of one of these classes, where the modification enhances Fc function. modifying, e.g., enhancing or reducing effector function or binding to Fc receptors It is decorated with seaweed.

[0109] As used herein, "K" assoc " or "K a " is a specific antibody-antibody Whereas "K" as used herein is intended to refer to the association rate of an atomic interaction di s " or "K d The term "antibody-antigen interaction" refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, "K" D The term "K d K a Ratio to (i.e. K d / K a ) and expressed as a molar concentration (M) Antibody K D Values ​​can be determined using methods well established in the art Antibody K D Methods for determining the β-amyloid ... Surface plasmon resonance is measured using a sensor system or by solution equilibrium titration (SE T) to measure affinity in solution.

[0110] As used herein, a "monoclonal antibody" or "monoclonal antibody composition" refers to a The term refers to a preparation of antibody molecules of single molecular composition. Each antibody displays a single binding specificity and affinity for a particular epitope.

[0111] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide." Deoxyribonucleotides or ribonucleotides and their single-stranded or The term "nucleic acid" refers to a polymer of the well-known nucleotides Nucleic acids containing analogs or modified backbone residues or modified linkages, including synthetic nucleic acids, naturally occurring nucleic acids, and Native nucleic acids and non-naturally occurring nucleic acids that have similar binding properties to the reference nucleic acid and are Examples of such analogs include, but are not limited to, nucleic acids that are metabolized in a manner similar to leutidine. Specifically, phosphorothioates, phosphoramidates, methylphosphonates, chiral methyl These include arylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0112] Unless otherwise indicated, a particular nucleic acid sequence also refers to conservatively modified versions thereof. Variants (e.g., degenerate codon substitutions) and complementary sequences are implicitly and explicitly included. In particular, as detailed below, degenerate codon substitutions are The third position of one or more selected (or all) codons may contain mixed bases and / or This can be achieved by creating sequences in which the amino acid sequence is substituted with α- or α-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).

[0113] The term "operably linked" refers to two or more polynucleotides (e.g., DNA) A) refers to the functional relationship between the segments. The term "operably linked" refers to the functional relationship between the segments. Typically, the term refers to the functional relationship of a regulatory sequence to a transcribed sequence. The promoter or enhancer sequence may be selected so that it is suitable for use in an appropriate host cell or other expression system. operably linked to a coding sequence when it stimulates or modulates transcription of the coding sequence within Generally, a promoter transcriptional regulatory sequence operably linked to the sequence to be transcribed The enhancer is physically adjacent to the transcribed sequence, i.e., it is cis-acting. Some transcriptional regulatory sequences, such as transcriptional sensors, are physically located to the coding sequences whose transcription they enhance. They do not need to be adjacent or closely spaced.

[0114] As used herein, the term "optimized" refers to a nucleotide sequence that is optimized for the production cell. or production organisms, generally eukaryotic cells, such as Pichia cells, Chinese ha amino acid sequences using preferred codons in mouse ovary cells (CHO) or human cells Optimized nucleotide sequence means that the nucleotide sequence is modified to encode the nucleotide sequence. is the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. The amino acid sequence is engineered to be retained entirely, or as much as possible. The sequences have been engineered to have codons that are preferred in mammalian cells. However, the present disclosure also provides optimized versions of these sequences in other eukaryotic or prokaryotic cells. Expression of the amino acid sequence encoded by the optimized nucleotide sequence is also contemplated. It is also referred to as being optimized.

[0115] As used herein, the terms "polypeptide" and "protein" refer to a polypeptide consisting of a sequence of amino acid residues. The terms "polypeptide" and "protein" are used interchangeably to refer to polymers. The term "amino acid" refers to an amino acid in which one or more amino acid residues are replaced by an artificial chemical equivalent of the corresponding naturally occurring amino acid. Amino acid polymers that are structural mimetics, as well as naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, the term "polypeptide" also applies to amino acid polymers of any particular A peptide sequence also implicitly encompasses conservatively modified variants thereof.

[0116] As used herein, the term "recombinant human antibody" refers to an antibody synthesized using human immunoglobulin genes. Animals (e.g., mice) that are transgenic or transchromosomal for or expressing an antibody, human antibody, isolated from the hybridoma prepared thereby. The antibody, composition, isolated from a host cell transformed in such a manner, e.g., a transfectoma, Antibodies isolated from recombinant combinatorial human antibody libraries, and human immunoglobulins Other sequences involving splicing of all or part of the purine gene sequence to other DNA sequences Antibodies, such as antibodies prepared, expressed, created, or isolated by any means. prepared, expressed, created, or isolated by recombinant means, such as This includes all human antibodies. Such recombinant human antibodies include the framework regions and CDRs. regions having variable regions derived from human germline immunoglobulin sequences, but In certain embodiments, such recombinant human antibodies are prepared by in vitro mutagenesis. (or in animals transgenic for human Ig sequences, In vivo somatic mutagenesis) and thus the VH of the recombinant antibody The amino acid sequences of the VH and VL regions are derived from human germline VH and VL sequences. These arise from and are related to, but not limited to, the germline repertoire of human antibodies in vivo. It is a sequence that may not occur in nature.

[0117] The term "recombinant host cell" (or simply, "host cell") refers to a cell that contains a recombinant expression vector. Such terms are intended to refer only to the particular subject cell. It is understood that the term "cell" is not intended to refer to the cells of a mammalian subject, but also to the progeny of such cells. In subsequent generations, certain modifications occur due to mutations or environmental influences. Therefore, such progeny may not in fact be identical to the parent cell. , also included within the scope of the term "host cell" as used herein.

[0118] The term "subject" includes human and non-human animals. Non-human animals include non-human primates. (e.g., cynomolgus monkeys), sheep, rabbits, dogs, cattle, chickens, amphibians, and reptiles Includes all vertebrates (e.g., mammals and non-mammals), including insects. Except where otherwise noted, the terms "patient" and "subject" are used interchangeably herein. As used herein, "cyno" or "cynomolgus" refers to The term "cynomolgus monkey" refers to the cynomolgus monkey (Macaca fascicularis )). In a specific embodiment, the patient or subject is a human.

[0119] In one embodiment, any disease or disorder (e.g., thromboembolic disorders) as used herein. "Treating" or these terms "treatment" means modifying a disease or disorder. improving (i.e., slowing the onset of the disease or at least one of its clinical symptoms) In another embodiment, the term "treating" refers to "treating" or "halting" a disease. "Treatment" or "treatment" includes parameters that may not be discernible by the patient. It refers to the alleviation or improvement of at least one physical parameter. In this context, "treating" or "treatment" refers to treating a disease or disorder by physically modifying it. It may be either accelerating (e.g., stabilizing discernible symptoms) or physiologically modulating (e.g., (e.g., stabilization of physical parameters) or modulating both physical and physiological In yet another embodiment, "treating" or "treatment" refers to a disease or refers to preventing or delaying the onset or development or progression of a disorder.

[0120] "Prevention" in the context of the indications described herein, including, for example, thromboembolic disorders thromboembolic disease progression, as described below, in patients at risk of such progression. "Abnormal" refers to any effect that prevents or slows the deterioration of a parameter.

[0121] The term "vector" refers to a molecule capable of transporting another polynucleotide to which it has been linked. A vector is intended to refer to a polynucleotide molecule that can be used to create a vector. The term "protease" refers to a circular double-stranded DNA loop into which a DNA segment consisting of Another type of vector is a plasmid, which inserts additional DNA segments into the viral genome. Adeno-associated viral vectors (AAV or AAV2) that can be ligated into the Which viral vectors? Certain vectors (e.g., bacterial vectors with a bacterial origin of replication) vectors and mammalian episomal vectors) in the host cells into which they are introduced. Other vectors (e.g., non-mammalian episomal vectors) are capable of self-replication. When introduced into a host cell, it can integrate into the genome of the host cell, thereby Moreover, certain vectors replicate together with the main genome. Such vectors are capable of directing the expression of genes. Generally, recombinant DNA vectors are referred to as recombinant expression vectors (or simply "expression vectors"). Expression vectors useful in Method A are often in the form of plasmids. The terms "plasmid" and "plasmid-like" are used herein as they are the most commonly used forms of vector. The terms "vector" and "vector" can be used interchangeably. However, the present invention does not contemplate the use of any vector that provides equivalent functions. viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adenoviruses) It is intended to include other forms of expression vectors, such as vector-associated viruses. [Brief explanation of the drawings]

[0122] [Figure 1-1] 1A-1C show the effect of NOV1401 on FeCl-induced thrombosis in FXI- / - mice reconstituted with human FXI protein. NOV1401 inhibited thrombosis in a dose-dependent manner. The antibody prolonged the aPTT to the same extent as in untreated FXI- / - mice. [Figure 1-2] (As mentioned above.) [Figure 2] Figures 2A-2B 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 their relationship to total plasma NOV1401 levels (squares) in cynomolgus monkeys. A single dose of 3 mg / kg approximately doubled the aPTT, which was maintained over 5-6 weeks. All doses tested prolonged the aPTT to a similar extent, and higher doses did not appear to increase the magnitude of the aPTT prolongation observed with the 3 mg / kg dose. [Figure 3]Figures 3A-3B show the effect of multiple iv (A; N=2) or sc (B; N=2) administrations of NOV1401 at 3 mg / kg, 10 mg / kg, and 30 mg / kg on plasma free FXI (squares) and its relationship to aPTT (diamonds) in cynomolgus monkeys. A single dose of 3 mg / kg reduced free FXI by approximately 90% over a 5-6 week period. All doses tested reduced free FXI to a similar extent, and higher doses tested did not appear to increase the magnitude of the reduction in free FXI observed with the 3 mg / kg dose. [Figure 4] Figures 4A-4B show the X-ray structure of the Fab of the NOV1401 antibody of the present invention bound to FXI. Figure 4A shows the X-ray structure of the NOV1401 Fab-FXI CD complex. The FXI catalytic domain is shown as a gray surface, and the Fab is shown as light gray (light chain) and dark gray (heavy chain) ribbons. Figure 4B shows the X-ray structure of the NOV1401 Fab-FXI CD complex superimposed with zymogen FXI. The FXI catalytic domain is shown as a gray ribbon. The variable domains of the Fab are shown as light gray (VL) and dark gray (VH) ribbons. The zymogen structure, including the four apple domains, is superimposed as a dark gray ribbon at the bottom of the structure (PDB 2F83). The activation cleavage site (Ile370) is indicated. [Figure 5] Figures 5A-5B show the conformational changes of FXIa upon binding of NOV1401 Fab. Figure 5A shows a perspective view of the active site of FXIa before Fab binding. FXIa is represented as a ribbon with a transparent surface. Cross sections of the structure that change conformation upon Fab binding are labeled (loop 145, loop 188, and loop 220). The S1 and S1 subpockets are indicated. Figure 5B shows the inactive conformation of FXI within the Fab complex. (Fab not shown). [Figure 6-1]Figures 6A-6C show composite response curves for anti-FXI / FXIa antibodies. Figure 6A shows the inhibition of factor XIa activity by NOV1401. A representative composite response curve for NOV1401, an antibody that inhibits the enzymatic activity of full-length human FXIa. The assay measures cleavage of a fluorescently labeled peptide, as described in Example 3. Nonlinear curve fitting using a logistic fit model (y = A2 + (A1 - A2) / (1 + (x / IC50)p), where y is the % inhibition at inhibitor concentration x, A1 is the minimum inhibition value, and A2 is the maximum inhibition value, and the exponent p is the Hill coefficient) on this representative data set yields an IC50 value of 160 pM. Figure 6B shows a composite response curve for aPTT. A representative composite response curve for the prolongation of clotting time by antibody NOV1401 in an aPTT assay using pooled human plasma. The assay measures clotting time after initiating the intrinsic clotting cascade in the presence of different concentrations of NOV1401, as described in Example 4. The black line represents a fit using a logistic nonlinear fitting model. The dotted line represents the baseline clotting time of pooled human plasma in the absence of NOV1401. The baseline clotting time was 32.3 seconds and is indicated in the graph by the gray dashed line. The gray dotted line indicates the antibody concentration at which the clotting time doubled compared to baseline, i.e., 2× the aPTT value, which is 14 nM. Figure 6C shows a TGA response curve. A representative composite response curve for the inhibition of thrombin generation by the antibody NOV1401 in a TGA with pooled human plasma is shown. The assay, as described in Example 4, measures the effect of different concentrations of NOV1401 on thrombin generation that can be induced by very low concentrations of tissue factor (TF) via the so-called thrombin → FXIa feed-forward loop. The black line represents a fit using a four-parameter dose-response curve model. The dotted line represents the residual thrombin concentration resulting from thrombin generation induced by small amounts of TF. An IC50 value of 24 nM and a residual thrombin concentration of 159 nM (dotted line) were calculated for this composite response curve. [Figure 6-2] (As mentioned above.) [Figure 7-1] Figures 7A-7B show the effects of NOV1401 administered sc at 10 mg / kg (N=3) and 100 mg / kg (N=5) weekly for 13 weeks (14 doses) or iv at 50 mg / kg (N=3) weekly for 4 weeks (5 doses) on aPTT and FXI activity (FXI:C). Figure 7A shows the effect on aPTT measured on days 2, 23, and 79 of the study. aPTT was increased 2.1- to 3-fold in all animals receiving NOV1401 and remained elevated throughout the dosing phase of the study. No dose-dependence was observed, and no gender-related differences were noted. Figure 7B shows the effect on FXI:C measured on days 2, 23, and 79 of the study, plotted as a percent of plasma FXI activity. FXI:C decreased to levels of 5-12% in all animals receiving NOV1401 and remained at these levels throughout the treatment phase of the study. No dose-dependence or gender-related differences were observed. [Figure 7-2] (As mentioned above.)

[0123] Detailed Description The present invention provides, in part, antibody molecules that specifically bind to FXIa and inhibit its biological activity. The present invention is based on the discovery of antibodies in full-length IgG format, as well as Fab fragments (e.g., and its antigen-binding fragments, such as antibodies NOV1090 and NOV1401. do.

[0124] Thus, the present invention provides a method for the production of FXI and / or FXIa (e.g., human, rabbit, and and pharmaceutical compositions specifically binding to FXI and / or FXIa of cynomolgus monkeys and methods for making and using such antibodies and compositions.

[0125] Factor XI FXI is a clotting factor that regulates both the intrinsic and extrinsic coagulation pathways, as well as plasma-mediated coagulation. It plays an important role in bridging the initiation and amplification phases of hemostasis. Both thrombin and FXI activate FXI, resulting in sustained thrombin generation and FXI may result in the inhibition of fibrinolysis and fibrinolysis in a tissue factor-rich environment after vascular injury. Although it plays a minor role in normal hemostasis, it is thought to play a key role in thrombosis. Severe factor XI deficiency is associated with a reduced incidence of ischemic stroke and venous thromboembolic events. and (Salomon et al. 2008; Salomon, et al. (2011) Thromb Haemost.; 105:269- 73) Bleeding episodes in subjects with severe factor XI deficiency are infrequent, mild, and It is often injury-induced, resulting in increased fibrinolytic activity in areas such as the oral mucosa, nasal mucosa, and urinary tract. It is preferable to affect affected tissues (Salomon et al. 2011). Hemorrhage in vital organs is , are extremely rare or not recognized.

[0126] Plasma coagulation is a sequential process in which coagulation factors in the blood interact and activate, ultimately resulting in Ultimately, this results in the production of fibrin and the formation of a blood clot. In the card model, the process of fibrin generation involves two distinct pathways: the intrinsic pathway and the It can be initiated by both the intrinsic and extrinsic pathways (Mackman, 2008).

[0127] In the extrinsic pathway, vascular injury induces the release of extravascular tissue factor (TF) and factor VII (FVII). interacts with and activates factor X and prothrombin, Active thrombin finally converts soluble fibrinogen into fibrinogen. The extrinsic pathway is central to hemostasis and has an effect on the clotting factors in this pathway. Interference results in the risk of bleeding.

[0128] In the intrinsic pathway, factor XII is sometimes activated by a process called contact activation. The generation of activated factor XIIa is the result of the sequential activation of factors XI and IX. Factor IXa activates factor X, which allows the extrinsic pathway and the intrinsic pathway to be activated. The two pathways merge at this stage (common pathway). Thrombin activity is initiated by thrombin activating factor XI. This occurs via a feed-forward loop that activates the factor XII-independent This feedforward loop is boosted by amplifying its own generation. Contributes to persistent thrombus growth, but clot formation requires potent activation by extravascular tissue factor Therefore, the intrinsic pathway plays a minimal role in hemostasis. It is not substantially involved in the blood (Gailani and Renne (2007) Arterioscler Thromb Vasc Biol 2007, 27(12):2507-13, Muller, Gailiani, and Renne 2011).

[0129] Preclinical studies to inhibit FXI or FXIa across a range of species using a variety of approaches FXI- / - mice are resistant to experimental venous thrombosis (Wa ng, et al. (2006) J Thromb Haemost; 4:1982-8) and arterial thrombosis (Wang, et al. (20 05) J Thromb Haemost; 3:695-702) and FXIIa-mediated inhibition of FXI. Treatment of mice with an activation-blocking antibody (Ab; 14E11) reduced experimental thrombosis. This results in inhibition of the vasopressin receptor agonist (Cheng, et al. (2010) Blood, 116:3981-9), which may contribute to ischemic stroke. reduced cerebral infarct size in a mouse model of cerebral ischemia (Leung, et al. (2012) Transl Stroke Res 2012; 3:381-9). Anti-FXI blocks the binding and activation of FIX by FXIa. In baboons administered Ab, on collagen-coated vascular grafts, A reduction in platelet-rich thrombus growth was observed (Tucker, et al. (2009) Blood 2009; 113:93 6-44), and similar results were found with 14E11 in this model (Cheng 2010). No excessive bleeding was observed in any of these studies.

[0130] 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) Arteri In oscler Thromb Vasc Biol 2013; 33:1670-8), antisense oligonucleotides Blocking FXI synthesis by steroids provides antithrombotic and anticoagulant effects without excessive bleeding. Furthermore, in rats (Schumacher, et al. (2007) Eur J Pharmacol 2007; 570:167-74) and rabbits (Wong, et al. (2011) J Thromb Thromboly sis 2011; 32:129-37) in venous thrombosis model and arterial thrombosis model, Blocking FXIa with inhibitors has a similar effect.

[0131] Patients with severe FXI deficiency rarely bleed spontaneously and have high fibrinolytic activity. The rare condition of severe FXI deficiency is characterized by only mild trauma-induced bleeding, except in certain tissues. The significance of this is that population studies are needed to clarify the thrombotic profile of these patients compared with the general population. In particular, such studies will necessitate the use of ischemic stroke in these patients. (Salomon 2008) and deep vein thrombosis (DVT) (Salomon, et al. (2011) Blood 200 8; 111: 4113-17). The number of ischemic strokes observed in 115 patients with deficiency (N=1) was The incidence rate was lower than expected in the general population (N=8.6) (p<0.003). The incidence of DVT in patients with severe FXI deficiency (N=0) was significantly higher than in the control population. The number of cases was lower than the predicted number (N=4.7) (p<0.019). Individuals with a BELL above the 90th percentile had twice the risk of developing DVT (Meijer (2000) N Engl J Med. 2000; 342:696-701).

[0132] Recently, patients undergoing total knee replacement, a procedure that predisposes to DVT, have been screened for FXI anemia. Patients were treated with either antisense therapy or standard therapy (enoxaparin). g) a seven-fold reduction in the incidence of venous thrombosis and fewer bleeding events compared to standard care (Not significant) (Buller et al, (2014) N Engl J Med. 372(3):232-40. do i: 10.1056 / NEJMoa1405760. Epub 2014 Dec 7).

[0133] Taken together, the above studies strongly support FXI as a valid target for antithrombotic therapy.

[0134] FXIa antibodies and antigen-binding fragments The present invention provides antibodies that specifically bind to FXI and / or FXIa. In some embodiments, the present invention provides human, rabbit, and cynomolgus monkey FXI and / or The antibody of the present invention specifically binds to FXIa or FXIa. Human monoclonal antibodies and Fabs isolated as described herein include, but are not limited to, human monoclonal antibodies and Fabs isolated as described herein. stomach.

[0135] The present invention relates to FXI and / or FXIa proteins (e.g., human, Antibodies that specifically bind to FXI and / or FXIa in herons and cynomolgus monkeys and an antibody comprising a VH domain having the amino acid sequence of SEQ ID NOs: 9 and 29. The present invention also provides a method for specifically binding to FXI protein and / or FXIa protein. an antibody that binds to any one of the VH CDRs listed in Table 1 below; The present invention also provides antibodies comprising VH CDRs having the sequence of Protein and / or FXIa protein (e.g., human, rabbit, and cynomolgus monkey FXIa protein) XI and / or FXIa), and One, two, three or more VH CDRs having any amino acid sequence of any of the VH CDRs Antibodies comprising (or alternatively consisting of) the CDRs are provided.

[0136] The present invention provides an antibody that specifically binds to FXIa protein, comprising SEQ ID NO: 19 or The present invention also provides an antibody comprising a VL domain having an amino acid sequence of 39. protein and / or FXIa protein (e.g., human, rabbit, and cynomolgus monkey Antibodies that specifically bind to monkey FXI and / or FXIa) are listed in Table 1 below. It contains a VL CDR having the amino acid sequence of any one of the VL CDRs listed. In particular, the present invention provides antibodies containing FXIa proteins (e.g., human, rabbit, and and cynomolgus monkey FXI and / or FXIa), one, two or more having the amino acid sequence of any of the VL CDRs listed in Table 1 below and providing antibodies comprising (or alternatively consisting of) three or more VL CDRs. do.

