RU2817219C2 - Novel humanised antibodies to factor xi with antithrombotic and anti-inflammatory action and use thereof - Google Patents

Abstract

FIELD: immunology.

SUBSTANCE: disclosed is a monoclonal antibody or its antigen-binding fragment, specifically binding human factor XI or human factor XIa. Also disclosed is a polynucleotide encoding a monoclonal antibody or antigen-binding fragment, a vector, a host cell, a method of producing the monoclonal antibody or antigen-binding fragment, compositions, methods and kit using monoclonal antibody or its antigen-binding fragment. Antibody or its antigen-binding fragment is able to bind to the A2 domain of factor XI and form an immune complex therewith, thereby disrupting the contact activation molecular complex, without affecting activity or activation of haemostatic factor XI.

EFFECT: inventions can be used for safe inhibition of thrombosis and inflammation without haemostasis disturbance.

25 cl, 10 dwg, 2 tbl

Description

RELATED APPLICATIONS

This application claims benefit from U.S. Provisional Patent Application No. 62/794,987, filed January 21, 2019, the disclosure of which is incorporated by reference in its entirety.

LIST OF SEQUENCES

The application currently pending contains a sequence listing provided electronically in ASCII format and incorporated by reference in its entirety. The ASCII copy created on January 15, 2020 is signed as ARB002PCT_SL.txt. Its size is 18337 bytes.

GOVERNMENT SUPPORT

The United States Government has provided support, at least in part, for the invention disclosed herein through grant numbers R44AI088937, R44HL128016, R43NS077600, and R44HL106919 from the United States Department of Health and Human Services (HHS), National Institutes of Health. The US Government has certain rights in the inventions based on this disclosure.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to antibodies, and specifically to humanized monoclonal antibodies capable of binding to factor XI, as well as methods of using them, including methods of using antithrombotic and anti-inflammatory agents that do not impair hemostasis.

BACKGROUND OF THE INVENTION

Thromboembolic disorders, including both venous and arterial thrombosis, are a leading cause of significant chronic disease and mortality in developed countries around the world. These disorders are caused by the formation of abnormal blood clots (thrombi), which result in the accumulation of fibrin, platelets and other red blood cells inside the blood vessel, leading to blockage of the blood vessels, tissue ischemia and, in some cases, embolization due to the movement and migration of punctate blood vessels. fragments from a blood clot.

Under typical circumstances, blood clotting mediates hemostasis, which is an important mechanism that prevents blood loss at sites of organ and tissue damage by stimulating platelet activation and fibrin formation. At the mechanical level, hemostasis occurs through two separate processes. In primary hemostasis, blood that comes into contact with the extraluminal environment is activated by the major hemostatic protein, tissue factor (TF), resulting in the immediate release of the clotting enzyme, thrombin. In turn, thrombin activates other coagulation factors (F), such as fibrinogen (FI), FXIII, FV and others. In addition, platelets adhere to the site of injury and are also activated, in most cases by thrombin, and eventually aggregate by binding to each other to form a platelet plug. Platelet plug formation is enhanced and stabilized during secondary hemostasis as a result of a series of continuous platelet activation and enzymatic reactions, including the coagulation proteins FXI, FIX, FX, FVIII, FVII, FV, FXIII, Fll (prothrombin) and FI, resulting in more stable hemostatic a plug that eventually seals the rupture in the walls of the blood vessels, preventing blood loss and death.

Since 1964, when McFarlane (Nature, May 2, 1964; 202:498-9) introduced the cascade model of the blood clotting process, much more has been known about the mechanisms and function of blood clotting in the body. Over the past ten years, the theory that two separate pathways, the so-called extrinsic and intrinsic pathways of activation of blood coagulation, initiate blood clotting and combine into a common pathway, ultimately leading to thrombin release and fibrin deposition, has been revised and challenged.

In one model, which has been generally accepted by many in the medical field, initiation of thrombin release during the hemostatic process occurs when factor VII (FVIIa), activated by circulating plasma proteases, comes into contact with a cofactor, TF, and thus forms nim complex. This TF-FVIIa complex converts the zymogens FIX and FX into their active (a) forms FIXa and FXa. In turn, FIXa can activate additional FX in the presence of a cofactor, FVIIIa, and FXa can then activate prothrombin (FII) to form thrombin (Flla) in the presence of a cofactor, FVa, respectively. Thrombin, a key element in coagulation, can in turn catalyze the conversion of fibrinogen to fibrin and cleave protease-activated receptors (PAR) 1 and 4 on platelets, resulting in platelet activation. Activated platelets in combination with fibrin are very important for the formation of a hemostatic plug and, therefore, are the main elements of normal hemostasis.