[0137] Other antibodies of the invention are mutated but still contain the amino acids listed in Table 1 within the CDR regions. The CDR regions depicted in the sequence shown and at least 60, 70, 80, 85, 90, or In some embodiments, the present invention provides a method for the preparation of a nucleotide sequence comprising: Other antibodies of the present invention have, within the CDR regions, the CDR regions depicted in the sequences set forth in Table 1. In comparison, one, two, three, four, or five or fewer amino acids are mutated. The amino acid sequence of the variant is included.

[0138] The present invention also provides FXI and / or FXIa proteins (e.g., human, VH, VL, and full-length heavy chains of antibodies that specifically bind to rabbit and cynomolgus monkey FXIa Nucleic acid sequences encoding the full-length light chain and the full-length light chain are also provided. The antibodies of the present invention can be optimized for expression in cells (e.g., Table 1). The optimized nucleic acid sequences for the light and low chains are shown.

[0139] [Table 1-1]

[0140] [Table 1-2]

[0141] [Table 1-3]

[0142] [Table 1-4]

[0143] [Table 1-5]

[0144] [Table 1-6]

[0145] Other antibodies of the invention may have mutated amino acids or nucleic acids encoding amino acids. However, the sequences listed in Table 1 may be at least 60, 65, 70, 75, 80, 85, Some embodiments include antibodies with 90, 95, or 100 percent identity to the variable Within the region, a sequence depicted within the sequence set forth in Table 1 may be used while retaining substantially the same antigen binding activity. No more than one, two, three, four, or five amino acids when compared to the indicated variable region. The amino acid sequence of the mutant is mutated.

[0146] Each of these antibodies can bind to FXI and / or FXIa and therefore has the VH sequence , VL sequence, full-length light chain sequence, and full-length heavy chain sequence (amino acid sequence and and "mix and match" the FXI-binding antibodies (with the corresponding nucleotide sequences) to produce other FXI-binding antibodies of the present invention. Such "mixed and matched" antibodies and / or FXIa-binding antibodies can be produced. "Matched" FXI-binding antibodies and / or FXIa-binding antibodies are known in the art. Known binding assays (e.g., ELISA and other assays described in the Examples section) can be used. When these chains are mixed and matched, a particular VH / V The VH sequence derived from the L pairing is replaced with a structurally similar VH sequence. The full-length heavy chain sequence derived from a specific full-length heavy chain / full-length light chain pairing is then compared with a structurally similar full-length heavy chain. Similarly, the VL sequence from a particular VH / VL pairing is A structurally similar VL sequence is intended to be substituted. Similarly, a particular full-length heavy chain / full-length light chain The full-length light chain sequence resulting from the pairing is replaced with a structurally similar full-length light chain sequence. .

[0147] Thus, in one aspect, the present invention provides a method for the treatment of a leukemia comprising administering to a subject a leukemia patient a method for the treatment of a leukemia patient ... a heavy chain variable domain comprising the amino acid sequence SEQ ID NO: 19 and SEQ ID NO: 39 Isolated antibody having a light chain variable domain comprising a selected amino acid sequence or its antigen-binding properties In this case, the antibody is FXI and / or FXIa (e.g., human, It specifically binds to FXIa in herons and cynomolgus monkeys.

[0148] More specifically, in certain embodiments, the present invention provides SEQ ID NOs: 9 and 29; or 19 and and 39, respectively. The present invention provides an isolated antibody or antigen-binding region thereof having a domain.

[0149] In a specific embodiment, the present invention provides a method for the preparation of a human FXI antibody that specifically binds to human FXI and / or FXIa. The antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence of SEQ ID NO: 9. It comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0150] In a specific embodiment, the present invention provides a method for the preparation of a human FXI antibody that specifically binds to human FXI and / or FXIa. The antibody or antigen-binding fragment thereof provided herein comprises 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.

[0151] In another aspect, the present invention provides (i) amino acids optimized for expression in mammalian cells. an amino acid sequence selected from the group consisting of SEQ ID NO: 11 or 31; A full-length heavy chain containing the amino acid sequence optimized for expression in mammalian cells. and obtaining a full-length light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21 or 41. or (ii) a functional protein comprising an antigen-binding portion thereof. More specifically, in certain embodiments, the present invention provides SEQ ID NOs: 11 and 31; or 19 and and 39. An isolated antibody having a heavy chain and a light chain comprising an amino acid sequence selected from or an antigen-binding region thereof.

[0152] In a specific embodiment, the present invention provides a method for the preparation of a human FXI antibody that specifically binds to human FXI and / or FXIa. The antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence of SEQ ID NO: 11. and a light chain comprising the amino acid sequence of SEQ ID NO:21.

[0153] In a specific embodiment, the present invention provides a method for the preparation of a human FXI antibody that specifically binds to human FXI and / or FXIa. The antibody or antigen-binding fragment thereof provided herein comprises 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.

[0154] As used herein, the terms "complementarity determining region" and "CDR" refer to a region that is capable of determining antigen specificity. and refers to the sequence of amino acids within the antibody variable region that confers antigen-binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3). There are three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). be.

[0155] The precise amino acid sequence boundaries of a given CDR can be determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National In Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), Lefranc e t al., (2003) Dev. Comp. Immunol., 27, 55-77 ("IMGT" numbering scheme) Many well-known schemes, including those described in the You can easily use either of them to make a decision.

[0156] For example, according to Kabat, NOV109, an antibody in the heavy chain variable domain (VH), The CDR amino acid residues of HCDR0 are 31 to 35 (HCDR1), 50 to 66 (HCDR2), and and 99-111 (HCDR3), which are located within the light chain variable domain (VL). CDR amino acid residues are 22 to 35 (LCDR1), 51 to 57 (LCDR2), and According to Chothia, the VH The CDR amino acids are 26-32 (HCDR1), 52-57 (HCDR2), and 99 The amino acid residues in VL are numbered 25 to 33 (HCDR3). Numbered LCDR1), 51-53 (LCDR2), and 92-99 (LCDR3) By combining both Kabat and Chothia CDR definitions, Therefore, the CDRs are amino acid residues 26 to 35 (HCDR1), 50 to 66 (H CDR2), and 99-111 (HCDR3), and amino acid residues 2 in human VL 2-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3) By combining both Kabat and Chothia CDR definitions, Therefore, the "combined" CDRs consist of amino acid residues 26-35 (HCDR1), 50 (HCDR2) and 51 (HCDR3) in human VH. ~66 (HCDR2), and 99–108 (HCDR3), as well as amino acids in human VL. The acid residues 24-38 (LCDR1), 54-60 (LCDR2), and 93-101 ( LCDR3). Another example, according to IMGT, is a heavy chain variable domain (VH) The CDR amino acid residues are 26 to 33 (HCDR1), 51 to 58 (HCDR2), and and 97-108 (HCDR3), and C in the light chain variable domain (VL). The DR amino acid residues are 27–36 (LCDR1), 54–56 (LCDR2), and 9 Table 1 shows anti-FXI / FXIa antibodies, e.g., For example, for NOV1090 and NOV1401, Kabat, Chothia, and Exemplary HCDR1, HCDR2, HCDR3, LCDR by combination and IMGT In another aspect, the present invention provides a method for producing a medicament comprising administering to a subject a medicament therapies listed in Table 1, comprising administering to a subject a medicament therapies therapies the invention provides to a subject a medicament ... CDR1, CDR2, and CDR3 of the heavy and light chains, or a combination thereof, The amino acid sequence of the VH CDR1 of the antibody is provided by the sequence The amino acid sequences of the VH CDR2 of the antibody are shown in SEQ ID NOs: 4 and 23. The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NOs: 5 and 25. The amino acid sequences of the VL CDR1 of the antibody are shown in SEQ ID NOs: 13 and 33. The amino acid sequences of the VL CDR2 of the antibody are shown in SEQ ID NOs: 14 and 34. The amino acid sequences of the VL CDR3 of the antibody are shown in SEQ ID NOs: 15 and 35. These CDR regions are represented using the Kabat system.

[0157] Alternatively, the Chothia system (Al-Lazikani et al., (1997), JMB 273 927-948) The amino acid sequence of the VH CDR1 of the antibody is SEQ ID NO:6, as defined using 26. The amino acid sequence of the VH CDR2 of the antibody is shown in SEQ ID NOs: 7 and 27. The amino acid sequences of the VH CDR3 of the antibody are shown in SEQ ID NOs: 8 and 28. The amino acid sequences of the VL CDR1 of the antibody are shown in SEQ ID NOs: 16 and 36. The amino acid sequences of the VL CDR2 of the antibody are shown in SEQ ID NOs: 17 and 37. The amino acid sequences of the VL CDR3 of the antibodies are shown in SEQ ID NOs: 18 and 38.

[0158] Alternatively, the amino acid sequence of the VH CDR1 of the antibody may be determined using a combinatorial system. The amino acid sequence is shown in SEQ ID NO: 46. The amino acid sequence of the VH CDR2 of the antibody is shown in the sequence The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NO:5. The amino acid sequence of the VL CDR1 of the antibody is shown in SEQ ID NO: 33. The amino acid sequence of the VL CDR2 is shown in SEQ ID NO: 14. The amino acid sequence is shown in SEQ ID NO:15.

[0159] Alternatively, the VH CDRs of the antibody as defined using the IMGT numbering scheme The amino acid sequence of antibody VH CDR2 is shown in SEQ ID NO: 43. The sequence is shown in SEQ ID NO: 44. The amino acid sequence of the VH CDR3 of the antibody is shown in SEQ ID NO: The amino acid sequence of the VL CDR1 of the antibody is shown in SEQ ID NO:47. The amino acid sequence of the VL CDR2 of the antibody is shown in SEQ ID NO: 37. The amino acid sequence of the L CDR3 is shown in SEQ ID NO:15.

[0160] Each of these antibodies is capable of binding to FXI and / or FXIa. The antigen binding specificity is mainly determined by the CDR1, CDR2, and CDR3 regions. Given that each antibody is derived from other FXI-binding molecules of the present invention and / or To create an FXIa binding molecule, VH CDR1, VH CDR2, and VH CDR3 are used. and VL CDR1, VL CDR2, and VL CDR3. Preferably, the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence are and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence are referred to as " It is possible to "mix and match" (i.e., to combine CDRs from different antibodies (These "mixed and matched" FXI-binding molecules The antibody and / or FXIa-binding antibody may be assayed using binding assays and techniques known in the art. Binding assays described in the Examples (e.g., ELISA, SET, BIACORE™ assays) When VH CDR sequences are mixed and matched, The CDR1, CDR2, and / or CDR3 sequences derived from a particular VH sequence are The VL CDR sequences are replaced with structurally similar CDR sequences. When mixing and matching, the CDR1 and CDR2 sequences derived from a particular VL sequence are , and / or CDR3 sequences are replaced with structurally similar CDR sequences. Those skilled in the art will appreciate that novel VH and VL sequences can be generated by combining one or more VH CDR region sequences. The VL and / or VL CDR region sequences are used in the methods of the present invention for the monoclonal antibodies of the present invention. Created by substituting structurally similar sequences derived from the CDR sequences shown in the subtitles In addition to the foregoing, in one embodiment, The antigen-binding fragment of the antibody to be analyzed comprises VH CDR1, VH CDR2, and VH CDR3. 3, or VL CDR1, VL CDR2, and VL CDR3. In this case, the fragments may be linked to FXI and / or FXIa as single variable domains. and combine them.

[0161] In certain embodiments of the invention, the antibodies or antigen-binding fragments thereof are those listed in Table 1. More specifically, the antibody or its antigen may have the heavy and light chain sequences of the Fab. The binding fragments can have the heavy and light chain sequences of NOV1090 and NOV1401.

[0162] In another embodiment of the invention, an antibody that specifically binds to FXI and / or FXIa Alternatively, the antigen-binding fragment may comprise a heavy chain variable fragment as defined by Kabat and listed in Table 1. CDR1 of the heavy chain variable region, CDR2 of the heavy chain variable region, CDR3 of the light chain variable region The present invention further comprises CDR1, CDR2 of the light chain variable region, and CDR3 of the light chain variable region. In another embodiment, an antibody or antigen that specifically binds to FXI and / or FXIa The binding fragments are heavy chain variable region fragments as defined by Chothia and listed in Table 1. CDR1 of the heavy chain variable region, CDR2 of the heavy chain variable region, CDR3 of the light chain variable region In another embodiment, the CDR comprises R1, CDR2 of the light chain variable region, and CDR3 of the light chain variable region. The antibodies or antigen-binding fragments that specifically bind to FXI and / or FXIa are CDR1 of the heavy chain variable region, defined by the matching system and listed in Table 1; CDR2 of the heavy chain variable region, CDR3 of the heavy chain variable region, CDR1 of the light chain variable region, In yet another embodiment of the present invention, the CDR2 of the light chain variable region and the CDR3 of the light chain variable region are included. Antibodies or antigen-binding fragments that specifically bind to FXI and / or FXIa are available from IM CDR1 of the heavy chain variable region as defined by GT and listed in Table 1, CDR2 of the heavy chain variable region, CDR3 of the light chain variable region, CDR1 of the light chain variable region, R2, and CDR3 of the light chain variable region.

[0163] In a specific embodiment, the present invention provides antibodies that specifically bind to FXI and / or FXIa. An antibody comprising: CDR1 of the heavy chain variable region of SEQ ID NO: 3; CDR2 of the heavy chain variable region of SEQ ID NO: 4 R2; CDR3 of the heavy chain variable region of SEQ ID NO: 5; CDR1 of the light chain variable region of SEQ ID NO: 13; CDR2 of the light chain variable region of SEQ ID NO: 14; and CDR3 of the light chain variable region of SEQ ID NO: 15 The antibody comprises:

[0164] In a specific embodiment, the present invention provides antibodies that specifically bind to FXI and / or FXIa. an antibody comprising CDR1 of the heavy chain variable region of SEQ ID NO: 23, CDR2 of the heavy chain variable region of SEQ ID NO: 24, CDR2 of the heavy chain variable region of SEQ ID NO: 25, CDR3 of the light chain variable region of SEQ ID NO: 33 DR1, CDR2 of the light chain variable region of SEQ ID NO: 34, and CDR3 of the light chain variable region of SEQ ID NO: 35 Includes antibodies containing CDR3.

[0165] In a specific embodiment, the present invention provides antibodies that specifically bind to FXI and / or FXIa. an antibody comprising CDR1 of the heavy chain variable region of SEQ ID NO: 6, CDR2 of the heavy chain variable region of SEQ ID NO: 7, DR2, CDR3 of the heavy chain variable region of SEQ ID NO: 8, CDR1 of the light chain variable region of SEQ ID NO: 16 , CDR2 of the light chain variable region of SEQ ID NO: 17, and CDR3 of the light chain variable region of SEQ ID NO: 18 3, including antibodies.

[0166] In a specific embodiment, the present invention provides antibodies that specifically bind to FXI and / or FXIa. an antibody comprising CDR1 of the heavy chain variable region of SEQ ID NO: 26, CDR2 of the heavy chain variable region of SEQ ID NO: 27, CDR2 of the heavy chain variable region of SEQ ID NO: 28, CDR3 of the light chain variable region of SEQ ID NO: 36 DR1, CDR2 of the light chain variable region of SEQ ID NO: 37, and CDR3 of the light chain variable region of SEQ ID NO: 38 Includes antibodies containing CDR3.

[0167] In specific embodiments herein, specific binding to FXI and / or FXIa an antibody comprising: CDR1 of the heavy chain variable region of SEQ ID NO: 43; CDR2; CDR3 of the heavy chain variable region of SEQ ID NO: 45; CDR4 of the light chain variable region of SEQ ID NO: 47 R1; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 15 Antibodies containing DR3 are displayed.

[0168] In specific embodiments herein, specific binding to FXI and / or FXIa An antibody comprising: CDR1 of the heavy chain variable region of SEQ ID NO: 46; CDR2 of the heavy chain variable region of SEQ ID NO: 4 DR2; CDR3 of the heavy chain variable region of SEQ ID NO: 5; CDR1 of the light chain variable region of SEQ ID NO: 33 CDR2 of the light chain variable region of SEQ ID NO: 14; and CDR3 of the light chain variable region of SEQ ID NO: 15 Antibodies containing 3 are presented.

[0169] In certain embodiments, the present invention provides a method for the treatment of FXI and / or FX, as described in Table 1. In a preferred embodiment, the antibody or antigen-binding fragment specifically binds to FX Ia. Antibodies or antigen-binding fragments that bind to FXIa and / or FXIa include NOV1090 and and NOV1401.

[0170] The variable regions or full-length chains of the antibody use human germline immunoglobulin genes As used herein, a human antibody refers to a "product of" a particular germline sequence when derived from a human lineage. A heavy or light chain variable region or a full-length heavy chain or Such systems include transgenic mice carrying human immunoglobulin genes. A mouse is immunized with an antigen of interest or is presented on a phage with the antigen of interest. This involves screening a library of human immunoglobulin genes. Human antibodies that are "the product of" or "derived from" immunoglobulin sequences of cell lines The amino acid sequences of human antibodies are compared with the amino acid sequences of human germline immunoglobulins. , the human germline sequence whose sequence is most similar to the sequence of a human antibody (i.e., has the highest percent identity) By selecting immunoglobulin sequences of the cell lineage, it can be identified as such. do.

[0171] "A product of" or "derived from" a specific human germline immunoglobulin sequence Human antibodies that are "humanized" include antibodies that have undergone, for example, naturally occurring somatic mutations or site-specific mutations. may contain amino acid differences compared to the germline sequence due to genetic introduction; , in the VH framework region or VL framework region, the selected human antibody The amino acid sequence is an amino acid sequence encoded by a human germline immunoglobulin gene. The sequences are at least 90% identical to those of the germline immunoglobulins of other species, making human antibodies identified as human when compared to the human amino acid sequence (e.g., mouse germline sequence) Typically, human antibodies contain amino acid residues that correspond to the amino acid sequence of the target protein. the amino acid sequence is an amino acid sequence encoded by a germline immunoglobulin gene, At least 60%, 70%, 80%, 90%, or at least 95%, or even less They may be at least 96%, 97%, 98%, or 99% identical.

[0172] Recombinant human antibodies may contain human amino acids within the VH or VL framework regions. The amino acid sequence encoded by the germline immunoglobulin gene is a 10-amino acid sequence. Typically, human antibodies exhibit the following differences: The amino acid sequence encoded by the immunoglobulin gene of the same series is 5 amino acids or less, Alternatively, they may exhibit no more than 4, 3, 2, or 1 amino acid difference. Examples of immunoglobulin genes include the germline variable domain fragments described below, as well as , DP47 and DPK9.

[0173] homologous antibody In yet another embodiment, the present invention provides a method for the production of a nucleic acid molecule comprising the sequence set forth in Table 1 (e.g., SEQ ID NO:2). 9, 31, 39, or 41) Although fragments are provided, antibodies may be directed to FXI and / or FXIa proteins (e.g., For example, human FXIa, rabbit FXIa, and cynomolgus monkey FXIa) and NOV1 The antibodies listed in Table 1, such as 090 and NOV1401, retain the desired functional properties. In a specific embodiment, such homologous antibodies have the CDR amino acids listed in Table 1. Acid sequences (e.g., Kabat CDR, Chothia CDR, IMGT CDR, or or combination CDR).

[0174] For example, the present invention provides an isolated antibody or antibody fragment comprising a heavy chain variable domain and a light chain variable domain. wherein the heavy chain variable domain is selected from the group consisting of SEQ ID NO: 9 and 29 and at least 80%, at least 90%, or an amino acid sequence at least 95% identical thereto; the light chain variable domain comprises an amino acid sequence of SEQ ID NO: 19 and 39, and at least 80%, at least 9 100%, or at least 95% identical amino acid sequence; the antibody may be FXI and / or to FXIa (e.g., human FXIa, rabbit FXIa, and cynomolgus monkey FXIa). In one embodiment, the isolated antibody or functional antigen-binding fragment thereof specifically binds to a The heavy chain variable domain comprises a heavy chain variable domain and a light chain variable domain, in which case the heavy chain variable domain is 9 and at least 80%, at least 90%, or at least 95% the light chain variable domain contains at least one amino acid sequence identical to the amino acid sequence of SEQ ID NO: 19; comprises an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical; The antibody may be FXI and / or FXIa (e.g., human FXIa, rabbit FXIa, and In one embodiment, the isolated antibody or its function is A functional antigen-binding fragment comprises a heavy chain variable domain and a light chain variable domain, where: The heavy chain variable domain has at least 80%, at least 90%, and 0%, or at least 95% identical amino acid sequence; the light chain variable domain comprises SEQ ID NO: No. 39 and at least 80%, at least 90%, or at least 95% identical % identical amino acid sequence; the antibody may be FXI and / or FXIa (e.g., human F XIa, rabbit FXIa, and cynomolgus monkey FXIa). In certain embodiments, the heavy and light chain sequences are HCDR1 sequences as defined by Kabat. column, HCDR2 array, HCDR3 array, LCDR1 array, LCDR2 array, and LCD R3 sequences, e.g., SEQ ID NOs: 3, 4, 5, 13, 14, and 15, respectively. In certain other aspects of the invention, the heavy and light chain sequences are selected from the group consisting of: Defined HCDR1 sequence, HCDR2 sequence, HCDR3 sequence, LCDR1 sequence, LC DR2 sequences, and LCDR3 sequences, e.g., SEQ ID NOs: 6, 7, 8, 16, 1 In certain other embodiments, the heavy and light chain sequences are The HCDR1 sequence, HCDR2 sequence, HCDR3 sequence, and LC sequence are defined by the system. DR1 sequence, LCDR2 sequence, and LCDR3 sequence, e.g., SEQ ID NO: 46, respectively. , 4, 5, 33, 14, and 15. In certain other embodiments, the heavy chain sequences and The light chain sequence is a sequence of HCDR1, HCDR2, and HCDR3 as defined by IMGT. 3 sequence, LCDR1 sequence, LCDR2 sequence, and LCDR3 sequence, e.g., Further included are SEQ ID NOs: 43, 44, 45, 47, 37, and 15.

[0175] In other embodiments, the VH and / or VL amino acid sequences are selected from the group consisting of those shown in Table 1. Sequences that are 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, and 98% %, or 99% identical. In other embodiments, the VH amino acid sequence and / or The VL amino acid sequence may have amino acid substitutions at no more than 1, 2, 3, 4, or 5 amino acid positions. The VH and VL regions of the antibodies listed in Table 1 may be identical except for amino acid substitutions. Antibodies with VH and VL regions that share a large (i.e., 80% or more) identity are , SEQ ID NO: 10 or 30, and nucleic acids encoding SEQ ID NO: 20 and 40, respectively. Mutagenesis of the molecule (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) The encoded modified antibodies are then assayed for functionality using the functional assays described herein. This can be obtained by examining retention.

[0176] In other embodiments, the full-length heavy chain amino acid sequence and / or the full-length light chain amino acid sequence are 1 and (e.g., SEQ ID NOs: 11 and / or 21, or 31 and / or Or 41), 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The full length of either SEQ ID NO: 11 or 31 heavy chain, and a full-length light chain of either SEQ ID NO: 21 or 41 and a large (i.e., Antibodies having full-length heavy chains and full-length light chains with identity (80% or more) are suitable for such polyclonal antibodies. Mutagenesis of a nucleic acid molecule encoding a peptide (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) and functional assays described herein were used to identify the coding region. The resulting modified antibodies can be tested for retention of function.

[0177] In one aspect herein, an isolated antibody comprising a heavy chain and a light chain or a functional antigen-binding domain thereof is provided. and wherein the heavy chain is an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 31. amino acid sequence at least 80%, at least 90%, or at least 95% identical to the amino acid sequence the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 and 41; amino acids that are at least 80%, at least 90%, or at least 95% identical to the sequence The antibody may comprise a sequence encoding FXI and / or FXIa (e.g., human FXIa, rabbit FXIa, In one embodiment, the isolated antibody specifically binds to a specific antibody against a specific target of the antibody, such as a cynomolgus FXIa or a cynomolgus FXIa. or a functional antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain has the sequence 11 and at least 80%, at least 90%, or at least 9 the light chain comprises an amino acid sequence that is at least 8.5% identical to the amino acid sequence of SEQ ID NO: 21; 0%, at least 90%, or at least 95% identical amino acid sequence; FXI and / or FXIa (e.g., human FXIa, rabbit FXIa, and cyno FXIa) In one embodiment, the isolated antibody or functional antibody thereof specifically binds to the IgG1a (monkey FXIa). The original binding fragment comprises a heavy chain and a light chain, wherein the heavy chain has the amino acid sequence of SEQ ID NO:31. amino acid sequences at least 80%, at least 90%, or at least 95% identical to the sequence the light chain comprises at least 80%, at least 90%, or both the amino acid sequence of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42; %, or at least 95% identical amino acid sequence to FXI; the antibody Specific for FXIa (e.g., human FXIa, rabbit FXIa, and cynomolgus monkey FXIa) In certain embodiments of the invention, the heavy and light chain sequences are HCDR1 sequence, HCDR2 sequence, HCDR3 sequence, LCDR1 sequence, LCDR2 sequences, and LCDR3 sequences, e.g., SEQ ID NOs: 3, 4, 5, and 13, respectively , 14, and 15. In certain other embodiments of the invention, the heavy chain sequences and the light chain sequences The sequences are the HCDR1 sequence, HCDR2 sequence, HCDR3 sequence, and HCDR4 sequence as defined by Chothia. 3 sequence, LCDR1 sequence, LCDR2 sequence, and LCDR3 sequence, e.g., Further included are SEQ ID NOs: 6, 7, 8, 16, 17, and 18. In certain other embodiments, The HCDR1 sequence, HCDR2 sequence, and the light chain sequence are defined by a combination system. 2 sequence, HCDR3 sequence, LCDR1 sequence, LCDR2 sequence, and LCDR3 sequence, e.g. For example, further include SEQ ID NOs: 46, 4, 5, 33, 14, and 15, respectively. In other embodiments, the heavy and light chain sequences comprise HCDR1 sequences as defined by IMGT. column, HCDR2 array, HCDR3 array, LCDR1 array, LCDR2 array, and LCD R3 sequences, e.g., SEQ ID NOS: 43, 44, 45, 47, 37, and 15, respectively. This includes:

[0178] In other embodiments, the nucleotide sequences of the full-length heavy chain and / or full-length light chain are as set forth in Table 1. The sequences to be analyzed (e.g., SEQ ID NOs: 12 and / or 22, or 32 and / or 42) and 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% % may be identical.

[0179] In other embodiments, the nucleotide sequence of the heavy chain variable region and / or the light chain variable region is The sequences shown in Table 1 (e.g., SEQ ID NOs: 10 and / or 20, or 30 and / or or 40) and 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.

[0180] As used herein, percent identity between two sequences refers to the best fit between the two sequences. Consider the number of gaps that need to be introduced for the alignment and the length of each gap. is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions) (number of positions / total number of positions × 100). Sequence comparison and percent identity between two sequences. Determination of is accomplished using a mathematical algorithm, described in the non-limiting examples below. It is possible.

[0181] Additionally or alternatively, the protein sequences of the present invention may be identified in the context of public databases. and perform a search using the sequence as a "query sequence" to identify related sequences. For example, such a search can be performed using the method of Altschul et al., 1990 J. Mol. Biol. This was performed using the BLAST program (version 2.0) according to 215:403-10. can be done.

[0182] Antibodies with conservative modifications In certain embodiments, an antibody of the invention comprises a CDR1 sequence, a CDR2 sequence, and a CDR a heavy chain variable region comprising three sequences, and a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence wherein one or more of these CDR sequences is The antibodies described herein or conservative modifications thereof are based on designated amino acid sequences. and the antibody retains the desired functional properties of the FXIa-binding antibodies of the present invention.

[0183] Thus, the present invention provides a heavy chain antibody comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence. a light chain variable region comprising a variable region, and a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence An isolated antibody or antigen-binding fragment thereof comprising a CDR1 amino acid sequence of a heavy chain variable region. The amino acid sequence is selected from the group consisting of SEQ ID NOs: 3 and 23, and conservative modifications thereof. The CDR2 amino acid sequences of the heavy chain variable region are set forth in SEQ ID NOs: 4 and 24, and their respective conserved sequences. and the CDR3 amino acid sequence of the heavy chain variable region is selected from the group consisting of: and 25, and conservative modifications thereof, The R1 amino acid sequence is selected from the group consisting of SEQ ID NOs: 13 and 33, and conservative modifications thereof and the CDR2 amino acid sequence of the light chain variable region is selected from SEQ ID NOs: 14 and 34. and conservative modifications thereof, is selected from the group consisting of SEQ ID NOs: 15 and 35, and conservative modifications thereof; An antibody or antigen-binding fragment thereof that specifically binds to OX-1 is provided.

[0184] In one embodiment herein, a heavy chain comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence a light chain variable region comprising a variable region, and a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence An isolated antibody or antigen-binding fragment thereof comprising a CDR1 amino acid sequence of a heavy chain variable region. The amino acid sequence is selected from the group consisting of the amino acid sequences set forth in Table 1 and conservative modifications thereof. the CDR2 amino acid sequence of the heavy chain variable region is the amino acid sequence shown in Table 1 and and conservative modifications thereof; wherein the CDR3 amino acid sequence of the heavy chain variable region is selected from the group consisting of: 1 and conservative modifications thereof; The CDR1 amino acid sequence of the variable region is the amino acid sequence shown in Table 1 and its conservative sequences. the CDR2 amino acid sequence of the light chain variable region is selected from the group consisting of: and a light chain variable region CD4A1, CD4B1, CD4C1, CD4D1, CD4E1, CD4F1, and CD4F2 are selected from the group consisting of the amino acid sequences of the light chain variable region and conservative modifications thereof; The R3 amino acid sequence consists of the amino acid sequence set forth in Table 1 and conservative modifications thereof the antibody or antigen-binding fragment thereof specifically binds to FXIa.

[0185] In other embodiments, the antibodies of the invention are optimized for expression in mammalian cells. and has a full-length heavy chain sequence and a full-length light chain sequence, one or more of which sequences are , an amino acid sequence designated based on an antibody described herein or a conservative modification thereof and the antibody retains the desired functional properties of the FXIa-binding antibodies of the present invention. Therefore, the present invention provides a method for the expression of a full-length heavy chain and a full-length light chain in mammalian cells. 1. An optimized isolated antibody, wherein the full length heavy chain is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 31, and their derivatives. and the full length light chain has an amino acid sequence selected from the group of SEQ ID NO: 21 or 4, 1, and conservative modifications thereof, and / or specific for FXIa (e.g., human, rabbit, and cynomolgus monkey FXIa) The present invention provides an antibody that binds to

[0186] Antibodies that bind to the same epitope The present invention relates to the FXI-binding antibodies and / or FXIa-binding antibodies listed in Table 1. Thus, antibodies that bind to the same epitope as FXI and / or FX Ia binding assays (such as the FXI and / or FXIa binding assays described in the Examples section) competes with other antibodies of the present invention (e.g., inhibits the binding of other antibodies of the present invention in the same manner as Statistically significant competitive inhibition by binding to epitopes or overlapping epitopes Further antibodies can be identified based on their ability to inhibit the activity of the antibodies of the present invention. the ability of the test antibody to inhibit binding to the FXI protein and / or FXIa protein The test antibody competes with the antibody for binding to FXI and / or FXIa. Such antibodies may bind to FXI protein, according to one non-limiting theory. The antibody is either the same as or related to the competing antibody on the protein and / or FXIa protein. capable of binding to related (e.g., structurally similar or spatially proximate) epitopes In certain embodiments, the antibodies of the present invention may be used in combination with the FXI and / or The antibody that binds to an epitope on FXIa is a human monoclonal antibody. Human monoclonal antibodies can be prepared and isolated as described herein. Cut.

[0187] As used herein, in the presence of an equimolar concentration of a competing antibody, the competing antibody is an antibody capable of binding to a target protein of the present invention. FX identical to the antibody or antigen-binding fragment (e.g., NOV1401 or NOV1090) I epitope and / or FXIa epitope, and The binding of the active fragment to FXI and / or FXIa is increased by more than 50% (e.g., 80% , 85%, 90%, 95%, 98%, or 99% inhibition of binding. This can be achieved, for example, by any of the methods well known to those skilled in the art. The amount of ATP can be determined in a combined assay.

[0188] As used herein, an antibody or antigen-binding fragment thereof refers to a competing antibody or its antigen. The binding fragment may bind to the same FXI epitope and / or antigen as the antibody or antigen-binding fragment of the present invention. or FXIa epitopes, or overlapping FXI and / or FX epitopes The FXI antibodies and / or FXI antibodies of the present invention, as long as they do not bind to the Ia epitope, a) Competing with an antibody or antigen-binding fragment (e.g., NOV1401 or NOV1090) As used herein, a competing antibody or antigen-binding fragment thereof refers to (i) an antibody or antigen-binding fragment thereof of the present invention sterically blocking (e.g., blocking) an antibody or antigen-binding fragment from binding to its target The competing antibodies are located adjacent to non-overlapping FXI and / or FXIa epitopes. and physically prevent the antibody or antigen-binding fragment of the invention from binding to its target. and / or (ii) different, non-overlapping FXI epitopes and / or binds to the FXIa epitope and binds to the protein, and the FXI antibody and / or The FXIa antibody or antigen-binding fragment exhibits the same activity as that observed in the absence of the conformational change. The FXI protein and / or does not include a competing antibody or antigen-binding fragment thereof directed against the FXIa protein.

[0189] Engineered and modified antibodies As a starting material for engineering modified antibodies with altered properties from the starting antibody, Antibodies having one or more of the VH and / or VL sequences of the present invention can be used to Further, antibodies of this type can be prepared by isolating one or both variable regions (i.e. , VH and / or VL), e.g., within one or more CDR regions, and / or or by modifying one or more residues in one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant region. For example, the effector functions of the antibody can also be modified. .

[0190] One type of variable region engineering that can be performed is CDR grafting. , primarily through amino acid residues located within the six heavy and light chain complementarity-determining regions (CDRs) For this reason, the amino acid sequences within the CDRs are highly consistent between individual antibodies. The diversity of CDR sequences is greater than that of sequences outside the CDRs. CDR sequences are responsible for most antibody-antigen interactions. This contributes to the use of the framework sequences from different antibodies with different properties. Constructing an expression vector containing CDR sequences derived from a specific naturally occurring antibody that has been rafted By using recombinant antibodies, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies. (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; Win ter, U.S. Pat. No. 5,225,539, and Queen et al., U.S. Pat. Patent No. 5,530,101; Patent No. 5,585,089; Patent No. 5,693 ,762; and 6,180,370).

[0191] Therefore, another embodiment of the present invention is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23. and CDR1 sequences having amino acid sequences selected from the group consisting of SEQ ID NOs: 4 and 24. a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 25; a heavy chain variable region comprising each of the CDR3 sequences having the amino acid sequence of SEQ ID NO: 13 and 33, a CDR1 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 14, and 34, and a CDR2 sequence having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 each of the CDR3 sequences consisting of an amino acid sequence selected from the group consisting of 5 and 35 and a light chain variable region having the following structure: Such antibodies include the VH and VL CDR sequences of a monoclonal antibody. The antibodies may have the same or different framework sequences as those of the antibodies described above, but may contain different framework sequences from these antibodies.

[0192] Such framework sequences are based on published D sequences, including germline antibody gene sequences. The sequences can be obtained from the NA database or published references. For example, human heavy chains and The germline DNA sequences of the human and light chain variable region genes were obtained from the human germline sequence database. VBase, a database available on the internet at mrc-cpe.cam.ac.uk / vbase ), as well as Kaba t, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed., US Depa rtment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M. . et al., 1992 J. Mol. Biol. 227:776-798; and Cox, JPL et al., 1994 Eur. J Immunol . 24:827-836.

[0193] Examples of framework sequences for use in the antibodies of the invention are: The framework sequences used by the antibody, for example, the framework sequences used by the monoclonal antibodies of the present invention, The consensus sequence and / or framework sequence used in the present study are structurally similar to the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence. The sequences, as well as the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence are , the sequences found in the germline immunoglobulin genes from which the framework sequences are derived. Alternatively, the CDR sequences can be grafted into framework regions having the same sequence as the CDR sequences. , a framework region containing one or more mutations compared to the germline sequence For example, in certain cases, the amino acid sequence may be grafted into a framework region. It is believed to be beneficial to mutate residues to maintain or enhance the antigen-binding ability of the antibody. (e.g., Queen et al., U.S. Pat. No. 5,530,101; Nos. 5,585,089; 5,693,762; and 6,18 (See US Pat. No. 5,370,030.) The antibodies and antigen-binding fragments described herein The frameworks that can be used as a scaffolding on which to build the VH1A, VH1B, and VH1C are These include, but are not limited to, H3, Vk1, Vl2, and Vk2. Further frameworks are known, for example on the Internet at vbase.mrc-cpe.cam. ac.uk / index.php?&MMN_position=1:1 can.

[0194] Thus, an embodiment of the present invention is an antibody selected from the group consisting of SEQ ID NOs: 9 and 29. amino acid sequence, or 1, 2, 3, 4, or Contains an amino acid sequence with five amino acid substitutions, deletions, or additions. an amino acid sequence selected from the group consisting of SEQ ID NO: 19 or 39, comprising a heavy chain variable region comprising or 1, 2, 3, 4, or 5 amino acids within the framework regions of such sequences. A light chain having an amino acid sequence with amino acid substitutions, deletions, or additions may be used. The present invention relates to an isolated FXIa-binding antibody or antigen-binding fragment thereof, which further comprises a variable region.

[0195] Another type of variable region modification is known as "affinity maturation," which involves the modification of the VH CDR1 region. within the VH CDR2 region, and / or within the VH CDR3 region, and / or is within the VL CDR1 region, the VL CDR2 region, and / or the VL CDR3 region mutating amino acid residues within the The goal is to improve the affinity (e.g., affinity) of the target gene. CR-mediated mutagenesis can be performed to introduce mutations, and the methods described herein , in vitro assays or in vivo assays, as also presented in the Examples section. In this manner, the effect on antibody binding or other functional properties of interest can be assessed. Conservative modifications (discussed above) can be introduced. Mutations can be made by substitution of amino acids. In addition, there may be additions or deletions within the CDR regions. No more than one, two, three, four, or five residues are altered.

[0196] Thus, in another embodiment, the present invention provides a method for the preparation of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23. 1, 2, 3, 4, or 5, as compared to the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 23. indicates an amino acid sequence with five amino acid substitutions, deletions, or additions. a VH CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 24; 1, 2, 3, 4, or 5 amino acid substitutions compared to the sequence of SEQ ID NOs: 4 and 24 VH CD having an amino acid sequence with amino acid substitutions, deletions, or additions R2 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 25, or SEQ ID NOs: 1, 2, 3, 4, or 5 amino acid substitutions compared to Nos. 5 and 25; VH CDR3 region having an amino acid sequence with deletion or addition of amino acids, SEQ ID NO: an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 and 33, or SEQ ID NOs: 13 and 3 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or is a VL CDR1 region having an amino acid sequence with an amino acid addition, SEQ ID NO: 14, and 34, or compared to SEQ ID NOs: 14 and 34 , 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions and a VL CDR2 region having an amino acid sequence having the amino acid sequence of SEQ ID NOs: 15 and 35. or SEQ ID NOs: 15 and 35, , 2, 3, 4, or 5 amino acid substitutions, deletions, or additions An isolated FX comprising a heavy chain variable region having a VL CDR3 region having an amino acid sequence Ia-binding antibodies or antigen-binding fragments thereof are provided.

[0197] Thus, in another embodiment, the present invention provides a method for the preparation of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26. 1, 2, 3, 4, or 5 amino acid sequences as compared to the amino acid sequence of SEQ ID NO: 6 and SEQ ID NO: 26. indicates an amino acid sequence with five amino acid substitutions, deletions, or additions. a VH CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 27; 1, 2, 3, 4, or 5 amino acid substitutions compared to the sequence of SEQ ID NOs: 7 and 27 VH CD having an amino acid sequence with amino acid substitutions, deletions, or additions R2 region and an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 28, has 1, 2, 3, 4, or 5 amino acid substitutions compared to SEQ ID NOs: 8 and 28; VH CDR3 region with an amino acid sequence containing amino acid deletions or additions a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 and 36; 1, 2, 3, 4, or 5 amino acid sequences compared to the sequence or SEQ ID NOs: 16 and 36. VL C having an amino acid sequence with an amino acid substitution, deletion, or addition DR1 region, an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 37, or a sequence Compared with columns 17 and 37, 1, 2, 3, 4, or 5 amino acid substitutions, a VL CDR2 region having an amino acid sequence with an amino acid deletion or an amino acid addition; and an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 and 38, or SEQ ID NOs: Compared with Nos. 18 and 38, 1, 2, 3, 4, or 5 amino acid substitutions, VL CDR3 region with amino acid sequence containing amino acid deletion or addition and a light chain variable region containing a FXIa-binding antibody or an antigen-binding fragment thereof. do.

[0198] Grafting antigen-binding domains onto alternative frameworks or scaffolds The resulting polypeptide has at least one bond that specifically binds to FXIa. A wide variety of antibody / immunoglobulin framework or Such frameworks or scaffolds can be used to identify human immunoglobulins. The five major idiotypes of phosphodiesterases or fragments thereof, preferably with humanized aspects. In this respect, the immunoglobulins identified in camelids include those of other animal species that have Single heavy chain antibodies, such as single heavy chain antibodies, are of particular interest. New frameworks, scaffolds, and fragments continue to be discovered and developed.

[0199] In one aspect, the present invention provides a method for the preparation of CDRs of the present invention using a non-immunoglobulin scaffold onto which the CDRs of the present invention can be grafted. This involves generating non-immunoglobulin-based antibodies that target FXI. as long as it contains a binding domain specific for the protein and / or FXIa protein. , utilizing known or future non-immunoglobulin frameworks and non-immunoglobulin scaffolds Known non-immunoglobulin frameworks and scaffolds was fibronectin (Compound Therapeutics, Inc., Wa ltham, MA), ankyrin (Molecular Partners AG, Zu rich, Switzerland), domain antibody (Domantis, Ltd., C ambridge, MA; and Ablynx nv, Zwijnaarde, Belgium ium), lipocalin (Pieris Proteolab AG, Freising, Germany), small molecule modular immunotherapeutics (Trubion Pharmaceut icals Inc., Seattle, WA), Maxibody (Avidia, Inc. ., Mountain View, CA), Protein A (Affibody AG, S weden), and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany) I can't.