It is well known that FXI is a plasma serine protease zymogen that plays a minor role in coupling the contact phase and the enhancement phase of thrombin release in vitro and in vivo (Davey E.W. et al., Biochemistry, October 29, 1991: 30(43):10363-70, Gheilani D. and Drose G. J., Science. August 23, 1991; 253(5022): 909-12; Jul 9, 2009;114(2):452-8). Physiological hemostatic and pathological prothrombotic release of thrombin involves FXI activation and FXIa activity.

However, data demonstrate that inherited FXI deficiency in humans or other mammals does not typically result in spontaneous bleeding, indicating that FXI is not a determining element in the hemostatic release of thrombin. FXI deficiency, hemophilia C, is associated with an increased risk of bleeding with specific stress on the hemostatic system, such as certain types of surgical procedures or trauma, although the severity of bleeding is little dependent on plasma levels or FXI activity. However, acute FXI deficiency in humans has been reported to have some protective effects against thrombotic diseases, including ischemic stroke and deep venous thrombosis (DVT) (Salomon O. et al., Thromb Haemost. February 2011;105(2): 269-73; Salomon O et al., Blood 15 Apr 2008;lll(8):4113-7; high levels of FXI are associated with thrombotic complications and are reported to pose a high risk of DVT, myocardial infarction (MI), and stroke (Majers JK et al. N Engl J Med. Mar 9, 2000;342(10): 696-701, Berliner JI et al, Thromb Res Jul 15, 2002;107(l-2):55-60, Young DT et al, Am J Clin Pathol. 2006;126(3):411-5). It has previously been suggested that pharmacological targeting of FXI may be safer than conventional coagulation inhibition, and primate studies have provided evidence for this hypothesis (Gruber A and Henson CR, Blood. Aug 1, 2003;102(3 ):953-955, Tucker EI et al., Blood 22 Jan 2009; 113(4): 936-944.

Overall, theoretical considerations and previous studies indicate that FXI plays a minor role in maintaining hemostasis but is an important element in the pathogenesis of thrombosis, making FXI a promising target for safe antithrombotic therapy. Non-clinical and clinical data support this concept. Currently available antithrombotic drugs either target the building blocks of thrombin (fibrin and platelets) or keep molecules (clotting factors) and cells (platelet) from participating in the process of clot formation and hemostatic plug. Antithrombotic, profibrinolytic and anticoagulant agents have been the basis for the treatment and prevention of thromboembolic diseases for decades and are among the most commonly prescribed drugs in clinical practice. Until now, most of these substances can completely block thrombosis and hemostasis when administered in effective doses, so they have dose-limiting antihemostatic toxicity.

As a result, physicians administer modern antithrombotic agents at doses that are lower than full effective doses, carefully attempting to balance the benefit of antithrombotic action with the potential for serious and even fatal bleeding.

To date, one of the few examples of anti-FXI antibodies demonstrating therapeutic potential is the murine antibody 1A6 (also called aximab) as published by Tucker et al. (Prevention of vascular graft occlusion and thrombin release from thrombus formation by inhibiting factor XI. Eric I. Tucker, Ulla M. Marcek, Tara K. White, Savan Hurst, Sandra Rugani, Oweg J.T. McCarthy, David Gailani, Andras Gruber and Stephen R. Henson, Blood 22 Jan 2009;113(4):936-944). Antibody 1A6 is also disclosed in US Pat. No. 9,125,895, which is incorporated herein by reference in its entirety.