[0200] Fibronectin scaffolds can be made of type III fibronectin domains (e.g., type III fibronectin domains). Based on the 10th module of fibronectin (Fn3 domain). Ionectin domains pack against each other to form the core of the protein It also contains loops that connect the beta strands to each other and are exposed to the solvent (CDRs and similar), with seven or eight beta strands distributed between two beta sheets There are at least three such loops on each edge of the beta-sheet sandwich. , edges are protein boundaries perpendicular to the direction of the beta strands (US 6,818, 418). The overall fold is similar to that of antigen recognition in camel and llama IgG. It is closely related to the heavy chain variable region fold, which is the smallest functional antibody fragment containing the whole unit. However, these fibronectin-based scaffolds are not immunoglobulins. Therefore, non-immunoglobulin antibodies have properties and affinities that are different from those of antibodies. These scaffolds mimic the antigen-binding properties of antibodies in vivo. Randomization of loops in vitro, similar to the process of affinity maturation in vivo. These fibronectin can be used in hybridization and shuffling strategies. The cDNA-based molecules can be cloned using standard cloning methods to clone the loop regions of the molecule into the CDNA of the present invention. It can be used as a scaffolding that can be replaced by DR.

[0201] Ankyrin technology is a method for synthesizing proteins containing ankyrin-derived repeat modules in heterologous and heterologous DNA. The method is based on using a scaffold that carries variable regions that can be used to bind to different targets. The kyrin repeat module consists of two antiparallel α-helices and a β-tandem repeat. It is a 33 amino acid polypeptide consisting of the variable region. The use of sprays is largely optimized.

[0202] Avimers are derived from naturally occurring A domain-containing proteins, such as LRP-1. Domains are primarily used for protein-protein interactions, and in humans, there are over 250 proteins The proteins are structurally based on the A domain. Avimers are linked via amino acid linkers. It consists of multiple (2-10) different "A domain" monomers that can bind to target antigens. Avimers are described, for example, in U.S. Patent Application Publication Nos. 20040175756; Nos. 50053973; 20050048512; and 2006 These can be created using the methods described in US Pat. No. 6,844,644.

[0203] Affibodies, which are affinity ligands, are the IgG-binding domains of Protein A. It is a small, simple protein consisting of a three-helix bundle based on the scaffold of one of the Protein A is a substance found in the bacterium Staphylococcus aureus. These scaffolding domains are surface proteins derived from the ribosomal proteins. Thirteen of these domains are involved in the formation of numerous ribosomal proteins. 58 randomized affibody libraries with nucleotide variants were generated. (See, e.g., US 5,831,012). Affibody molecules mimics an antibody and has a molecular weight of 6 kDa compared to the molecular weight of an antibody, which is 150 kDa. Despite their small size, the binding site of affibody molecules is similar to that of antibodies. It seems that

[0204] Anticalins are synthetic proteins developed by Pieris ProteoLab AG. Anticalins are generally used to transport chemically sensitive or insoluble compounds to the physiological Liposomes are a diverse group of small, robust proteins involved in biotransport or storage. Several natural lipocalins occur in human tissues or in human body fluids. Protein architecture involves hypervariable loops on top of a rigid framework Reminiscent of immunoglobulins, but in contrast to antibodies or their recombinant fragments, Pocaridin is a 160-180 amino acid sequence barely larger than a single immunoglobulin domain. It consists of a single polypeptide chain with four loops that form a binding pocket. The set of α- and β-glycosides exhibits remarkable structural plasticity and tolerates a variety of side chains. binding sites to recognize defined target molecules with high affinity and specificity. can be reshaped in a unique process. By mutagenizing a set of four loops, To develop anticalins, a protein from the lipocalin family was used. , used by bilin-binding protein (BBP) of the large white butterfly (Pieris brassicae) One example of a patent application describing anticalins is PCT Publication WO 2004 / 023990. Found in brochure no. 199916873.

[0205] Affilin molecules are molecules that bind to proteins and small molecules due to their specific affinity. The new affilin molecule is a small non-immunoglobulin protein designed to Each of them was highly purified from two libraries based on different human-derived scaffold proteins. Affilin molecules can be rapidly selected for specific binding to immunoglobulin proteins. There are currently two affilin scaffolds in use, which do not show any structural homology. One of them is gamma crystallin, a structural protein of the human lens, and the other is , a "ubiquitin" superfamily protein. Both human scaffolds are very small. It exhibits high thermal stability and is nearly resistant to pH changes and denaturing agents. The stability is mainly due to the unfolding of the beta-sheet structure of the protein. Examples of proteins derived from ubiquitin are described in WO200104144 and are referred to as "ubiquitins." Examples of "anti-inflammatory" proteins are described in WO2004106368.

[0206] Protein epitope mimics (PEMs) are key molecules involved in protein-protein interactions. A medium-sized secondary structure mimicking the beta-hairpin secondary structure of proteins. It is a cyclic peptide-like molecule (molecular weight: 1-2 kDa).

[0207] The present invention provides fully human antibodies that specifically bind to the FXIa protein. In comparison to human or humanized antibodies, the human FXIa-binding antibodies of the present invention are The antigenicity is further reduced when

[0208] Camelid antibodies Llama species (alpacas, llamas, and vicuñas) Camels and dromedaries (French camels), including New World members such as the camel and the dromedary bactrian camel (Camelus bactrianus) and dromedary (Calelus dromaderius) The antibody proteins obtained from members of the family vary in size, structural complexity, and The naturally occurring members of this family have been characterized for their antigenicity to target subjects. Certain IgG antibodies derived from mammals lack light chains and therefore are different from those derived from other animals. The typical four-chain quaternary structure of an antibody is two heavy chains and two light chains. are structurally different. PCT / EP93 / 02214 (published March 3, 1994) See WO 94 / 04678 (published on 2006 / 022222).

[0209] The region of camelid antibodies is a small single variable domain identified as VHH. This results in small proteins with high affinity for the target, Resulting in low molecular weight antibody-derived proteins known as "Cedrus nanobodies" It can be obtained by genetic engineering. U.S. Patent No. 5,747,297 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 Bioco njugate 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. Camelid antibodies and engineered libraries of antibody fragments, available from, for example, Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, camelid antibodies have the amino acid sequence The sequence can be modified by recombinant means to obtain a sequence that more closely resembles the human sequence. Nanobodies can be "humanized" and thus can be used for human immunodeficiency. The naturally low antigenicity of cucurbit antibodies can be further reduced.

[0210] The molecular weight of camelid nanobodies is approximately one-tenth that of human IgG molecules. Proteins have a physical diameter of only a few nanometers. One consequence of their small size is , camelid natto that binds to antigenic sites that are functionally invisible to larger antibody proteins. The ability of nanobodies, i.e. camelid nanobodies, to overcome classical immunological techniques It is useful as a reagent for detecting otherwise hidden antigens, and is possible Thus, a further consequence of their small size is that they are useful as therapeutic agents. The dandelion nanobodies target specific regions within the groove or narrow cleft of the target protein. As a result of binding to the site, it is possible to inhibit the target protein, and thus In their ability to more closely mimic the function of classical small molecular weight drugs than that of classical antibodies It can be used.

[0211] The low molecular weight and compact size also give it exceptional thermal stability and resistance to extremes of pH and Camelid nanobody that is stable to bacterial and proteolytic digestion and has low antigenicity Another consequence is that camelid nanobodies are more likely to be transported from the circulation to tissues. It travels easily, even crosses the blood-brain barrier, and can treat disorders that affect nervous tissue. Nanobodies may also facilitate drug transport across the blood-brain barrier. See U.S. Patent Application No. 20040161738, published August 19, 2004. These features, combined with low antigenicity to humans, allow for a large Furthermore, these molecules can be expressed in prokaryotic cells such as E. coli, indicating therapeutic potential. It can be expressed entirely intracellularly and as a fusion protein with a bacteriophage. It is expressive and functional.

[0212] Thus, a feature of the present invention is the ability to provide antibodies with high affinity for FXI and / or FXIa. A camelid antibody or camelid nanobody having the antibody function described herein is a camelid antibody or camelid nanobody having the antibody function described herein. In embodiments, the camelid antibody or camelid nanobody is naturally derived from a camelid. produced in animals, i.e., using the techniques described herein for other antibodies immunization with FXI and / or FXIa or peptide fragments thereof, Alternatively, FXI and / or FXIa binding camelids Nanobodies are engineered, i.e., for example, in the examples herein The disclosed FXI and / or FXIa as targets, and / or Use panning procedures involving domains and / or peptide fragments to appropriately mutate Selection from a phage library displaying induced camelid nanobody proteins Engineered nanobodies are further engineered to target the recipient The half-life in the target can be customized to range from 45 minutes to 2 weeks. In specific embodiments, the camelid antibody or camelid nanobody is administered via, for example, As described in PCT / EP93 / 02214, the heavy chain or Glycated light or dark chain CDR sequences into nanobody or single domain antibody framework sequences. Obtained by rafting.

[0213] Bispecific molecules and multivalent antibodies In another aspect, the present invention provides a FXI-binding antibody and / or a FXIa-binding antibody of the present invention. The present invention also features bispecific or multispecific molecules comprising antibodies or fragments thereof. or its antigen-binding region binds to at least two different binding sites or target molecules. They can also be derivatized to create bispecific molecules that bind to other functional molecules, e.g. linked to a peptide or protein of interest (e.g., another antibody or ligand for the receptor) The antibodies of the invention may in fact have three or more distinct binding sites and / or They can also be derivatized to create multispecific molecules that bind to a target molecule and two or more other molecules. Such multispecific molecules can also be linked to the above functional molecules, and such multispecific molecules are also used herein. These are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, the antibodies of the invention can be combined with one another to form a bispecific molecule, such that the resulting molecule is a bispecific molecule. one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic operably linked to a molecule (e.g., by chemical coupling, genetic fusion, non-covalent association) (by combination or otherwise).

[0214] Thus, the present invention provides at least one target molecule for FXI and / or FXIa. Bispecific molecules comprising one binding specificity and a second binding specificity for a second target epitope. For example, the second target epitope may be a different epitope from the first target epitope, such as FXI or and / or another epitope of FXIa.

[0215] Additionally, in the present invention, where the bispecific molecule is multispecific, the molecule may target a first target epitope and a second target epitope. and a second target epitope, it may further comprise a third binding specificity.

[0216] In one embodiment, the bispecific molecules of the invention have as their binding specificities, e.g., Fab, Fa at least one antibody or its derivatives, including F(ab'), F(ab'), Fv, or single-chain Fv; Antibodies also include antibody fragments. Antibodies may also be dimers of light or heavy chains, or fragments of antibodies as described by Ladner et al., US Pat. Fv or single chain constructs, such as those described in Patent No. 4,946,778 It can be any smallest fragment thereof.

[0217] Diabodies are molecules with chains that are too short to allow pairing between two domains on the same chain. The VH and VL domains are expressed on a single polypeptide chain, connected by a linker. The VH and VL domains are expressed as counterparts of different chains. The complementary domains pair together, thereby creating two antigen-binding sites (e.g., Holliday et al., 1999). Iger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994 Structure 2:1121-1123). The diabodies are VHA-VLB and VHB-VLA. structure (VH-VL configuration), or VLA-VHB and VLB-VHA structures (VL- produced by expressing two polypeptide chains with the VH configuration in the same cell Most diabodies can be expressed in soluble form in bacteria. Single-chain diabodies (scDbs) are composed of two diabody-forming polypeptide chains separated by approximately 1 It is constructed by connecting the two with a linker of five amino acid residues (Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3~4):128~30; Wu et al., 1996 Immunotechnology, 2 (1):21-36). The scDb is expressed in a soluble, active, monomeric form in bacteria. (Holliger and Winter, 1997 Cancer Immunol. Immunother., 45( 34):128-30; Wu et al., 1996 Immunotechnology, 2(1):21-36; Pluckthun and Pack, 1 997 Immunotechnology, 3(2):83~105; Ridgway et al., 1996 Protein Eng., 9(7):617~ 21). Diabodies can be fused to Fc to create "diabodies." (See Lu et al., 2004 J. Biol. Chem., 279(4):2856-65).

[0218] Other antibodies that may be incorporated within the bispecific molecules of the invention include murine monoclonal antibodies, chimeric monoclonal antibodies, and humanized monoclonal antibodies.

[0219] Bispecific molecules can be prepared by combining the constituent binding specificities using methods known in the art. For example, bispecific molecules can be prepared by conjugating each binding specificity of the molecule. The isomers can be generated separately and then conjugated to each other. If the material is a protein or peptide, various coupling or cross-linking agents can be used. Examples of cross-linking agents include protein A, Rubodimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5 ,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (S PDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane -1-carboxylate (sulfo-SMCC) (e.g., Karpovsky et al., 1984 J.E. xp. Med. 160:1686; Liu, MA et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648 Other methods are described in Paulus, 1985 Behring Ins. Mitt. 78, 118-132; Brennan et al., 1985 985 Science 229:81-83; and Glennie et al., 1987 J. Immunol. 139:2367-2375 The conjugating agents are SATA and sulfo-SMCC. Both are manufactured by Pierce Chemical Co. (Rockford, IL). It is available from

[0220] When the binding specificity is an antibody, they bind to the C-terminal hinge regions of the two heavy chains via sulfhydryl bonds. In certain embodiments, the conjugation can be achieved by a drill bond. Prior to conjugation, an odd number of sulfhydryl residues, e.g., one sulfhydryl The hinge region is modified to contain residues.

[0221] Alternatively, both binding specificities can be encoded in the same vector and expressed in the same host cell. This method allows the bispecific molecule to be expressed and assembled in a mAb x mAb Fusion proteins, mAb x Fab fusion proteins, Fab x F(ab')2 fusion proteins The present invention is particularly useful when the antibody is a fusion protein or a ligand x Fab fusion protein. The specificity molecule may be a single chain molecule containing one single chain antibody and a binding determinant, and two binding Bispecific molecules may also be single-chain bispecific molecules containing a determinant. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. 0,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881, 175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,51 No. 3; U.S. Patent No. 5,476,786; U.S. Patent No. 5,013,653 U.S. Patent No. 5,258,498; and U.S. Patent No. 5,482,858 No. 5,999,299.

[0222] The binding of bispecific molecules to their specific targets can be assessed by, for example, enzyme-linked immunosorbent assays. (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g. This can be confirmed by a growth inhibition assay or Western blot assay. Each of these assays generally involves the use of a labeled reagent ( For example, by using antibodies, the presence of protein-antibody complexes of particular interest can be detected. is detected.

[0223] In another aspect, the present invention provides a method for the preparation of at least two identical antibodies of the present invention that bind to FXIa. or different antigen-binding moieties. , linked together via protein fusion or covalent or non-covalent linkage. Alternatively, linking methods for bispecific molecules have also been described. The compound can be, for example, a compound prepared by combining an antibody of the present invention with an antibody that binds to the constant region of the antibody of the present invention, such as F This can be obtained by cross-linking the c region or hinge region.

[0224] Trimerization domains are described, for example, in the Borean patent EP10122. 80B1. The pentamerization module is described, for example, in PCT / E P97 / 05897.

[0225] Antibodies with extended half-lives The present invention provides a method for producing FXIa protein-specific antibodies with an extended half-life in vivo. A binding antibody is provided.

[0226] Many factors can affect the half-life of a protein in vivo. For example, filtration in the kidney, metabolism in the liver, and degradation by proteolytic enzymes (proteases) degradation by the immune system, and immunogenic responses (e.g., neutralization of proteins by antibodies and macrophage activation). Various strategies can be used to extend the half-life of the antibodies of the invention. For example, polyethylene glycol (PEG), reCODE PEG , antibody scaffold, polysialic acid (PSA), hydroxyethyl starch (HES), albumin Chemical linkage to albumin, IgG, and IgG-binding ligands and carbohydrate shields It has bonds with proteins that bind to serum proteins such as FcRn and transferrin. Gene fusions allow the creation of nanobodies, Fabs, DARPins, avimers, affibodies, and Coupling with other binding moieties, such as anticalins, that bind to serum proteins (gene By (physical or chemical) combining rPEG, albumin, albumin domains, albumin by genetic fusion with amine-binding proteins and Fc, or by nanocarriers, sustained-release formulations, Alternatively, the half-life of the antibody of the present invention can be extended by incorporating it into a medical device. .

[0227] To prolong the serum circulation of antibodies in vivo, undesirable PEGs, such as high molecular weight PEG, are used. The active polymer molecule is site-specifically conjugated to the N- or C-terminus of the antibody via PEG. via gation or via the epsilon-amino group present on lysine residues. to antibodies or fragments thereof, with or without functional linkers To PEGylate an antibody, the antibody or fragment thereof can be attached to a polyethylene glycol (PEG)-based polymer. PEG, such as a reactive ester or aldehyde derivative of PEG, and one Alternatively, the reaction is typically carried out under conditions in which multiple PEG groups are attached to the antibody or antibody fragment. PEGylation is the process of combining reactive PEG molecules (or similar reactive water-soluble polymers) with This can be carried out by an acylation reaction or an alkylation reaction. The term "polyethylene glycol" refers to a mono(C1-C10)alkoxy-polyethylene glycol. Polyethylene glycol or aryloxy-polyethylene glycol or polyethylene glycol PEG, which has been used to derivatize other proteins, such as PEG-maleimide In certain embodiments, P The antibody to be EG is a deglycosylated antibody. Linear or branched polymer Derivatizations that result in minimal loss of biological activity are used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry. Conjugation of PEG molecules to antibodies can be monitored periodically to confirm proper conjugation. Unreacted PEG can be purified by size exclusion or ion exchange chromatography. The antibody-PEG conjugate can be separated by chromatography. Derivatized antibodies can be prepared using methods well known to those of skill in the art, for example, by immunoprecipitation using the immunoglobulins described herein. Assays can be used to assess binding activity and in vivo efficacy. Methods for PEGylating proteins are known in the art, and can be used to PEGylate the antibodies of the invention. For example, EP0154316 by Nishimura et al. and See EP0401384 by Ishikawa et al.

[0228] Another modified PEGylation technique involves the use of a reconstituted system containing tRNA synthetase and tRNA. Direct reconstitution of chemically defined side chains into biosynthetic proteins via reconstituting chemically orthogonal directed engineering ( This technology involves the synthesis of over 30 new amino acids in Escherichia coli ( Incorporation into biosynthetic proteins in E. coli, yeast, and mammalian cells tRNA can transfer unnatural amino acids to any position where an amber codon is located. and the termination amber codon is used to generate the incorporation signal for the chemically specified amino acid. Converts to a codon that transmits.

[0229] Recombinant PEGylation technology (rPEG) can also be used to extend serum half-life This technology allows the unstructured protein tails of 300-600 amino acids to be synthesized using existing pharmaceutical tails. This involves genetically fusing the unstructured protein chains into the target protein. The molecular weight is approximately 15 times its actual molecular weight, so the serum half-life of the protein is significantly In contrast to traditional PEGylation, which requires chemical conjugation and repurification In addition, the manufacturing process is greatly simplified and the product is homogeneous.

[0230] Polysialylation is the process of using the natural polymer polysialic acid (PSA) to enhance the activity of sialic acid. Another technique to extend the life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for drug delivery, upon conjugation, polysialic acid creates a protective microenvironment This extends the active life of the therapeutic protein in the circulation, allowing it to be detected by the immune system. PSA polymers are naturally found in the human body. SAs have evolved over millions of years to coat their walls with PSA. These naturally polysialylated bacteria were then molecularly Mimicry makes it possible to disable the body's defenses. Nature's ultimate stealth technique. PSA, which is readily isolated from such bacteria in large quantities and with certain physical characteristics, Bacterial PSA is chemically identical to PSA in humans, so , and is still completely non-immunogenic when coupled to proteins.

[0231] Another technique involves the use of hydroxyethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy corn starch. HES solutions are typically used to compensate for blood volume deficiency and improve blood circulation. The hydroxylation of antibodies increases the stability of the molecule. In addition to this, the reduction of renal clearance also allows for an extension of the circulating half-life. This results in increased biological activity. A wide range of HES antibody conjugates can be customized by modifying can.

[0232] Antibodies with increased in vivo half-lives may also be modified with one or more amino acids. (i.e., substitutions, insertions, or deletions) to the IgG constant domain or its FcRn binding It can also be produced by introducing it into a functional fragment (preferably an Fc domain fragment or a hinge Fc domain fragment). For example, International Patent Publication No. 98 / 23289, International Patent Publication No. 97 / 34631; and U.S. Patent No. 6,277,375 Please refer to the book.

[0233] Furthermore, the antibody or antibody fragment may be rendered more stable in vivo or To extend the half-life in vivo, the antibody may be covalently bound to albumin (e.g., human serum albumin). The conjugation of the hydroxybenzoates to serum albumin (HSA) is also possible. are well known, and are described, for example, in International Patent Publication Nos. 93 / 15199 and 93 / 1 Nos. 5200 and 01 / 77137; and European Patents See, for example, US Pat. No. 413,622. In addition, in the context of bispecific antibodies as described above, So, the specificity of the antibody is such that one binding domain of the antibody binds to FXIa, while The second binding domain of the antibody is designed to bind to serum albumin, preferably HSA. You can also design it.

[0234] Strategies for extending half-life include those where an increase in in vivo half-life is desired. Nanobodies, fibronectin-based binders, and other antibodies or proteins This is particularly useful.