However, antibody 1A6 is a murine antibody and is not suitable for human treatment, especially for chronic use such as antithrombotic therapy. Thus, another example of an anti-FXI antibody demonstrating therapeutic potential is the selective murine antibody 14E11 (also called xisomab), according to Cheng et al., A role for factor Xlla-mediated factor XI activation in thrombus formation in vivo. activation of Xlla-mediated factor XI in thrombus formation in the body"), Cheng K.I., Tucker E.I., Pine M.S., Sisler I., Matafonov A, Sun M.F., White-Adams T. K., Smith SA, Henson SR, McCarthy OJ, Renne T, Gruber A, Gailani D, Blood. November 11, 2010; 116(19):3981 -3989; Luo D. et al., (2012), Journal of Infect Immun. 80(1):9109; Tucker E. et al., (2012), Journal of Blood. 119(20):4762-8). Antibody 14E11 is also disclosed in US Patents 9,637,550, 8,940,883 and 8,388,959 (the "14E11 Patents").

One method of converting a murine antibody into an acceptable therapeutic antibody is humanization. Standard techniques are known to one skilled in the art, such as those described in: O'Brien, S., Jones, T. (2001) Humanizing Antibodies by CDR Grafting, Kontermann, R., Dubel S. (Ed.) Antibody Engineering. pp. 567-590. Springer Lab Manuals. Springer, Berlin, Heidelberg), in Hwang, Almago, Buss, Tan and Foote (2005). Use of human germline genes in a CDR homology-based approach to antibody humanization. Methods (36(l):35-42) and referenced documents. To further reduce the immunogenicity potential of humanized antibodies, sequence optimization and germline targeting of antibodies may be required.

These standard methods were used to humanize and optimize muscle antibody 14E11, the resulting recombinant humanized antibody, hereinafter referred to as AB023, which demonstrated binding activity in a biomechanical assay compared to its murine precursor. Through sequence changes, humanized variants of the mouse 14E11 antibody, such as AB023, have been generated that show comparable biochemical profiles and antithrombotic effects in the body.

Cardiovascular disease and venous thromboembolism (VIE) remain the leading causes of death. Primary prevention, acute treatment, and secondary prevention strategies such as anticoagulation and antiplatelet therapy are effective but generally increase the risk of bleeding. Thus, the present invention satisfies current medical needs for safe and at the same time effective substances for antithrombotic and anti-inflammatory therapy and treatment without weakening of hemostasis.

SUMMARY OF THE INVENTION:

The invention disclosed herein overcomes existing disadvantages and prior art solutions by providing binding molecules, compositions, methods and kits for inhibiting thrombosis without compromising hemostasis. The compositions of the present invention include apple domain 2 binding molecules of a recombinant, humanized anti-FXI antibody, binding fragments, variants, derivatives thereof, cell lines and nucleic acid molecules, acid-encoding amino acid sequence binding molecules. The present invention further includes pharmaceutical compositions comprising a therapeutically effective amount of binding molecules, binding fragments, variants and derivatives on a pharmaceutically acceptable carrier and methods of using them. The methods of the present invention include administering the compositions of the present invention to a patient in need thereof for the purpose of inhibiting thrombosis, preventing thrombosis, or treating inflammation through, for example, antithrombotic and anti-inflammatory activity by blocking Xlla-mediated FXI activation without inhibiting FXI activation by thrombin or the procoagulant function of FXIa . Methods for preparing binding molecules, binding moieties, variants, and derivatives are also provided.

In a preferred aspect, the present invention provides a binding molecule comprising:

Light chain CDR1 comprising the sequence KASQDVSTAVA (SEQ ID NO: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2);

Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3);

Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4);

Heavy chain CDR2 comprising the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); And

Heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6).

The binding molecule may include a _VH region as set forth in SEQ ID No.: 8 and/or a VL region as set forth in SEQ ID No.: 9.

The binding molecule may include a light chain, as set forth in SEQ ID No.: 10, or may be encoded by SEQ ID No.: 12, and/or a heavy chain, as set forth in SEQ ID No.: 11, or may be encoded by SEQ ID No.: 13.

The binding molecule of the present invention is capable of binding to FXI and/or FXla of a mammal, including human or non-human primate FXI and/or human or non-human primate FXla.

Specifically, the binding molecule is capable of binding to and forming a therapeutic immune complex with the amino acid sequence corresponding to the A2 domain of FXI, including amino acids 91-175 of SEQ ID No: 7. In this case, the amino acid numbering of human FXI includes the signal sequence starting with methionine at position -18 to -1 and then from glutamine at position 1. It is contemplated that the binding molecule may be an antibody, an antigen binding fragment, variant or derivative thereof, including a humanized monoclonal antibody, an antigen binding fragment, variant or derivative thereof, for example an IgG antibody.