[0235] antibody conjugates The present invention relates to the use of heterologous proteins or polypeptides to create fusion proteins. to (or a fragment thereof, preferably at least 10, at least 20, at least 30 , at least 40, at least 50, at least 60, at least 70, at least 80 , or a polypeptide of at least 90, or at least 100 amino acids), fused or chemically conjugated (covalent conjugation and (including both covalent and non-covalent conjugation), specifically binds to the FXIa protein. In particular, the present invention provides antibodies or fragments thereof that bind to the antibodies described herein. antigen-binding fragments (e.g., Fab fragments, Fd fragments, Fv fragments, F(ab)2 fragments, VH domain, VH CDR, VL domain, or VL CDR) and a heterologous protein, heterologous Fusion proteins comprising a heterologous polypeptide or a heterologous peptide are provided. The present invention relates to the fusion or covalent coupling of a protein, polypeptide, or peptide with an antibody or antibody fragment. For example, U.S. Pat. No. 5,336,603 discloses a method for conjugating a hydroxyl group to a hydroxyl group. Specification No. 5,622,929, Specification No. 5,359,046, No. 5,34 Nos. 9,053, 5,447,851, and 5,112,946 No.; European Patent Nos. 307,434 and EP 367,166; International Patent Publication No. 96 / 04388 and International Patent Publication No. 91 / 06570 Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 19 95, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89 Please refer to :11337~11341.

[0236] Further fusion proteins have been developed using gene shuffling, motif shuffling, and exon shuffling. shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling") DNA shuffling can be used to create the present invention. modifying the activity of the antibody or fragment thereof (e.g., increasing affinity, decreasing dissociation rate, etc.) Generally, U.S. Patent No. 5,605,777, Specification No. 93, Specification No. 5,811,238, Specification No. 5,830,721, Specification No. 5,830,721, Nos. 5,834,252 and 5,837,458; Patten et al., 1999 97, 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 Bla sco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications is incorporated by reference) (which are incorporated herein by reference in their entirety). The fragments, or the encoded antibodies or fragments thereof, may be free of errors prior to recombination. Random mutagenesis by prone PCR, random nucleotide insertion, or other methods The antibody specifically binds to the FXIa protein. The polynucleotide encoding the polypeptide or a fragment thereof may be incorporated into one or more heterologous molecules. or multiple components, motifs, sections, parts, domains, fragments, etc. .

[0237] Additionally, antibodies or fragments thereof may be linked to marker sequences, such as peptides, to facilitate purification. In a preferred embodiment, the amino acid sequence of the marker is Many of these vectors are commercially available, including the pQE vector (QIAGEN, Inc. , 9259 Eton Avenue, Chatsworth, CA 91311 A hexa-histidine peptide (SEQ ID NO: 48), such as the tag provided in Gent As described in Z. et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, e.g., For example, hexa-histidine (SEQ ID NO: 48) provides for convenient purification of the fusion protein. Another peptide tag useful for purification is derived from the influenza hemagglutinin protein Hemagglutinin ("HA") tag corresponding to the epitope (Wilson et al., 1984, Cell 37:767). "flag" tags, and "flag" tags.

[0238] In other embodiments, the antibodies of the invention or fragments thereof can be used to bind to diagnostic or detectable agents. Such antibodies are useful in preventing the onset, development, progression, and progression of a disease or disorder. and / or severity as part of a clinical investigation procedure, such as to determine the effectiveness of a particular treatment. Such diagnosis and detection may be useful for monitoring or prognostic diagnosis. Antibodies were stained with horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and various enzymes, such as, but not limited to, acetylcholinesterase, or acetylcholinesterase including, but not limited to, streptavidin / biotin and avidin / biotin Unknown prosthetic group: Umbelliferone, fluorescein, fluorescein isothiocyanate fluorescein, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, is a fluorescent material such as, but not limited to, phycoerythrin; luminol, etc. Luminescent materials, including but not limited to luciferase, luciferin, and aequorin bioluminescent materials such as, but not limited to; iodine (I, I, I) , and 121I), carbon (14C), sulfur (35S), tritium (3H), indium In (115In, 113In, 112In, and 111In), technetium (99T c), thallium (201Tl), gallium (68Ga, 67Ga), palladium (103 Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153 Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 9 0Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68G e, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54 Radioactive materials such as, but not limited to, Mn, 75Se, 113Sn, and 117Tin emitting materials; and positron-emitting metals and non-radiative materials using various positron emission tomography techniques. coupling to a detectable substance, including but not limited to a paramagnetic metal ion This can be achieved by:

[0239] The present invention further relates to the use of antibodies or fragments thereof conjugated to therapeutic moieties. The antibody or fragment thereof may be a cytotoxin, e.g., a cytostatic or cytocidal agent. , conjugation to a therapeutic moiety such as a therapeutic agent or radioactive metal ion, e.g., an alpha emitter. A cytotoxin or cytotoxic agent is any agent that is detrimental to cells. include.

[0240] Additionally, antibodies or fragments thereof may be used as therapeutic moieties or drugs that modify a given biological response. Therapeutic or drug moieties can also be conjugated to a moiety using classical chemistry. For example, the drug moiety may be a compound that induces a desired biological effect. The target protein may be a protein, peptide, or polypeptide possessing a therapeutic activity. Proteins include, for example, abrin, ricin A, Pseudomonas exotoxin, cholera toxin, and toxins such as diphtheria toxin; tumor necrosis factor, α-interferon, β-interferon ron, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptosis or proteins such as biological response modifiers, e.g., lymphokines. It may include quality.

[0241] Additionally, antibodies can be used with radioactive metal ions, such as alpha emitters such as 213Bi, 131I Radiation including but not limited to n, 131LU, 131Y, 131Ho, 131Sm Macrocyclic chelating agents useful for conjugating reactive metal ions to polypeptides It can be conjugated to any therapeutic moiety. In certain embodiments, macrocyclic The chelator may be attached to the antibody via a linker molecule, such as 1,4,7,10-tetrazole. The compound is azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA). Such linker molecules are generally known in the art and are each incorporated herein by reference. Incorporated in its entirety, 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.

[0242] Techniques for conjugating therapeutic moieties to antibodies are well known and are described, for example, in Arno n et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, M Onoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Li ss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” 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”, Monoclo nal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), 475~ 506 pages (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Us” e Of Radiolabeled Antibodies In Cancer Therapy”, Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and and Thorpe et al., 1982, Immunol. Rev. 62:119-58.

[0243] Antibodies can also be attached to solid supports, which are particularly useful in immunoassays or purification of the target antigen. Such solid supports include glass, cellulose, polyacrylamide, and the like. including, but not limited to, polyethylene, nylon, polystyrene, polyvinyl chloride, or polypropylene Not limited.

[0244] How to generate antibodies Nucleic acid encoding the antibody The present invention relates to polypeptides comprising segments or domains of the FXIa-binding antibody chains described above. The present invention provides a substantially purified nucleic acid molecule encoding a polypeptide. The nucleotide sequence encoding the heavy chain variable region shown in SEQ ID NO: 10 or 30 and / or a nucleotide sequence encoding the light chain variable region set forth in SEQ ID NO: 20 or 40. In a specific embodiment, the nucleic acid molecule comprises a nucleic acid molecule identified in Table 1. Some other nucleic acid molecules of the present invention are nucleotide equivalents of the nucleic acid molecules identified in Table 1. Substantially (e.g., at least 65, 80%, 95%, or 99%) identical to a nucleotide sequence When expressed from an appropriate expression vector, these polynucleotides The polypeptide encoded by the peptide binds to the FXI and / or FXIa antigens. It is possible to present Noh.

[0245] The present invention also provides at least one antibody derived from the heavy chain or light chain of the FXIa-binding antibody shown above. A polynucleotide encoding one CDR region and usually all three CDR regions. Several other polynucleotides are also provided that bind to the FXIa-binding antibodies set forth above. The coding sequence for the heavy and / or light chain variable regions of the vector is substantially the same as that of the vector. Due to the degeneracy of the code, a variety of nucleic acid sequences can encode each of the immunoglobulin amino acid sequences. It will be.

[0246] The nucleic acid molecules of the present invention can encode both the variable and constant regions of an antibody. Some of the nucleic acid sequences are substantially similar to the heavy chain sequences shown in SEQ ID NO: 11 or 31. For example, nucleic acids encoding heavy chain sequences at least 80%, 90%, or 99% identical Some other nucleic acid sequences include the light chain sequence shown in SEQ ID NO: 21 or 41. and encoding a light chain sequence substantially (e.g., at least 80%, 90%, or 99%) identical to It contains nucleotides that modulate the nucleotide sequence.

[0247] The polynucleotide sequences can be synthesized by de novo solid phase DNA synthesis or by the synthesis of FXIa-binding antibodies or to an existing sequence encoding the binding fragment (e.g., the sequence described in the Examples below). Direct chemical synthesis of nucleic acids can be achieved by PCR-mediated mutagenesis. , Narang et al., 1979, Meth. Enzymol. 68:90; the phosphotriester method by Brown et al., Meth. Enzymol. 68:109, 1979; phosphodiester method by Beaucage et al., Tetra. Lett., 22:185 9, 1981; and U.S. Pat. No. 4,458,066. This can be achieved by methods known in the art, such as solid support methods according to the specification. Introduction of mutations into a polynucleotide sequence by PCR can be achieved, for example, by PCR Technology. In: Principles and Applications for DNA Amplification, HA Erlich (ed.), Freeman. Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (eds.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:96 7, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991. It can be carried out as is.

[0248] The present invention also provides the above-described FXI-binding antibody and / or FXIa-binding antibody. Expression vectors and host cells for producing the vectors are also provided. The present invention relates to the expression of polynucleotides encoding FXIa-binding antibody chains or binding fragments. Both viral and non-viral expression vectors can be used. Any of these vectors can be used to produce antibodies in mammalian host cells. Transgenic and non-viral systems use plasmids, typically expressing proteins or RNA. episomal vectors with expression cassettes for the expression of genomic DNA, and human artificial chromosomes (e.g., See, Harrington et al., Nat Genet 15:345, 1997). For example, mammals (e.g., FXIa-binding polynucleotide and LOX-1-binding polypeptide in human cells Non-viral vectors useful for expression of ThioHis include pThioHis A, pThioHis B, and pThioHis C, pcDNA3.1 / His, pEBVHis A, pEB VHis B, and pEBVHis C (Invitrogen, San Diego) , CA), MPSV vectors, as well as those in the art for expressing other proteins. Useful viral vectors include retroviruses, adenoviruses, and many other vectors known in the art. vectors based on SV40, adenovirus, adeno-associated virus, and herpesvirus <Vector, papillomavirus, HBP, Epstein-Barr virus, vaccinia virus and Semliki Forest virus (SFV). Brent et al., supra; Smith, See Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992. I want to be.

[0249] The choice of expression vector will depend on the intended host cell in which the vector will be expressed. The expression vector contains a polynucleotide encoding a FXIa-binding antibody chain or fragment. Contains an operably linked promoter and other regulatory sequences (e.g., enhancers) In some embodiments, except under inducing conditions, the inserted sequence To prevent expression of the gene, an inducible promoter is used. , arabinose, lacZ, metallothionein promoter, or heat shock promoter Cultures of transformed organisms contain the expression products of the transformed organisms, which are then transformed into the host cells. They can be grown under non-inducing conditions without biasing the population of permissive coding sequences. In addition to the promoter, other regulatory elements may also be involved in the expression of the FXIa-binding antibody chain or These elements may be required or desired for efficient expression of the fragment. It typically contains a TG initiation codon and adjacent ribosome binding site or other sequence. The efficiency of expression can be increased by incorporating enhancers appropriate for the cell line used. (See, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and (See, for example, SV40 enzyme and Bittner et al., Meth. Enzymol., 153:516, 1987). enhancer or CMV enhancer to increase expression in mammalian host cells. It is possible.

[0250] The expression vector also contains the polypeptide encoded by the inserted FXIa-binding antibody sequence. The location of a secretory signal sequence may also be provided to form a fusion protein with the insert. The inserted FXI-binding antibody sequence and / or FXIa-binding antibody sequence are FXI-binding antibodies and / or or sequences encoding the light chain variable domain and heavy chain variable domain of an FXIa-binding antibody. The vector used to receive the vector optionally also contains a constant region or part thereof. Such vectors also encode the variable region as a fusion protein with the constant region. Expression is permitted, resulting in the production of an intact antibody or a fragment thereof. Such constant regions are typically human constant regions.

[0251] The antibody possesses and expresses an FXI-binding antibody chain and / or an FXIa-binding antibody chain. Host cells for the production of E. coli can be prokaryotic or eukaryotic. ) is one prokaryotic organism useful for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use are bacillus bacteria such as Bacillus subtilis. Bacillus subtilis, Salmonella spp., Serratia spp., and various Schizosaccharomyces cerevisiae These prokaryotic hosts include other Enterobacteriaceae, such as Pseudomonas species. The vector also typically contains expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, expression vectors using the lactose promoter system, tryptophan, trp promoter system, beta-lactamase promoter system, or Fa There are also any number of different well-known promoters, such as promoter systems derived from dilambda. The promoter optionally controls expression typically through an operator sequence. ribosome binding site sequences for initiating and terminating transcription and translation, Other microorganisms, such as yeast, may also be used to express the FXIa-binding polypeptides of the present invention. In addition, the use of insect cells in combination with baculovirus vectors can also be used. Cells can also be used.

[0252] In some preferred embodiments, the FXI binding polypeptides and / or uses mammalian host cells to express and produce FXIa-binding polypeptides The mammalian host cell may be any normal non-immortalized animal cell or normal immortalized animal cell or These include non-normal immortalized animal or human cells, such as CHO cell lines, various Cos cells, Intact immunoglobulins, including IgG1, HeLa cells, myeloma cell lines, and transformed B cells A number of suitable host cell lines capable of secreting ribonucleotides have been developed. For the use of mammalian tissue cell cultures to express As generally discussed in From Genes to Clones, VCH Publishers, New York, NY, 1987. Expression vectors for mammalian host cells include an origin of replication, a promoter, and an enhancer. Expression control sequences such as enzymes (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986) (referred to as "ribosome binding site", "RNA splice site", "polyadenylation site"), , and may contain necessary processing information sites, such as a transcription terminator sequence.

[0253] These expression vectors typically contain promoters derived from mammalian genes or Suitable promoters include constitutive promoters, including promoters derived from viruses. -, cell type-specific promoters, stage-specific promoters, and / or modulating Useful promoters can be regulated or regulatable promoters. Tarothionein promoter, constitutive adenovirus major late promoter, dexamethasone Zone-inducible MMTV promoter, SV40 promoter, MRP pol III promoter promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoter (human Constitutive CMV promoters, and those known in the art, such as the immediate early CMV promoter, These include, but are not limited to, known promoter-enhancer combinations.

[0254] Methods for introducing expression vectors containing polynucleotide sequences of interest include the introduction of vectors into cell host species. For example, calcium chloride transfection is commonly used in prokaryotic cells. whereas calcium phosphate treatment or electroporation can be used for other cell hosts. (See generally Sambrook et al., supra.) Other methods include, for example, electrochemical Perforation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection ion, gene gun method, virosomes, immunoliposomes, polycation:nucleic acid conjugates Gate, naked DNA, artificial virion, herpesvirus structural protein VP 22 (Elliot and O'Hare, Cell 88:223, 1997), drug-enhanced uptake of DNA Long-term high-quality recombinant protein production For yield production, stable expression is often desired. For example, FXIa-binding antibodies Cell lines stably expressing the chains or binding fragments can be derived from viral origins of replication or endogenous expression. The expression vector of the present invention contains a selection marker gene and a gene encoding the expression vector. After the vector is introduced, the cells are cultured in enriched medium for 1-2 days. Once grown, the cells can be switched to selective media. The purpose of the selectable marker is to allow the cells to Its presence allows cells to grow, thereby protecting the cells from the introduced sequence. The goal is to successfully express the sequence in selective medium. The transplanted cells can be grown using tissue culture methods appropriate to the cell type.

[0255] Framework or Fc operations Engineered antibodies of the invention may, for example, have modified VH and / or VL sequences to improve the properties of the antibody. This includes engineered antibodies in which modifications have been made to framework residues within the antibody. The decoration is typically done to reduce the immunogenicity of the antibody. For example, one technique reversion of one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation are mutated to the extent that they are not mutated. These residues may contain framework residues that differ from the germline sequence. Framework sequences are identified by comparison to the germline sequences from which the antibody is derived. To restore the framework region sequences to their germline configuration, For example, somatic mutations can be converted to germline sequences by site-directed mutagenesis. Such "backmutated" antibodies can also be are intended to be encompassed by the present invention.

[0256] Another type of framework modification is a modification of one or more residues within the framework region, Alternatively, residues within one or more CDR regions may be mutated to generate T cell epitopes. This involves removing the fragments, thereby reducing the potential immunogenicity of the antibody. Also referred to as "deimmunization," Carr et al., U.S. Patent Publication No. 20030153043 This is described in further detail in the specification.

[0257] In addition to or alternatively to modifications made within the framework or CDR regions The antibodies of the invention may contain modifications within the Fc region to typically improve serum half-life, complement fixation, Fc c receptor binding, and / or antigen-dependent cellular cytotoxicity, Additionally, the antibodies of the present invention may be chemically engineered to modify their functional properties. The antibody may also be further modified (e.g., one or more chemical moieties may be attached to the antibody). (which can be used to modify the glycosylation of the antibody, again to improve one or more functional characteristics of the antibody. Each of these embodiments is described below. The numbering of residues in the Fc region is based on the EU index according to Kabat. It's a kus.

[0258] In one embodiment, the number of cysteine ​​residues in the hinge region is altered, e.g., increased or The hinge region of CH1 is modified to reduce the cleavage. This technique is based on the method described by Bodmer et al. Further described in U.S. Patent No. 5,677,425. CH1 Hinge The number of cysteine ​​residues in a region determines, for example, whether it facilitates assembly of the light and heavy chains. , or modified to increase or decrease the stability of the antibody.

[0259] In another embodiment, the Fc hinge region of the antibody is mutated to increase the biological half-life of the antibody. More specifically, the binding of antibodies to Staphylococcus aureus protein A (SpA) shortens the time to one or more Fc-hinge domains, such that binding to SpA is impaired compared to that of the natural Fc-hinge domain. Amino acid mutations in the CH2-CH3 domain interface region of the Fc-hinge fragment This method is described in US Patent No. 6,165,745 by Ward et al. Further details are provided in the accompanying text.

[0260] In another embodiment, the antibody is modified to increase its biological half-life. For example, in U.S. Pat. No. 6,277,375 to Ward, One or more of the mutations described above can be used. To extend biological half-life, antibodies can be prepared using methods such as those described in U.S. Patent No. 5,869 to Presta et al. ,046; and 6,121,022. Salvage receptor binding properties taken from two loops of the CH2 domain of the Fc region of glucagon-like protein It can also be modified within the CH1 or CL region to contain the epitope.

[0261] In yet other embodiments, at least one amino acid residue is modified to enhance the effector function of the antibody. The Fc region is modified by replacing the Fc region with a different amino acid residue that modifies the Fc region. For example, The antibody has altered affinity for the effector ligand but not the antigen of the parent antibody. One or more amino acids are replaced with different amino acid residues so that binding ability is maintained. The effector ligand for which the affinity is modified can be, for example, For example, the Fc receptor or the C1 component of complement. This approach is described in detail in Winter et al. U.S. Patent Nos. 5,624,821 and 5,648,260 to This is described in more detail in

[0262] In another embodiment, the antibody alters binding to C1q and / or complement dependent cytotoxicity. One or more amino acid residues selected from the group consisting of hydroxybenzoates, hydroxybenzoates, and hydroxybenzoates are selected to reduce or eliminate the adverse drug reactions (CDC). One or more amino acids can be replaced with different amino acid residues. As further detailed in U.S. Patent No. 6,194,551 to Dusogie et al. It is listed.

[0263] In another embodiment, one or more amino acid residues are modified to provide a polypeptide that binds complement. This technique is described in PCT Publication No. WO 2004 / 023990 by Bodmer et al. This is further described in brochure 94 / 29351.

[0264] In specific embodiments, the anti-FXI / FXIa antibodies described herein (e.g., NO V1401 VL CDR and VH CDR) to any surface-associated FXI. Two amino acids are added to reduce the possibility of ADCC or CDC caused by A human IgG (e.g., IgG1) Fc domain containing the substitutions D265A and P329A. These alanine substitutions have been shown to reduce ADCC and CDC. (e.g., Idosugie et al., J. Immunol. 164:4178-4184, 2000; Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

[0265] In yet another embodiment, antibody-dependent Increase the ability of the antibody to mediate anti-inflammatory cell-mediated cytotoxicity (ADCC) and / or inhibit Fcγ receptor activity. The Fc region is modified to increase the affinity of the antibody for the receptor. Presta in PCT Publication WO 00 / 42072 Furthermore, FcγRI, FcγRII, Fcγ The binding sites for RIII and FcRn have also been mapped, and mutations that improved binding were identified. Variants have also been described (see Shields, RL et al., 2001 J. Biol. Chen. 276:6591-6604). (I want to be).

[0266] In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated antibodies The antibody can be made to lack glycosylation. For example, the antibody may be modified to increase its affinity for an "antigen." Such carbohydrate modifications can occur, for example, by modifying one or more glycosylation sites within the antibody sequence. This can be achieved by modifying one or more variable region fragments. One or more amino acids resulting in the loss of a framework glycosylation site Substitutions can be made to eliminate glycosylation at that site Such deglycosylation can increase the affinity of the antibody for the antigen. Such techniques are described in U.S. Patent No. 5,714,350 to Co et al.; and more fully described in US Pat. No. 6,350,861.