In a further aspect, the present invention provides a polynucleotide encoding a binding molecule, as defined herein, and a vector, for example, a euspression vector, comprising the polynucleotide. The present invention also relates to a host cell including a vector or polynucleotide.

In an additional aspect, a process for producing binding molecules is provided, as described herein. The process involves culturing a host cell, as defined herein, under conditions allowing expression of the binding molecule and, in some cases, recovery of the resulting molecule from the culture.

Furthermore, the present invention provides a pharmaceutical composition comprising a binding molecule, polynucleotide, vector and/or host cell as defined herein, and, in some cases, one or more pharmaceutically acceptable excipients. The pharmaceutical composition may include one or more additional active ingredients, for example, antithrombotic and/or anticoagulants, or may be administered as part of a combination therapy with additional active ingredients.

According to the present invention, a binding molecule, polynucleotide, vector, host cell or pharmaceutical composition can be used in a method of inhibiting contact activation, blood coagulation, platelet aggregation and/or thrombosis in a patient, and is therefore useful for the treatment and/or prevention of disorders, for example, cardiovascular, infectious or inflammatory diseases, preferably thrombotic or thromboembolic disorders and/or thrombotic or thromboembolic complications.

This document further proposes the use of the binding molecule as an anticoagulant in blood samples, blood preservation, plasma preparations, biological samples or drug supplements, or as a coating on medical devices.

Moreover, the present invention provides a kit comprising a binding molecule, polynucleotide, vector, host cell or pharmaceutical composition as described herein.

DESCRIPTION OF DRAWINGS

The accompanying drawings are included in and form part of the specification for illustrating the present invention. These drawings, together with the description, serve to explain the principles of the invention. The drawings illustrate preferred and alternative examples and should not be construed as limiting the present invention to the examples shown. Other features and advantages will become apparent from the following more detailed description of various aspects, embodiments and configurations of the present invention, as illustrated in the drawings below.

In FIG. 1 is a graph showing the concentration-dependent effects of 14E11 (10 ^-5 to 10 ⁰ mmol) on activated partial thromboplastin time (aPTT) in mouse (open circles) and human (filled circles) plasma.

In FIG. 2A-F are blots and graphs demonstrating the binding properties of 14E11. In FIG. 2A shows a 10% Coomassie blue-stained polyacrylamide gel of recombinant human (H) and mouse (M) FXI; in FIG. 2B and C show Western blots of 10% polyacrylamide gels of normal (N) and FXI-deficient (XI-/-) mouse (B) and human (C) plasma under non-reducing conditions using biotinylated 14E11 for detection. FXI in panel B is a measure of the recombinant mouse FXI control; in FIG. 2D shows biotinylated antibody 14E11 to immobilize mouse FXI (open circles), human FXI (filled circles), or human FXIa (open squares); in FIG. 2E shows a Western blot of a 10% polyacrylamide gel of human FXI (hXI), human prokallikrein (PK), and human FXI under non-reducing conditions, with the A1, A2, A3, or A4 domain replaced by the corresponding domain from PK. The position for the FXI dimer is indicated on the right as "D" and for the monomeric PK as "M" (it is worth noting that FXI with an A4 PK domain is monomeric, since A4 mediates the formation of the FXI dimer); and in FIG. 2F shows a Western blot (left panel) of a 10% polyacrylamide gel of human FXI (hXI) and individual apple domains of human FXI under non-reducing conditions associated with tissue plasma activator (tPA); and the right panel is a stained gel showing the recombinant apple domain-tPA fusion protein (it is worth noting that A4 fusion proteins form a dimer). For panels A-C and E, the molecular weight standards in kDa are located on the left of the figures, and for panel F, on the right.

In FIG. 3A-B are graphs showing the effects of 14E11 (filled circles) and the humanized version, AB023 (open circles) on aPTT in vitro. In FIG. 3A shows exposure to 14E11 and AB023 in mixed human plasma; and in FIG. 3B shows the effects of 14E11 and AB023 in mixed baboon blood plasma. 14E11 and AB023 similarly prolong aPTT in both human and baboon plasma.

In FIG. 4A-F are blots and graphs demonstrating the binding properties of AB023. In FIG. 4A shows a Western blot of a 10% polyacrylamide gel of human FXI (track 1) and human FXI under non-reducing conditions where the A1, A2, A3 or A4 domain is replaced with the corresponding domain from human prokallikrein (PK) using biotinylated AB023 for detection; in FIG. 4B shows a Western blot of a 10% polyacrylamide gel of individual apple human FXI domains (A1-A4) under non-reducing conditions associated with tPA, where AB023 recognizes the A2 domain of human FXI; in FIG. 4C shows the binding of AB023 to human FXI (filled circles), human FXIa (open square), and mouse FXI (filled triangles); in FIG. 4D shows how AB023 inhibits FXlIa activation-FXI activation in a concentration-dependent manner; in FIG. 4E shows that AB023 does not prevent activation of FXI by thrombin; and in FIG. 4F shows that AB023 prolongs aPTT in a plasma concentration-dependent manner in humans (filled circles), baboon (open squares), cynomolgus monkey (open circles), and rats (open diamonds). *FXI/PKA4 fusion protein (A4*) is a dimeric molecule created by replacing Cys326 with alanine in the PK domain of A4.

In FIG. Figure 5 shows the effect of AB023-FXI complex formation on aPTT in vitro. These experiments were performed in sequential order using FIX-deficient plasma (George King, product no. 1100, lot 6538). Baseline aPTT in FIX-deficient plasma was determined to be 118.5 s (circles). Recombinant human FXI (Enzyme Research Labs, cat. no. HFXI1111) was added to FIX-deficient plasma for a final concentration of 10 mg/ml; aPTT was measured. Addition of FXI to FIX-deficient plasma shortens aPTT to 32.3 s (squares). Subsequently, AB023 antibody was added to FIX-deficient plasma + 10 mg/ml FXI mixture at a final antibody concentration of 100 mg/ml, aPTT was extended to 66.6 s (triangles). In the second experiment, AB023 antibody was added to FIX-deficient plasma for a final antibody concentration of 100 mg/ml; aPTT was measured. Addition of AB023 to FIX-deficient plasma did not change aPTT compared to baseline (117.7 s, inverted triangles). Addition of recombinant human FXI (final concentration 10 mg/ml) to this mixture shortens aPTT to 59.1 s (diamonds), similar to the results of the previous experiment. These data demonstrate that the anticoagulant effect of AB023 is only upon the formation of a complex between FXI and AB023.

In FIG. Figure 6 shows the effects of 14E11 and AB023 in a mouse model of experimentally induced arterial thrombosis. C57B1/6 mice (with or without administration of 14E11 (1.0 mg/kg, i.v.) or AB023 (1.0 mg/kg, i.v.) or FXI-/- mice were tested in the FeCl ₃ -induced carotid artery thrombosis model. At the carotid artery, FeCl ₃ was used with a concentration of 2.5% to 10%N; The time to occlusion was measured. The height of the bars indicates the percentage of mice in each group with unoccluded veins 30 minutes after FeCl ₃ application. Intravenous infusion of 1.0 mg/kg AB023 into wild mice (bars with circles) protected mice from carotid artery occlusion induced by 3.5%, 5.0%, and 7.5% _FeCl3 , compared with untreated wild mice. (columns with dots). These results are comparable to those from FXI-/- mice (open bar) and 14E11-treated mice (boxed bars) (n = 10/group).

In FIG. Figure 7 shows the relationship between plasma AB023 and aPTT concentrations in four baboons. Each graph represents the duration of plasma AB023 concentration (left y-axes, filled circles) and aPTT (right y-axes, open circles) in one baboon injected intravenously with AB023, 1.0 mg/kg. In each baboon, aPTT was prolonged until plasma AB023 concentrations fell below detectable levels (1000 ng/ml). A partially validated ELISA assay was used to determine free AB023 in baboon plasma. aPTT is shown as fold change from baseline.