[0267] Additionally or alternatively, hypofucosylated antibodies or bi-branched antibodies having reduced amounts of fucosyl residues. We have been producing antibodies with altered glycosylation types, such as antibodies with increased GlcNac structures. Such modification of the glycosylation pattern can also increase the ADCC ability of the antibody. Such carbohydrate modifications, for example, have been shown to enhance the glycosylation machinery. This can be achieved by expressing the antibody in a modified host cell. describes cells with modified glycosylation machinery, which can be used to express the recombinant antibodies of the invention. This allows the use of the host cells for producing antibodies with modified glycosylation. For example, EP 1,176,195 by Hang et al. The fucosyltransferase was co-expressed to ensure that the antibody expressed in the They describe a cell line in which the FUT8 gene, which encodes the ATP-dependent ATPase inhibitor, is functionally disrupted. PCT Publication No. WO 03 / 035835 brochure by the same author, describes the substitution of fucose with Asn( 297) Reduced ability to attach to linked carbohydrates and A mutant CHO cell line, Lecl, also results in hypofucosylation of the antibody. 3 cells (see also Shields, RL et al., 2002 J. Biol. Chem. 277:26733 (See also PCT Publication WO 99 / 54342 by Umana et al. The brochure demonstrates that antibodies expressed in engineered cell lines result in increased ADCC activity of the antibody. Glycoproteins were modified to display increased bisecting GlcNac structures, resulting in Glycosyltransferases (e.g., beta(1,4)-N-acetylglucosamine) Cell lines engineered to express gonadotropin dehydrogenase III (GnTIII) (See also Umana et al., 1999 Nat. Biotech. 17:176-180.) stomach).

[0268] Methods for engineering engineered antibodies As discussed above, the VH and VL sequences or full-length heavy chains and A FXIa-binding antibody having a full-length light chain sequence may be a FXIa-binding antibody having a full-length heavy chain sequence and / or a full-length light chain sequence. modifying the VH and / or VL sequences, or the constant regions attached thereto This can be used to generate new FXIa-binding antibodies. Therefore, in another aspect of the present invention, the structural features of the FXIa-binding antibodies of the present invention can be used to identify human FXIa-binding antibodies. Binding to FXIa and also inhibiting one or more functional properties of FXIa (e.g., For example, it inhibits the binding of FXIa to the FXIa receptor and inhibits FXIa-dependent cell proliferation. Structurally related FX that retain at least one functional property of the antibodies of the invention, such as Ia-binding antibodies are created.

[0269] For example, one or more CDR regions of the antibody of the present invention or mutations thereof can be recombinantly synthesized. Thus, recombinant CDRs can be combined with known framework regions and / or other CDRs.

[0013] The present invention provides further FXIa-binding antibodies, as discussed above, that are engineered by Other types of modifications include those described in the previous section. Starting Materials for the Procedure The material may be one or more of the VH and / or VL sequences presented herein. The engineered antibodies are generated by using the CDR regions of the engineered antibodies. one or more of the VH and / or VL sequences provided herein, and The method actually prepares an antibody having one or more of those CDR regions (i.e., It is not necessary for the sequence to be expressed as a protein. The information is used as starting material to create "second generation" sequences derived from the original sequence. The "second generation" sequence is then prepared and expressed as a protein.

[0270] Thus, in another embodiment, the present invention provides a method for the preparation of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 23. a CDR1 sequence selected from the group consisting of SEQ ID NOs: 4 and 24; and / or a duplicate having a CDR3 sequence selected from the group consisting of SEQ ID NOs: 5 and 25. an antibody sequence of the chain variable region; and 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 light chain variable region having a CDR3 sequence selected from the group consisting of SEQ ID NOs: 15 and 35 and preparing a FXIa-binding antibody comprising an antibody sequence of a heavy chain variable region and / or by modifying at least one amino acid residue in the antibody sequence of the light chain variable region to form at least Creating a modified antibody sequence; and methods for expressing the modified antibody sequence as a protein to provide.

[0271] Thus, in another embodiment, the present invention provides a method for the preparation of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 26. a CDR1 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. an antibody sequence of the chain variable region; and 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 light chain variable region having a CDR3 sequence selected from the group consisting of SEQ ID NOs: 18 and 38 and preparing a FXIa-binding antibody comprising an antibody sequence of a heavy chain variable region and / or by modifying at least one amino acid residue in the antibody sequence of the light chain variable region to form at least Creating a modified antibody sequence; and methods for expressing the modified antibody sequence as a protein to provide.

[0272] Thus, in another embodiment, the present invention provides a method for the production of a recombinant human ovarian protein optimized for expression in mammalian cells. a FXIa-binding antibody having a sequence selected from the group of SEQ ID NO: 11 or 31; a full-length heavy chain antibody sequence having a sequence selected from the group of SEQ ID NO: 21 or 41; and preparing a FXIa-binding antibody comprising a full-length heavy chain antibody sequence and / or a long light chain antibody sequence. At least one amino acid residue in the full-length light chain antibody sequence is modified to form at least one modified To provide methods for creating variant antibody sequences and expressing the variant antibody sequences as proteins. In one embodiment, the heavy or light chain modification is within the framework regions of the heavy or light chain. is.

[0273] The modified antibody sequence may also be the CDR3 sequence, or the CDR4 sequence described in US2005 / 0255552. The minimal essential binding determinants are fixed and the CDR1 and CDR2 sequences are It can also be prepared by screening a diverse antibody library. Screening can be performed by using techniques such as phage display to screen antibodies in antibody libraries. This may be done according to any screening technique appropriate for screening from can.

[0274] Standard molecular biology techniques can be used to prepare and express the modified antibody sequences. The antibodies encoded by the modified antibody sequences may be human, cynomolgus monkey, rat and / or specifically binds to mouse FXIa; and the antibody binds to F36E cell proliferation assay and / or FXIa-dependent cell proliferation in Ba / F3-FXIaR cell proliferation assays. The FXIa binding proteins described herein, including but not limited to, inhibiting cell proliferation, An antibody that retains one, some, or all of the functional properties of a synthetic antibody.

[0275] In certain embodiments of the methods of engineering antibodies of the invention, the FXIa-binding antibody coding sequence Mutations can be introduced randomly or selectively along all or part of the sequence. and the resulting modified FXIa-binding antibodies are tested for binding activity and and / or other functional properties. Mutational methods have been described, for example, in PCT Publication No. WO 02 / 02004 by Short. Brochure 92780 provides information on saturation mutagenesis, synthetic ligation assembly, and or combinations thereof to create and screen antibody mutations. Alternatively, Lazar et al., PCT Publication No. WO 03 / 030944, discloses a method for the preparation of a compound of formula (I) and (II). The 74679 pamphlet uses computer-assisted screening methods to identify antibodies Methods for optimizing physiochemical properties are described.

[0276] In certain embodiments of the invention, the antibody is engineered to remove deamidation sites. Deamidation induces structural and functional changes within a peptide or protein. Deamidation is known to cause not only a decrease in biological activity but also the degradation of protein pharmaceuticals. This may also result in altered pharmacokinetics and antigenicity of the drug (Anal Chem. 2005 March 1;77 (5):1432~9).

[0277] In certain embodiments of the invention, antibodies have an increased pI, improving their drug-like properties. The pI of a protein is key to the overall biophysical properties of the molecule. Antibodies with a low pI are less soluble, less stable, and more prone to aggregation. Furthermore, the purification of antibodies with small pI is particularly important for clinical use. Scaling up for practical use can be difficult and problematic. Increasing the pI of anti-FXI and / or anti-FXIa antibodies or Fabs This improves their solubility and allows antibodies to be formulated at high concentrations (>100 mg / ml). By formulating antibodies at high concentrations (e.g., >100 mg / ml), This offers the advantage of allowing high doses of antibody to be administered, which reduces the risk of thrombosis. and / or thromboembolic disorders. Increasing the pI also increases the IgG version of the antibody, allowing for a reduction in the frequency of administration. It also increases the FcRn-mediated recycling of the drug, which allows the drug to be released for a prolonged period of time. It may also be possible for the drug to remain in the body, reducing the number of injections required. Due to the increased stability, the overall stability of the antibody is significantly improved, resulting in a longer shelf life. and prolonged in vivo biological activity. pI is 8.2 or greater. It is preferable that:

[0278] The functional properties of the modified antibodies can be evaluated using assays such as those shown in the Examples (e.g., ELISA). standard algorithms available in the art and / or described herein, such as The assay can be used to assess the effect of ATP on the immune system.

[0279] Prophylactic and Therapeutic Uses Antibodies that bind to FXI and / or FXIa (e.g., NO) as described herein Table 1, including anti-FXI / FXIa antibodies containing the VL and VH CDRs of V1401 The antibody or antigen-binding fragment of the present invention may be administered in an amount of 100 mg / kg or more. administration to a subject in need thereof to treat a thromboembolic disease or disorder (e.g., Thrombotic stroke, atrial fibrillation, Prevention of Stroke in Atrial Fibrillation (SPAF), deep vein thrombosis , venous thromboembolism, pulmonary embolism, acute coronary syndrome (ACS), ischemic stroke, acute lower limb ischemia It is therapeutically effective for treating chronic pulmonary hypertension (CPH), chronic thromboembolic pulmonary hypertension, or systemic embolism. The present invention provides an effective amount of the antibody of the present invention to a patient in need thereof. and administering the compound to a subject suffering from a thromboembolic disorder (e.g., a thrombotic disorder). The present invention provides a method for administering an effective amount of an antibody of the present invention to a subject in need thereof. This may reduce the risk of thromboembolic disorders (e.g., thrombotic stroke, atrial fibrillation, and cerebral Prevention of stroke (SPAF), deep vein thrombosis, venous thromboembolism, pulmonary embolism, acute coronary syndrome group (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, or systemic The present invention provides a method for treating vascular embolism.

[0280] An antibody described herein (e.g., NOV1401, or the VL of NOV1401) Antibodies described in Table 1, such as anti-FXI / FXIa antibodies containing the CDRs and VH CDRs. The term "body" refers to, among other things, thrombotic disorders, including, but not limited to, thrombotic disorders, as described in more detail herein. It is used to treat, prevent, and ameliorate thromboembolic conditions or disorders, including but not limited to: It can be used.

[0281] The antibodies presented herein (e.g., the VL CDRs and VH CDRs of NOV1401) Antibodies listed in Table 1, such as anti-FXI / FXIa antibodies, including R, also have been shown to be effective in preventing thromboembolic events. It may also be used in combination with other drugs to prevent, treat, or ameliorate disorders. For example, statin therapy is used to treat patients with thrombotic and / or thromboembolic disorders. and to use in combination with the FXIa antibodies and antigen-binding fragments of the present invention to can be done.

[0282] In specific embodiments herein, a method for treating or preventing stroke in patients with atrial fibrillation is provided. A method for inhibiting FXI / FXIa comprising administering an effective amount of an anti-FXI / FXIa antibody described herein, e.g., , NOV1401, or an anti-F comprising the VL CDR and VH CDR of NOV1401 If necessary, use an anti-FXI / FXIa antibody listed in Table 1, such as an XI / FXIa antibody. A method is presented comprising administering to a patient:

[0283] In specific embodiments herein, the present invention provides a method for preventing embolic stroke and total stroke in patients with atrial fibrillation. To manage or prevent atrial fibrillation (AF) and associated risks or conditions, such as cardiac embolism The method comprises administering an effective amount of an anti-FXI / FXIa antibody described herein, e.g., NOV 1401, or anti-FXI / F containing the VL and VH CDRs of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, such as XIa antibodies, are administered to patients in need of them. A method is presented that includes administering to a subject.

[0284] In specific embodiments herein, the present invention provides a method for preventing embolic stroke and total stroke in patients with atrial fibrillation. In a method for treating, managing, or preventing atrial fibrillation (AF) and related conditions, such as cardiac embolism an effective amount of an anti-FXI / FXIa antibody described herein, e.g., NOV14 01, or anti-FXI / FXI containing the VL and VH CDRs of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, such as FXIa antibodies, are available to patients in need. In certain embodiments, the AF patient is treated with a method comprising administering High risk.

[0285] In specific embodiments herein, a subject (e.g., a patient suffering from or suffering from deep vein thrombosis) to treat deep vein thrombosis or related conditions in subjects at risk of developing it. A method for treating, managing, or preventing FXI / F syndrome, comprising administering to a subject an effective amount of an anti-FXI / F syndrome described herein. XIa antibody, e.g., NOV1401, or the VL CDRs and VH of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, including anti-FXI / FXIa antibodies containing the CDRs A method is presented that includes administering the antibody to a subject in need thereof.

[0286] In specific embodiments herein, a subject (e.g., a patient suffering from or suffering from venous thromboembolism) Venous thromboembolism (VTE) or related events in subjects at risk of developing VTE A method for treating, managing, or preventing a condition comprising administering an effective amount of an anti-inflammatory drug described herein. FXI / FXIa antibodies, e.g., NOV1401, or the VL CDRs of NOV1401 and anti-FXI / FXIa antibodies containing the VH CDRs, as listed in Table 1 / FXIa antibody to a subject in need thereof. In certain embodiments, subjects treated with the anti-FXI / FXIa antibodies provided herein 1) first-time spontaneous VTE with low bleeding risk, including cancer patients; 2) spontaneous VTE or 3) have experienced a VTE associated with a thrombophilia.

[0287] In specific embodiments herein, a subject (e.g., a patient suffering from or experiencing a pulmonary embolism) is to treat, manage, or prevent pulmonary embolism or related conditions in subjects at risk of developing pulmonary embolism A method for treating or preventing inflammatory bowel disease, comprising administering to a subject an effective amount of an anti-FXI / FXIa antibody described herein; For example, NOV1401, or a sequence containing the VL CDR and VH CDR of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, including anti-FXI / FXIa antibodies, to a subject in need thereof.

[0288] In specific embodiments herein, a method for treating acute coronary syndrome (ACS) or its associated comorbidities in a subject is provided. A method for treating, managing, or preventing a condition associated therewith, comprising administering an effective amount of a compound as described herein. The anti-FXI / FXIa antibody described herein, for example, NOV1401, or a V Anti-FXI / FXIa antibodies containing the VL CDR and VH CDR are listed in Table 1. and administering to a subject in need thereof an anti-FXI / FXIa antibody. It is presented.

[0289] In specific embodiments herein, a subject (e.g., suffering from or having an ischemic stroke) Methods for treating, managing, or preventing ischemic stroke in a subject at risk of developing an ischemic stroke an effective amount of an anti-FXI / FXIa antibody described herein, e.g., NOV1 401, or anti-FXI / FX containing the VL and VH CDRs of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, such as Ia antibodies, can be administered to patients in need of such antibodies. A method is presented comprising administering to

[0290] In specific embodiments herein, a method for treating, managing, or administering acute lower limb ischemia in a subject is provided. A method of preventing, comprising administering an effective amount of an anti-FXI / FXIa antibody described herein, e.g., For example, NOV1401 or an antibody containing the VL CDR and VH CDR of NOV1401. Anti-FXI / FXIa antibodies listed in Table 1, such as FXI / FXIa antibodies, should be used if necessary. A method is presented comprising administering to a subject in need thereof.

[0291] In specific embodiments herein, a method for treating and vascularizing chronic thromboembolic pulmonary hypertension in a subject is provided. A method for treating or preventing FXI / FXIa-mediated leukemia, comprising administering an effective amount of an anti-FXI / FXIa antibody described herein. An antibody, such as NOV1401, or the VL CDR and VH CDR of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, including anti-FXI / FXIa antibodies containing R and administering the compound to a subject in need thereof.

[0292] In specific embodiments herein, a subject (e.g., a patient suffering from or suffering from a systemic embolism) Methods for treating, managing, or preventing systemic embolism in subjects at risk of developing an effective amount of an anti-FXI / FXIa antibody described herein, e.g., NOV1 401, or anti-FXI / FX containing the VL and VH CDRs of NOV1401 Anti-FXI / FXIa antibodies listed in Table 1, such as Ia antibodies, can be administered to patients in need of such antibodies. A method is presented comprising administering to

[0293] In certain embodiments herein, the catheter may be a thrombosed catheter (e.g., Hickman catheter in cancer patients) related condition or tubing causing blood clots Treat, manage, or prevent thromboembolic conditions that may develop with extracorporeal membrane oxygenation (ECMO) A method for inhibiting FXI / FXIa comprising administering an effective amount of an anti-FXI / FXIa antibody described herein, e.g., , NOV1401, or an anti-F comprising the VL CDR and VH CDR of NOV1401 If necessary, use an anti-FXI / FXIa antibody listed in Table 1, such as an XI / FXIa antibody. A method is presented comprising administering to a subject

[0294] In certain embodiments, the anti-FXI / FXIa antibodies described herein, e.g., NOV 1401, or anti-FXI / F containing the VL and VH CDRs of NOV1401 Requires treatment with an anti-FXI / FXIa antibody, such as an XIa antibody, as listed in Table 1 The target is: - Patients with indications for chronic anticoagulation therapy (e.g., AF, left ventricular thrombosis, previous cardioembolic stroke) Subject to change; Subjects at moderate to high risk of major bleeding; Dual antiplatelet therapy (aspirin and P2Y1) to prevent stent thrombosis Elective percutaneous coronary intervention with stent placement may require administration of a thrombus-receptor antagonist (THR). Subjects undergoing percutaneous coronary intervention (PCI) or direct PCI may include:

[0295] In certain embodiments, the following conditions: Paroxysmal atrial fibrillation or paroxysmal atrial flutter, persistent atrial fibrillation or persistent atrial flutter, or suspected or confirmed cardiac arrhythmias such as permanent atrial fibrillation or permanent atrial flutter Thromboembolism in subjects who have been Subjects with Stroke Prevention in Atrial Fibrillation (SPAF) subpopulations Subjects who are AF patients undergoing percutaneous coronary intervention (PCI); Treatment of acute venous thromboembolic events (VTE) in patients at high risk for bleeding, and and long-term prevention of secondary VTE; In secondary prevention after transient ischemic attack (TIA) or non-disability-inducing stroke In the prevention of cerebral and cardiovascular events, as well as thromboembolic events in heart failure with sinus rhythm cerebral and cardiovascular events in Left atrium clot formation and thrombosis in subjects undergoing cardiac defibrillation for cardiac arrhythmias thrombosis; Thrombosis before, during, and after ablation procedures for cardiac arrhythmias; Venous thrombosis, which can be deep vein thrombosis or superficial vein thrombosis in the lower or upper extremities. thrombosis of the abdominal and thoracic veins, sinus thrombosis and jugular vein thrombosis including but not limited to treatment and secondary prevention; Thrombosis on any artificial surface within a vein, such as a catheter or pacemaker lead; Pulmonary embolism in patients with or without venous thrombosis; · Chronic thromboembolic pulmonary hypertension (CTEPH); Arterial thrombosis over ruptured atherosclerotic plaques, over intra-arterial prostheses or catheters thrombosis in the arteries of the thorax and in apparently normal arteries, which may be associated with acute coronary syndromes. group, ST-segment elevation myocardial infarction, non-ST-segment elevation myocardial infarction, unstable angina, stent thrombosis, arterial Thrombosis of any artificial surface in the system, and in subjects with or without pulmonary hypertension including but not limited to pulmonary artery thrombosis; Thrombosis and thrombosis in patients undergoing percutaneous coronary intervention (PCI) embolism; Cardioembolic and cryptogenic stroke; Thrombosis in patients with invasive and non-invasive cancer malignancies; Thrombosis across the indwelling catheter; · Thrombosis and thromboembolism in critically ill patients; Cardiac thrombosis and thromboembolism, which are cardiac thrombosis after myocardial infarction, cardiac artery conditions such as aneurysms, myocardial fibrosis, cardiac hypertrophy and dysfunction, myocarditis, and artificial surfaces within the heart. including, but not limited to, cardiothrombosis associated with pulmonary embolism; · Thromboembolism in patients with valvular heart disease, with or without atrial fibrillation; · Thromboembolism across mechanical or biological heart valve prostheses; - After cardiac repair surgery for simple or complex cardiac anomalies, natural or artificial cardiac pads injury or trauma in patients with arterial or venous conduits; Knee replacement, hip replacement, and orthopedic, thoracic, or abdominal surgery venous thrombosis and thromboembolism after After neurosurgery, including intracranial and spinal interventions arterial or venous thrombosis; Factor V Leiden, prothrombin mutations, antithrombin III, prothrombin Protein C deficiency and protein S deficiency, factor XIII mutations, familial fibrinogen deficiency Plasminogenemia, congenital plasminogen deficiency, elevated factor XI levels, sickle cell disease, Antiphospholipid syndrome, autoimmune diseases, chronic intestinal diseases, nephrotic syndrome, hemolytic uremia, bone marrow Proliferative disorders, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and heparin-induced hematoma congenital or acquired thrombophilia, including but not limited to thrombocytopenia; Thrombosis and thromboembolism in chronic kidney disease; and - Thrombosis and thrombosis in patients undergoing hemodialysis and extracorporeal membrane oxygenation Thromboembolism or one of the anti-FXI / FXIa antibodies described herein, e.g., NOV140. 1, or anti-FXI / FXIa containing the VL and VH CDRs of NOV1401 These can be treated or managed with anti-FXI / FXIa antibodies, such as those listed in Table 1. Cut.

[0296] In specific embodiments herein, the anti-FXI / FXIa antibodies provided herein (e.g., , an anti-FXI / FXIa antibody comprising the VL CDR and VH CDR of NOV1401, etc. Bleeding in patients treated with or receiving antibodies (antibodies listed in Table 1) A method for managing bleeding associated with, for example, trauma, surgery, menstruation, or postpartum, comprising administering an anticoagulant A method involving reversal of the vasodilatory effect is presented. FXI deficiency has been associated with spontaneous bleeding episodes. In particular embodiments, bleeding is associated with trauma, surgery, menstruation, or postpartum. Prolonged bleeding is most typically seen after major trauma or in the oral mucosa, nasal mucosa, May occur after surgery involving organs with areas of high fibrinolysis, such as the genital or urinary mucosa Tooth extractions, tonsillectomies, and uterine or prostate ablations carry a significant risk of bleeding. People with disabilities are also prone to developing nosebleeds and ecchymoses. FXI deficiency is highly prevalent and, in rare cases, can cause bleeding into the urine or intestines. The frequency of spontaneous muscle or joint bleeding and intracranial bleeding is not increased in patients with idiopathic pulmonary edema. Pulse puncture is not usually associated with prolonged bleeding. Other gene mutations associated with FXI deficiency This may contribute to the heterogeneous and unexpected bleeding tendency in patients with severe FXI deficiency. The concomitant use of platelet agents, other anticoagulants, and fibrinolytic agents may increase the risk of bleeding.

[0297] In certain embodiments herein, the anti-FXI / FXIa antibodies provided herein (e.g., For example, an anti-FXI / FXIa antibody containing the VL CDR and VH CDR of NOV1401. 1. A method for managing bleeding in a patient treated with an antibody (such as an antibody listed in Table 1). A method involving the reversal of the primary anticoagulant effect for a period of time sufficient to control bleeding is presented. In a specific embodiment, the step of reversing the anticoagulant effect comprises (i) administering a colloid, crystalline Fluid replacement using plasma, human plasma, or plasma proteins such as albumin; or (ii) transfusion with packed red blood cells or whole blood. In certain embodiments, e.g. In severe emergency cases, a therapeutic agent for reversing the effects of anticoagulants is fresh frozen plasma (FFP). FP), prothrombin complex concentrate (PCC), and activated PCC [(APCC); For example, factor VIII inhibitor bypass activity (FEIBA)] and recombination activity Prohemostatic blood components, including, but not limited to, recombinant factor VII (rFVIIa) In one embodiment, administration of rFVIIa at a dose of 30 μg / kg is followed by 6 hourly for 5-7 days, plus 1g of tranexamic acid every 2-4 hours plus 15mg of tranexamic acid every 2-4 hours. A regimen involving administration of rFVIIa at a dose of ~30 μg / kg over 24–48 hours undergoing major surgery, the use of anti-FXI / FXIa antibodies presented herein (e.g., NOV 1401 or NOV1401) restore hemostasis in subjects with active bleeding and inaccessible bleeding sites; For example, Riddelle et al. reported their experience in four patients undergoing surgery with severe FXI deficiency ( Riddell et al., 2011, Thromb. Haemost.; 106: 521-527), patients received 30 μg / kg rFVIIa and 1 g of tranexamic acid were administered intravenously at the time of induction of anesthesia. , bolus administration of 15–30 μg / kg rFVIIa is effective in Rotational Th As indicated by the results of romboelastometry (ROTEM), Patients were treated with the above doses of rFVIIa over 24-48 hours. Tranexamic acid, 1 g every 6 hours, was continued for 5 days. In small series, low doses of 15–30 μg / kg in combination with tranexamic acid rFVIIa was shown to be a safe and effective treatment for correcting hemostatic abnormalities in severe FXI deficiency in this study. Inhibitors (usually administered via transfusion or in patients with severe FXI deficiency) were safe and effective. presents with severe FXI deficiency associated with autologous FXI neutralizing antibodies acquired after administration of blood products. In another study involving four patients who underwent five surgeries, the authors (Livnat et al., 2009, T hromb. Haemost.; 102: 487-492) applied the following protocol: 2 hours before surgery Oral tranexamic acid 1 g in the 1990s, then immediately before the intervention, patients were given another The animals were given 1 g of intravenous tranexamic acid. FVIIa was infused at the completion of surgery, followed by oral tranexamic acid 1 g for 6 hours. Fibrin glue was applied for at least 7 days after each extraction in one patient. This protocol was used in patients with severe FXI deficiency associated with inhibitors. Normal hemostasis was achieved in patients with pulmonary embolism.

[0298] In one embodiment, fibrin glue is used to improve dental prosthesis function in patients with FXI deficiency. It can restore local hemostasis during surgery (Bolton-Maggs (2000) Haemophilia; 6 (S1): 100-9). The anti-FXI / FXIa antibodies presented herein (e.g., NOV1401) In certain embodiments of the methods for managing bleeding in a treated patient, the method comprises administering a fibrinogen associated with the use of anesthetic adhesives, consisting of tranexamic acid 1 g every 6 hours for 5 to 7 days. The regimen is suitable for subjects undergoing minor surgery and for bleeding events in the oral and nasal cavities. may be used to establish local hemostasis in subjects where the bleeding site is accessible. do.

[0299] Pharmaceutical Composition The present invention relates to a FXIa-binding antibody (interacting antibody) formulated together with a pharmaceutically acceptable carrier. The compositions may also be used to treat, for example, thrombus formation. one or more other therapies suitable for the treatment or prevention of an embolic disorder (e.g., a thrombotic disorder); Pharmaceutically acceptable carriers may also contain agents that enhance or stabilize the composition. Alternatively, it may be used to facilitate the preparation of the composition. The carrier may contain physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic These include agents that delay absorption and agents that delay absorption.

[0300] The pharmaceutical compositions of the present invention can be administered by a variety of methods known in the art. The route and / or mode of administration will vary depending on the desired results. administration (iv), intramuscular (im), intraperitoneal (ip), or subcutaneous (sc) or administration in close proximity to the target site. Acceptable carriers include those suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration. It should be suitable for administration (e.g., by injection or infusion). Depending on the route of administration, activation Antibodies, i.e., bispecific and multispecific molecules, can bind compounds and block compounds. It can be coated with a material that protects it from the action of activating acids and other natural conditions. Cut.

[0301] In certain embodiments, the anti-FXI / FXIa antibodies described herein (e.g., NOV14 01, or antibodies containing the LCDR and HCDR of NOV1401, as described in Table 1 Approximately 75 mg to 1 mL of the antibody (antibody) is added to a fluid vial for subcutaneous injection. In certain embodiments, the pharmaceutical composition is formulated at a concentration of about 200 mg per 1000 mg of the active ingredient. or pharmaceutical excipients, such as sucrose and polysorbate 20. In the present invention, 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.

[0302] In certain embodiments, the anti-FXI / FXIa antibodies described herein (e.g., NOV14 01, or antibodies containing the LCDR and HCDR of NOV1401, as described in Table 1 The antibody (antibody containing the antibody) is prepared in a fluid vial for subcutaneous injection at a concentration of 150 mg per mL. In one embodiment, the 150 mg / mL fluid formulation is formulated at pH 5.5±0.5. 150 mg of anti-FXI / FXIa antibody, L-histidine, histidine HCl monohydrate , sucrose, and polysorbate 20. The composition is sterile and flowable. The appropriate fluidity can be achieved by using a coating such as lecithin. In the case of a dispersion, the required particle size can be maintained by This can be maintained by using surfactants. Often, the composition also contains an isotonicity agent, For example, sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride It is preferable that the injectable composition contains a compound that delays absorption. by incorporating agents such as aluminum monostearate or gelatin. can bring about.

[0303] The pharmaceutical compositions of the present invention can be prepared by methods well known and routinely practiced in the art. For example, it can be prepared according to the method described in Remington: The Science and Practice of Pharmacology. acy, Mack Publishing Co., 20th edition, 2000; and Sustained and Controlled Release Dru See "Delivery Systems," edited by J.R. Robinson, Marcel Dekker, Inc., New York, 1978. The pharmaceutical composition of the present invention is preferably manufactured under GMP conditions. Herein, the therapeutically effective dose or therapeutically effective dose of a FXIa-binding antibody is used. Typically, FXIa-binding antibodies can be produced in pharmaceutical formulations by conventional methods known to those skilled in the art. The dosage regimen is determined based on the optimal response desired (e.g., therapeutic efficacy). For example, a single bolus can be administered, or multiple doses can be administered. Several divided doses may be administered over a period of time, as indicated by the requirements of the therapeutic situation. Doses may also be proportionally reduced or increased as indicated. Parenteral compositions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to a dosage unit that is Each unit refers to a physically discrete unit suitable as a unit dosage for a desired pharmaceutical agent. It contains a predetermined amount of active compound, in association with a carrier, calculated to produce a desired therapeutic effect. Has.

[0304] The practical dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be adjusted to any level, particularly those that are not toxic to the patient. An effective amount of active ingredient is used to achieve the desired therapeutic response for a given patient, composition, and mode of administration. The dosage level selected can be varied to obtain an effective dose. The specific compositions of the invention, their activity, route of administration, timing of administration, and excretion of the specific compounds incorporated therein the rate of release, the duration of treatment, other drugs used in combination with the particular composition employed, the compound and / or material, the age, sex, weight, condition and general health of the patient to be treated; and medical history.

[0305] A physician may administer the antibodies of the present invention, incorporated into a pharmaceutical composition, to achieve the desired therapeutic effect. Start at levels below those required to achieve optimal efficacy and gradually increase the dosage until the desired effect is achieved. Generally, the thrombotic and / or thromboembolic The dosage of the compositions of the present invention that is effective to treat a disorder will vary depending on the means of administration, the target site, the patient's physiology, and the dosage. the clinical condition, other medications being administered, and whether the treatment is prophylactic or therapeutic Treatment dosages vary depending on many different factors. For systemic administration of antibodies, the dose should be adjusted to 100 mg / kg of host body weight. For antibody administration (e.g., subcutaneous administration), the dose is in the range of about 0.01 to 15 mg. can be in the range of 0.1 mg to 5 mg or 1 mg to 600 mg. The anti-FXI / FXIa antibodies described in mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / k g, 0.8mg / kg, 0.9mg / kg, 1.0mg / kg, 1.1mg / kg, 1. 2mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2.0mg / kg, 2 .1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4m g / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg , 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3 mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / k g, 4.8 mg / kg, 4.9 mg / kg, or 5.0 mg / kg. Exemplary treatment regimes include once every two weeks, or once a month, or every 3-6 days. An exemplary treatment regime involves systemic administration once every week, once every two weeks, or , once every 3 weeks, once a month, or once every 3-6 months, or as needed ( This involves systemic administration of PRN).

[0306] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) at 3 mg / kg, for example, by iv or sc Administer at a dose of

[0307] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) at 10 mg / kg, for example, by iv or sc Administer at a dose of 1 g.

[0308] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) at 30 mg / kg, for example, by iv or sc Administer at a dose of 1 g.

[0309] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) at 50 mg / kg, for example, by iv or sc Administer at a dose of 1 g.

[0310] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) administered intravenously or sc, for example, at 100 mg / Administer at a dose of kg.

[0311] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) are administered intravenously or intravenously for 5 min. Administer in doses ranging from 1000 mg to 600 mg.

[0312] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, 1) by the iv route or sc route, for example, mg, 10mg, 15mg, 20mg, 30mg, 40mg, 50mg, 60mg, 90 mg, 100mg, 120mg, 150mg, 180mg, 200mg, 210mg, 2 40mg, 250mg, 270mg, 300mg, 330mg, 350mg, 360mg , 390mg, 400mg, 420mg, 450mg, 480mg, 500mg, 510 It is administered in doses of 540 mg, 550 mg, 570 mg, or 600 mg.

[0313] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, antibodies listed in Table 1) at a dose of 5 mg, e.g., by sc route .

[0314] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) are administered, for example, by the sc route at a dose of 15 mg. do.

[0315] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) are administered, for example, by the sc route at a dose of 50 mg. do.

[0316] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, antibodies listed in Table 1) at a dose of 150 mg, e.g., by sc route do.

[0317] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, antibodies listed in Table 1) at a dose of 300 mg, e.g., by sc route do.

[0318] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, antibodies listed in Table 1) at a dose of 600 mg, e.g., by sc route do.

[0319] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) can be administered to patients via the aP route, for example, by the iv or sc route. The mean duration of TT was extended by more than two times for 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 administered in a dose sufficient to achieve this for a period not exceeding 50 days.

[0320] In certain embodiments, an anti-FXI / FXIa antibody described herein (e.g., NO V1401, or an antibody comprising the VL CDR and VH CDR of NOV1401, Antibodies listed in Table 1) can be administered to patients via the aP route, for example, by the iv or sc route. Achieved a 2-fold or greater increase in the mean duration of TT for a period not exceeding 42 days Administer in a dose sufficient to

[0321] The antibody is typically administered multiple times. The interval between single doses may be weekly, biweekly, monthly, or every The interval may also be years. It may also be irregular, as indicated by measuring blood levels of the antibody. Additionally, your doctor may determine an alternative dosing interval, such as monthly or as needed to be effective. In some methods of systemic administration, the dose is administered at intervals that achieve a plasma concentration of antibody of 1 to 1 Adjust to achieve 1000µg / mL or 1-1200µg / mL, and The method is adjusted to achieve a concentration of 25-500 μg / mL. Alternatively, the antibody may be administered as a sustained release It can also be administered as an excipient, which requires less frequent administration. The frequency and duration of antibody responses will vary depending on the half-life of the antibody and its target in the patient. and humanized antibodies have a longer half-life in humans than chimeric and non-human antibodies. The dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. Thus, relatively high doses are required at relatively short intervals until disease progression is reduced or terminated. and preferably until the patient shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient can be administered a prophylactic regimen. [Example]

[0322] The following examples are presented to further illustrate the present invention and should not be construed as limiting its scope. Other variations of the invention will be readily apparent to those skilled in the art. and is encompassed by the appended claims.

[0323] Example 1 Panning with a human Fab phage library To select antibodies that recognize human factor XI, multiple panning strategies were utilized. for different mutants of the catalytic domain proteins of human factor XI and rabbit factor XIa Therapeutic antibodies are derived from commercially available phase display libraries, such as Mo Using the rphosys HuCAL PLATINUM® library, The phage midi blocker was generated by selecting clones that bound to factor XI. Lee developed the HuCAL® concept (Knap et al.) to display Fab on the phage surface. CysDisplay™ based on the results of the study (Pik et al., 2000, J Mol Biol 296: 57-86). The present invention is based on the technology disclosed in International Publication No. WO 01 / 05950. To isolate the body phase, a panning strategy was employed.

[0324] Cross-reactivity analysis The purified Fab was assayed for human factor XI (factor XI, factor XIa, and and catalytic domain of factor XIa) and biotinylation of the catalytic domain of rabbit factor XIa The binding of the lysed protein to different mutants was investigated. p™ (Nunc) 384-well plates were filled with 10 μg / ml Ne in PBS at 4°C. The antigen was coated with NeutrAvidin overnight. Top, captured via biotin for 30 min at room temperature (RT). Binding was confirmed using the Attophos fluorescent substrate (Roche: product number 11681982001). was used to conjugate to alkaline phosphatase (diluted 1:5000) The fluorescence at 535 nm was detected by F(ab)2-specific goat anti-human IgG. , recorded with excitation at 430 nm.

[0325] IgG conversion and expression To express full-length IgG in CAP-T cells, the variable domain (VH) of the heavy chain was cleaved. The variable domain (VL) fragment of the Fab and light chain was synthesized from an expression vector for human IgG1 The fragments were subcloned into the appropriate pMorph®_hIg vector. The amino acid substitutions (D265A and P329A) were introduced into the Fc portion to allow for any surface interaction. These antibodies reduced the possibility of ADCC or CDC caused by FXI. Ranine substitutions have been shown to reduce ADCC and CDC (e.g., Idosugie et al., J. Immunol. 164:4178-4184, 2000; Shields et al., J. Biol. Chem. 276:659 (See, e.g., J. Immunol. 2001, 1-6604). Seven days after transfection, cell culture supernatants were collected. After sterile filtration, the solution was transferred to the process using a liquid handling station. The sample was subjected to tein A affinity chromatography. The product was eluted in 40 nM NaOH, the pH was neutralized with 1 M Tris buffer, and sterile filtered ( Protein concentration was determined by UV spectrophotometry at 280 nm. The purity of the IgG was analyzed under denaturing and reducing conditions in SDS-PAGE.

[0326] Example 2 Combined Data Surface plasmon resonance (SPR) analysis of the FXI catalytic domain SPR measurements were performed using BIACORE, a surface plasmon resonance-based optical biosensor. (Trademark) T200 (BIACORE (Trademark), GE Healthcare, Uppsa The experiment was carried out on a Series S sensor chip (CM5), immobilization kit, and The IgG and regeneration buffer were purchased from GE Healthcare (Uppsala). Alternatively, two different assay setups were performed depending on the Fab ligand format. First, N-hydroxysuccinimide (NHS) and N-(3-dimethylamino) Surface activation with N-ethyl-N-propylcarbodiimide hydrochloride (EDC) The amine coupling was carried out by standard amine coupling method (GE Healthcare, Uppsala, a) NOV1401-Fab was transferred to activated dextran matrix on a CM5 chip. The NOV1401-IgG was covalently attached to the antibody. Then, goat anti-human IgG-Fc antibody (JIR) was immobilized on the chip at 14,000 RU. The remaining active surface groups were deactivated with ethanolamine (EA). Prepare uninoculated reference cells and inoculate the system with 1x HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7 The mixture was equilibrated with 1000 kJ / min (Teknova H8022).

[0327] All binding experiments were performed using HBS-EP+ buffer at 25°C with a flow rate of 50 μL / min. For the capture assay, NOV1401-IgG was diluted to a level of 80 RU. For kinetic studies, the antibody was captured in the range of 0–200 nM in HBS-EP+ buffer. A dilution series of FXI catalytic domain was used at a concentration of 0.01 mg / mL. The association time was 120 seconds. The dissociation time was 180 seconds. The surface was then washed with a single injection of 10 mM glycine, pH 1.5 ( The data was regenerated by a 60-second contact time and a 120-second stabilization time. Processing and k on、 k off , and K. D The decision was made by the T200 BiaEvaluat This was achieved using the ion software version 1.0. A correction factor (subtraction of the injection) was applied to correct for bulk effects and other systematic artifacts. The 1:1 binding model (R max By applying The sargram was fitted.

[0328] Equilibrium titration in solution (SET) for FXI and FXIa Twenty-two serial 1.6-fold dilutions of the antigen were diluted in sample buffer (0.5% Prepared in PBS (pH 7.4 containing BSA and 0.02% Tween 20) A fixed concentration of NOV1401-Fab (200 pM for huFXI, hu For FXIa, the concentration was 500 pM) or NOV1401 antibody (huFXI and For huFXIa, 10 pM was added to each antigen concentration. The starting concentration of huFXIa was 100 nM and that of huFXI was 20 The concentration was 60 nM (Fab assay) or 1 nM (IgG assay) per well. μl aliquots of the diluted mix were dispensed in duplicate into 384-well polypropylene MTPs Sample buffer was used as a negative control, and a sample containing no antigen was used as a positive control (Bmax). The plates were sealed and insulated overnight at room temperature on a shaker. A standard 384-well MSD array MTP was incubated with 100% ribosomal RNA diluted in PBS. 0.1 μg / ml huFXIa (for huFXIa and huFXI) 1 well Coat with 30 μl per well, seal and incubate overnight at 4°C. I did.

[0329] Incubation and TBST (TBS containing 0.05% Tween 20) After washing three times with ), the antigen-coated MSD plate was Block with 50 μl of blocking buffer (PBS containing 5% BSA) per well. The plate was then incubated for 1 hour at room temperature on a shaker. 30 μl of Fab-antigen preparation / IgG-antigen preparation per well was added to polypropylene Transfer the antigen from the Renn MTP to the antigen-coated MSD plate and incubate at room temperature on a shaker. After a further washing step, the plate was diluted in sample buffer and incubated for 20 min. Diluted with 0.5 μg / ml of ECL-labeled goat anti-human IgG / Fab detection antibody (MSD 30 μl of the solution was added to each well and incubated at room temperature for 30 minutes with shaking. The plate was washed again three times, and then 35 μl of read buffer (M SD) was added to each well. Electrochemiluminescence (ECL) signal was generated and MSD Detection was performed using a Sector Imager 6000.

[0330] The mean ECL signal was calculated from duplicate measurements within each assay. The data points were adjusted for baseline by subtracting the lowest value and then plotted against the corresponding antigen concentration. and plotted. D The values ​​are used to split the plot into 1:1 (for Fab) or 1: 2 (for IgG) fitting model (following Piehler et al., 1997) This was decided as follows.

[0331] result The results are summarized in Tables 3 and 4. The mat also contains the FXI catalytic domain as determined by BIACORE™. For ATP, the K value is approximately 20 nM. D The values ​​obtained were: Fab, activated FXI, and zymogen F The affinity for both XI is in the pM range, and the affinity for the catalytic domain is Based on their high affinity, These interactions were measured by SET assay. Zymogen FXI (62 pM) vs. activated FXI (305 pM) The interaction may be influenced by avidity effects. Therefore, NOV1401-IgG is a dimeric zymogen FXI and an activated form of FXI. The affinity of FXI for either D The value is recorded as

[0332] To confirm that NOV2401 also binds to cynomolgus monkey FXI, SET Experiments were performed on cynomolgus monkey activated FXI and cynomolgus monkey zymogen FXI The results were 12.5±6.6pM (N=2) and 5.0±0.7pM, respectively. (N=2) apparent K D Therefore, the efficacy of NOV1401 against cynomolgus monkey FXI was The affinity for the protein (active form and zymogen) is shown to be related to the binding to human FXI. The affinity for the fusion protein is equivalent to that for the fusion protein (Table 3).

[0333] [Table 2]

[0334] [Table 3]

[0335] Example 3 Biochemical assay: Inhibition of FXIa in activity assays using fluorescent peptides as substrates harm Human FXIa (Kordia Life Science NL: Cat. No. HFXIa 1 The activity of 111a) was confirmed by the sequence D-Leu-Pro-Arg*Rh110-D-Pro (product No.: BS-2494; Biosyntan GmbH, Berlin, Germany The cleavage of the fluorescently labeled peptide is determined by monitoring the cleavage of the fluorescently labeled peptide with the In the substrate sequences shown, * denotes the scissile bond, D-Leu: D-leucine, Pro: Proline, Arg: arginine, Rh110: rhodamine 110, D-Pro: D-proline FXIa-mediated cleavage of the scissile bond of the peptide substrate is indicated by , rhodamine when using excitation and emission wavelengths of 485 nm and 535 nm. This results in an increase in fluorescence intensity of 110. The fluorescence intensity was measured at room temperature (RT) using a microtiter plate. Rate reader Safire2 (TECAN, Maennedorf, Switzerland) The assay buffer is 50 mM HE at pH 7.4. PES, 125 mM NaCl, 5 mM CaCl, and 0.05% (w / v) C The final activity assay was performed using human FXIa and the substrate BS- 2494 have assay concentrations of 0.1 nM and 0.5 μM, respectively. Under these conditions, the increase in fluorescence intensity over time is linear for at least 60 minutes. do.

[0336] To examine the inhibitory activity of the antibody, the antibody was incubated in a PBS containing 0.05% (w / v) CHAPS. BS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, Prepare a dilution series of the antibody in 1.8 mM KH2PO4. Add 2 μL of antibody solution to 1 Pre-incubated with 100 μL of FXIa solution (in assay buffer) at room temperature for 60 minutes. After the pre-incubation step, 10 μL of the substrate BS-2 494 (diluted in assay buffer) was added and the enzymatic reaction was allowed to proceed for 60 minutes. The fluorescence intensity is then measured. The signal from the control reaction (uninhibited reaction) is 0%. The signal from the reaction containing no enzyme is equivalent to 100% inhibition. ) and the following formula for transferring values: y=100%-[FI(x)-FI(min)] / [FI(max)-FI(min)] [where y is the % inhibition when the antibody concentration is x, and FI(x) is the FI (min) is the fluorescence intensity measured when the antibody is not present in the control reaction. FI(max) is the fluorescence intensity measured in the uninhibited control reaction; FI(max) is the fluorescence intensity measured in the uninhibited control reaction. The fluorescence intensity values ​​were calculated as percentage inhibition by using the formula [where γ is the fluorescence intensity measured at γ = 0.05, γ = 1.0, γ = 2.0 ... The data was converted into a graph using the Origin 7.5SR6 program (OriginLa b Analyzed using a 2000 serogroup (Chemicals Corporation, USA). IC50 based on average data The value is the logistic function: y = A2 + (A1 - A2) / (1 + (x / IC50) p ) [wherein y is the % inhibition when the antibody concentration is x, A1 is the minimum inhibition value, A2 is the maximum inhibition value. The exponent p is the Hill coefficient.

[0337] Figure 6A shows the inhibitory effect of antibody NOV1401 on the enzymatic activity of full-length human FXIa. Representative composite response curves for human full-length FXIa are shown. The enzyme activity was inhibited in a concentration-dependent manner (Fig. 6A). A rule fit gave an IC of approximately 160 pM. 50 Brings value.

[0338] Example 4 Anticoagulant activity of anti-FXIa Ab Activated partial thromboplastin time (aPTT) assay and thrombin generation assay By using TGA, the antibodies NOV1401 and NOV1090 The antithrombotic activity was investigated.

[0339] aPTT assay: Lyophilized normal human plasma "Coagulation Control N" (reference number: 5020050) was purchased from Techno The coagulation control N was purchased from Clone GmbH (Vienna, Austria). The normal plasma was pooled from citrated plasma of selected healthy donors. Clotting time reflects the normal concentrations of clotting factors involved in blood clotting. Freeze-dried plasma is stored at 4°C. Before use, the vial was carefully rotated and then left to stand at room temperature for 10 minutes. The plasma was resuspended in 1 mL of distilled water by holding.

[0340] The intrinsic pathway inducer "aPTT-s" (reference number: TE0350) is available from SYCO. med (Lemgo, Germany) and buffer (sodium chloride phospholipids in (polyethylene glycol 20000; sucrose, sodium azide) The solution was stored at 4°C.

[0341] Calcium chloride (reference number: C1016-500G; Sigma-Aldrich C hemie GmbH, Steinheim, Germany) in double-distilled water for 25 minutes. It was prepared at a stock concentration of 100 mM.

[0342] UltraPure Tris / HCl buffer at pH 7.5 (reference number: 15567-02 7; Life Technologies Corporation, NY, USA) and and phosphate-buffered saline (PBS; reference number: P4417-100TAB; Sigma -Aldrich Chemie GmbH, Steinheim, Germany) , was used as a composite diluent.

[0343] 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate dimethicone hydrate (CHAPS; reference number: C3023-25G) and anhydrous dimethyl sulfoxide DMSO (Ref: 276855-100ML) was purchased from Sigma-Aldrich It was purchased from Chemie GmbH (Steinheim, Germany).

[0344] The clotting time was measured using a semi-automated mechanical clot detection system, Amelung ba ll coagulometer model KC4A(SYCOmed, Lemgo The system was carried out using a stainless steel ball (purchased from Sigma-Aldrich GmbH, Germany). (Ref. AI5000; SYCOmed) Utilize I4000;SYCOmed).

[0345] The sample is added to the cuvette. After an appropriate incubation time, the cuvette is Place the sample in the measuring well of the Amelung ball coagulometer. The well is slowly rotated, causing the cuvette to rotate around its long axis. Because it is placed at a slight angle, gravity and inertia forces always keep the ball at the lowest point in the cuvette. The magnetic sensor is located opposite the ball position. After adding the inducer, A timer is started. As clotting occurs, fibrin strands are formed in the reaction mixture. The fibrin strands pull the ball away from its inertial position, thereby creating a magnetic field within the magnetic sensor. , triggers an impulse that electrically stops the timer. The feeding scheme was as follows (Table 4a):

[0346] [Table 4]

[0347] The samples were stored in duplicate in an Amelung ball coagulometer at 37°C. Measured in degrees.

[0348] Figure 6B shows the effect of NOV1401, an antibody that causes a concentration-dependent prolongation of the aPTT clotting time. Representative composite response curves are shown. Results show that NOV1401 inhibits the aPTT clotting time of human plasma. These results suggest that aPTT clotting time was prolonged at a concentration of approximately 14 nM. Compared to baseline, the IC 50 The value is approximately 13 nM. It was calculated that there was.

[0349] Thrombin Generation Assay (TGA): TGA lyophilized normal human plasma (clotting control N) was purchased from Technoclone GmbH ( Reference number: 5020040, Lot number: 1P37B00) and indicated by the manufacturer. Reconstitute in distilled water in the suggested volume.

[0350] Technoclone GmbH (Reference number: 5006230, Lot number: 8F4 The fluorescent substrate Z-Gly-Gly-Arg-AMC (manufactured by 1B00) was used to detect the substrate. The solution was prepared. Aliquots of the lyophilized substrate were kept at 4°C. Twenty minutes before use, the substrate was freshly dissolved in the volume of distilled water indicated on the vial. The resulting substrate solution contained 1 mM of fluorescent peptide and 15 mM of CaCl2. Contains.

[0351] Two different reagents, "T GA RD" (Reference number: 500622) and "TGA RC Low" (Reference number: 5006213) is a subsidiary of Technoclone GmbH, Vienna, Austria The induction reagent, PPP (platelet-poor plasma) - Reagent Low " is a Thrombinoscope (TS31.00, Lot Number: PPL1409 / 01) and reconstituted in distilled water as indicated on the vial. "t Low" contains a mixture of phospholipids and tissue factor at a very low concentration. Immediately before use, resuspend the solution in 80 mM Tris / HCl, pH 7.4, 0.05% (w / v) CHAP The solution was diluted 8-fold in S.

[0352] Samples were plated in a 96-well black clear bottom plate purchased from Costar (product number 3603). For automated transfer, samples were transferred into a V-bottom 96-well plate. The wells were placed in a well plate (Costar, 3894) and analyzed using a CyBio automated system (Anal The transfer was performed using a ytik Jena US (Woburn, MA, USA).

[0353] reconstituted human plasma, the triggering reagent "PPP-Reagent Low," and The substrate was preheated in a water bath at 37°C for 10 minutes. A dilution series of 5 μM (5x the final highest concentration of 1 μM) was prepared in a 96-well plate. A total of eight dilutions were prepared, starting with the NOV1401 concentration. The drug was mixed with 1108 μl of substrate solution to create a 10+50 inducer reagent substrate mix. For subsequent transfer using an automated system, 80 μl was added per well using a V-shaped The reagents were added to a 96-well plate. The plate was kept at 37°C. The reagents were as shown in Table 4b. The addition was carried out according to the scheme.

[0354] [Table 5]

[0355] Automation was used to transfer the inducer / substrate mixture. After adding the mixture, the Syner gy Neo instrument(BioTek Instrument Inc. , Winooski, VT, USA) at 360 nm and 460 nm, respectively. The excitation and emission of the sample were recorded immediately. The sample was incubated in a plate reader at 37°C. , measured in duplicate at 55-second intervals over a 90-minute period.

[0356] To generate peaks, the TGA evaluation software provided by Technoclone was used. The thrombin concentration data was processed using a software file. Use GraphPad software to generate a graph of thrombin concentration versus antibody The concentration data were fitted using GraphPad Prism5 software. GraphPad Software Inc., La Jolla, CA, USA A nonlinear regression model was fitted to the IC 50 The value is a built-in type of 4 parameters. Dose-response curve equation (variable slope): y = Bottom + (Top - Bottom) / (1 +10 (Log IC50-x)×HillSlope ) [wherein y is the inhibitor concentration is the maximum thrombin concentration formed when the concentration is x, and Top and Bottom are , the inhibitor-free thrombin concentration and the maximum inhibitor concentration, respectively. The thrombin concentration was determined using the following formula:

[0357] Figure 6C shows the concentration-dependent inhibition of thrombin generation in TGA by the antibody NO. A representative composite response curve for V1401 is shown. IC of 24 nM 50 Value and 159 Residual thrombin concentrations in nM (dotted line) were calculated for this composite response curve.

[0358] Example 5 Protein expression, complex formation, and binding of NOV1401(Fab)-FXI(catalytic domain) Crystallization and structure determination Antibody NOV1 obtained by papain cleavage in a complex with the Factor XI catalytic domain The structure of the Fab portion of 401 was obtained by co-crystallization at 2.04 Å resolution.

[0359] Protein expression: The expression construct for the FXI catalytic domain contained an unpaired cysteine ​​mutated to a cysteine. Amino acid residues 388 to 625 (Swissprot P0395) with C500 1), which is composed of the amino acid MGSS (SEQ ID NO: 49), an octahistidine tag (SEQ ID NO: 49), and No. 50), a PreScission™ cleavage site followed by an enterokinase cleavage site The construct was assembled by gene synthesis and cloned into pE E. coli (E. coli) was cloned into the T24a expression vector and grown in LB medium. The inclusion bodies were expressed in BL21(DE3) strain of 50 mM Tris / HCl pH 8.0, 6.0 M guanidinium chloride, 50 mM DTT for 2 hours The recombinant protein was solubilized and completely denatured. Buffer (0.5 M Tris pH 8.0, 0.9 M arginine HCl, 5 mM GSH) , 0.5 mM GSSG, 1 mM EDTA to the IB solution, and a final protein concentration of 50 The mixture was immediately added to 100 μg / ml and incubated at 4°C for 5 days. Bonding and disulfide bridge formation were performed in buffer A (50 mM Trisodium EDTA) for 3 days. This was achieved by dialysis with HCl (pH 8.0).

[0360] The refolded protein was then equilibrated with buffer A and washed with Q-Sep Anion exchange containing harose® FF (GE Healthcare) The unbound recombinant protein was collected and loaded onto a chromatography column. Enterokinase (Enterokinase: recombinant protein) was recovered from the flow-through and wash fractions. Protein ratio of 1:100 and incubation time of 2.5 hours Before removing the N-terminal tag sequence, the p The pH was adjusted to 7.4. The cleavage reaction was carried out by adding the sample to buffer B (50 mM Trisodium EDTA at pH 7.4). Benzamidine Sepharose equilibrated and washed with 0.5M NaCl Contains harose 4 FF (high sub) (GE Healthcare) The eluate was terminated by loading onto a benzamidine affinity column containing 1000 ribonucleotides (2000 ribonucleotides) containing 1000 ribonucleotides (2000 ribonucleotides), and then eluted with Buffer C ( Elution was performed with buffer B containing 50 mM benzamidine. Active FXI catalytic domain was equilibrated with 20 mM Na acetate, 75 mM NaCl, pH 5.3, XK 26 / 600 Superdex 75 size exclusion column (GE Healthcare) The final protein concentration was 1.07 mg / ml.

[0361] The Fab portion of NOV1401 was obtained by papain cleavage of IgG. The final Fab concentration was The digest was added to the antibody at a ratio of 1:100 (w / w). Papain (Roche Diagnostics: 108 014; 10 mg / ml) and in the presence of 1 mM cysteine ​​(added to the original IgG solution), Digestion was carried out overnight at 37°C. The digestion was carried out in the presence of 50 μM of the specific papain inhibitor E64 (N-[N-(L-3-trans-carboxylane-2-carbonyl)-L- The antibody was stopped by adding [[isocyl]-agmatine] and then digested with HCl to remove the Fc portion. The product was passed through a small Protein A column (5 mL). Fab was present in the flow-through. The solution is collected, dialyzed against PBS, concentrated to its final concentration by ultrafiltration, and sterile filtered. (0.22 μm).

[0362] Complexation, crystallization, and structural resolution: FXI catalytic domain and Fab were mixed in an equimolar ratio to a final concentration of approximately 9 mg / ml. Concentrated.

[0363] Crystals used for data collection were placed in 0.3 μL of reservoir solution (0.2 M ammonium chloride). monium, 20% PEG 3350), 0.2 μL of Fab-FXI complex, and 0.1 μL of crystal seeds derived from crystals obtained in the first round of crystal screening The results were obtained at 277K using the sitting drop vapor diffusion method.

[0364] For data collection, crystals were flash frozen directly in liquid nitrogen. K, using the Pilatus pixel detector (Dectris), 1.00002 Å The spectra were collected at the Swiss Light Source beamline X10SA at wavelengths of 1000 Hz. Data processing and scaling were performed with XDS and XSCALE (Kabsch , W. (2010) Acta Cryst. D66, 125-132). The crystals consist of one complex per asymmetric unit. The dime...

Claims

1. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to human factor XI (FXI) and / or activated factor XI (FXIa), the antibody or antigen-binding fragment: i. CDR1 of the heavy chain variable region of SEQ ID NO: 23; CDR2 of the heavy chain variable region of SEQ ID NO: 24; CDR3 of the heavy chain variable region of SEQ ID NO: 25; CDR1 of the light chain variable region of SEQ ID NO: 33; CDR2 of the light chain variable region of SEQ ID NO: 34; and CDR3 of the light chain variable region of SEQ ID NO: 35; ii. CDR1 of the heavy chain variable region of SEQ ID NO: 26; CDR2 of the heavy chain variable region of SEQ ID NO: 27; CDR3 of the heavy chain variable region of SEQ ID NO: 28; CDR1 of the light chain variable region of SEQ ID NO: 36; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 38; iii. CDR1 of the heavy chain variable region of SEQ ID NO: 43; CDR2 of the heavy chain variable region of SEQ ID NO: 44; CDR3 of the heavy chain variable region of SEQ ID NO: 45; CDR1 of the light chain variable region of SEQ ID NO: 47; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 15; or iv. CDR1 of the heavy chain variable region of SEQ ID NO: 46; CDR2 of the heavy chain variable region of SEQ ID NO: 4; CDR3 of the heavy chain variable region of SEQ ID NO: 5; CDR1 of the light chain variable region of SEQ ID NO: 33; CDR2 of the light chain variable region of SEQ ID NO: 14; and CDR3 of the light chain variable region of SEQ ID NO: 15 Including, the pharmaceutical composition is for use in a method for treating a thromboembolic disorder; The method comprises administering to a subject suffering from or at risk of developing a thromboembolic disorder an effective amount of the antibody or antigen-binding fragment thereof; Pharmaceutical compositions.

2. the antibody or antigen-binding fragment thereof (i) 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, or (ii) 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, or The antibody comprises (iii) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 31 and a light chain comprising the amino acid sequence of SEQ ID NO: 41, or (iv) an antibody comprising 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. The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 2, wherein the antibody comprises a D265A substitution and a P329A substitution in the Fc domain.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the subject is suffering from or at risk of developing a thromboembolic disorder selected from the group consisting of venous thromboembolism, paroxysmal atrial fibrillation or paroxysmal 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.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject suffers from one or more of a stroke and deep vein thrombosis associated with atrial fibrillation.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the subject is suffering from a stroke associated with atrial fibrillation or atrial flutter.

7. 7. The pharmaceutical composition of claim 6, wherein the atrial fibrillation or atrial flutter is paroxysmal atrial fibrillation (PAF).

8. The pharmaceutical composition of any one of claims 1 to 4, wherein the subject is suffering from or at risk of developing venous thromboembolism.

9. The pharmaceutical composition of any one of claims 1 to 5, wherein the subject is suffering from or at risk of developing deep vein thrombosis (DVT).

10. 10. The pharmaceutical composition of claim 8 or 9, wherein the method further comprises measuring efficacy by the incidence of DVT occurrence.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the subject is at high risk of bleeding.

12. 12. The pharmaceutical composition of any one of claims 1 to 4 or 8 to 11, wherein the subject has undergone a medical procedure selected from the group consisting of knee replacement, hip replacement, orthopedic surgery, pacemaker leads, catheter leads, thoracic surgery, and abdominal surgery.

13. 13. The pharmaceutical composition of claim 12, wherein the medical procedure is knee replacement surgery.

14. The pharmaceutical composition of any one of claims 1 to 7, wherein the at-risk subject suffers from one or more of hypertension, congestive heart failure, left ventricular hypertrophy, and diabetes.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the method further comprises assessing the effectiveness of the pharmaceutical composition by one or more biomarkers selected from the group consisting of free factor XI, total factor XI, factor XI clotting activity, activated partial thromboplastin time, and D-dimer.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the method further comprises assessing adverse events to the pharmaceutical composition by measuring bleeding events and / or the presence of anti-drug antibodies.

17. The method comprises administering to a patient experiencing an adverse event: (i) fluid replacement 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 concentrate (PCC), activated PCC (APCC) (e.g., factor VIII inhibitors) and / or recombinant activated factor VII; 17. The pharmaceutical composition of claim 16, further comprising administering one or more of the following:

18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the method further comprises administering to a subject suffering from or at risk of developing a thromboembolic disorder an effective amount of statin therapy.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein a dose of at least 5 mg and up to 600 mg of the antibody or antigen-binding fragment thereof is administered to the subject.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof is administered to the subject subcutaneously or intravenously.

21. The pharmaceutical composition of any one of claims 1 to 20, wherein the antibody or antigen-binding fragment thereof is administered to the subject once a month.

22. 22. The pharmaceutical composition of any one of claims 1 to 21, 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.

23. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to human factor XI (FXI) and / or activated factor XI (FXIa), the antibody or antigen-binding fragment thereof i. CDR1 of the heavy chain variable region of SEQ ID NO: 23; CDR2 of the heavy chain variable region of SEQ ID NO: 24; CDR3 of the heavy chain variable region of SEQ ID NO: 25; CDR1 of the light chain variable region of SEQ ID NO: 33; CDR2 of the light chain variable region of SEQ ID NO: 34; and CDR3 of the light chain variable region of SEQ ID NO: 35; ii. CDR1 of the heavy chain variable region of SEQ ID NO: 26; CDR2 of the heavy chain variable region of SEQ ID NO: 27; CDR3 of the heavy chain variable region of SEQ ID NO: 28; CDR1 of the light chain variable region of SEQ ID NO: 36; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 38; iii. CDR1 of the heavy chain variable region of SEQ ID NO: 43; CDR2 of the heavy chain variable region of SEQ ID NO: 44; CDR3 of the heavy chain variable region of SEQ ID NO: 45; CDR1 of the light chain variable region of SEQ ID NO: 47; CDR2 of the light chain variable region of SEQ ID NO: 37; and CDR3 of the light chain variable region of SEQ ID NO: 15; or iv. CDR1 of the heavy chain variable region of SEQ ID NO: 46; CDR2 of the heavy chain variable region of SEQ ID NO: 4; CDR3 of the heavy chain variable region of SEQ ID NO: 5; CDR1 of the light chain variable region of SEQ ID NO: 33; CDR2 of the light chain variable region of SEQ ID NO: 14; and CDR3 of the light chain variable region of SEQ ID NO: 15 Including, The pharmaceutical composition is for use in a method for treating a thromboembolic disorder in a subject in need thereof.

24. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) of SEQ ID NO: 29, or a VH of an amino acid sequence having at least 90% identity thereto; and a light chain variable region (VL) of SEQ ID NO: 39, or a VL of an amino acid sequence having at least 90% identity thereto.

25. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 90% identity thereto.

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