In FIG. 8A-D show the effect of AB023 on a baboon thrombosis model (graft + expansion chamber). AB023 reduces platelet-rich thrombus growth in a primate model of thrombosis. Effect of AB023 (0.2 mg/kg, IV) on platelet deposition (as shown in FIGS. 8A and 8C) in collagen-coated (4 mm diameter, 2 cm length) vascular grafts (FIG. 8A) and venous expansion chamber (diameter 9 mm, length 2 cm) (FIG. 8C). In FIG. 8B and 8D show fibrin deposition within the collagen graft (FIG. 8B), venous expansion chamber (FIG. 8D) in the control group (shaded bar) and after administration of AB023 (open bar). Values are mean ± SD, n = 7 trials/group with 4 animals for control group (including historical control groups from one trial performed with 14E11), n = 2 trials with 2 animals for AB023 administration. Values are mean ± SD.

In FIG. 9A-F show the effect of AB023 on platelet-rich thrombus growth in a baboon thrombosis model (collagen-coated graft). In FIG. 9A-9C shows the effect of AB023

(1.0 mg/kg, IV) to deposit platelets (FIG. 9A-C) in collagen-coated (4 mm diameter, 2 cm length) vascular grafts (FIG. 9A) and 10 cm downstream of the vascular graft ("tail" ") (FIG. 9B); in FIG. 9C shows platelet deposition in a graft and tail combination; in FIG. 9D-F depicts fibrin deposition within the collagen graft (FIG. 9D), tail (FIG. 9E), and graft-tail combination (FIG. 9F) in the control group (shaded bar) and after AB023 administration. Values are mean ± SD, n = 4 experiments/group with 4 animals. *p <0.05, **p <0.01, ***p <0.001 compared with the control group. Each animal was subjected to a control experiment, followed by an experiment with the introduction of AB023.

In FIG. 10A-D compare the effect of AB023 with that of the murine antibody 14E11. In FIG. 10A-B show the effect of 14E11 and ABQ23 on FXI autoactivation in the presence of dextran sulfate (FIG. 10A) and DNA (FIG. 10B) as a function of the concentration of antibodies 14E11 (dotted bars) and AB023 (shaded bars). Humanized antibody AB023 inhibits DNA-induced self-activation of purified human FXI in a concentration-dependent manner in vitro; in FIG. 10C shows the effects of 14E11 (open circles) and AB023 (filled squares) on inhibition of FXIIa activation of FXI compared to the control group (filled circles); and in FIG. 10D shows the effect of 14E11 and AB023 on FXII activation by FXIa in vitro. The graph displays FXIIa activity shown as a function of antibody concentration. A mixture of purified human FXII and FXIa was incubated with varying concentrations of 14E11 (dotted bars) or AB023 (shaded bars). The amidolytic activity of FXIIa was measured. AB023 inhibits activation of human FXII by FXIa in a concentration-dependent manner in vitro, whereas 14E11 does not.

DETAILED DESCRIPTION

Reference will now be made to the main embodiments of the present invention in more detail. Although the disclosed antibodies, fragments, variants and derivatives thereof will be described in relation to the listed embodiments, it will be understood that they are not intended to limit the present invention. To the contrary, the disclosed antibodies, fragments, variants and derivatives thereof are intended to cover modifications, alternatives and equivalent variants that may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be practiced within the scope of the present invention and which are included within this scope. The present invention is in no way limited to the methods and materials described.

All publications and patents referenced herein are incorporated by reference in their entirety for the purpose of describing and disclosing, for example, designs and techniques that are presented in the publications that may be used in connection with the present invention. The publications discussed throughout the text are presented to disclose the prior art up to the date of filing of this application. Nothing contained herein should be construed as an admission.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly used by those skilled in the art to whom the present invention pertains. The term "any" refers to one or more relevant objects, so it should be understood as, for example, "one or more" and "at least one", which can be used synonymously.

In the practical part of the present invention, unless otherwise indicated, auxiliary methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology are used within the limits of competence in the art. Full details of such methods can be found in publications such as: Sambrook et al., eds. (1989), Molecular Cloning A Laboratory Manual (2nd edition; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992), Molecular Cloning: A laboratory Manual (Cold Spring Harbor Laboratory, New York); D.N. Glover, ed., (1985), DNA Cloning, Volumes I and II; Gait, ed., (1984), Oligonucleotide Synthesis; Mulis et al., US Patent No. 4,683,195; Hames and Higgins, eds., (1984), Nudeic Acid Hybridization; Hames and Higgins, eds., (1984), Transcription and Translation; Freshney, (1987), Culture of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, (1984), A Practical Guide to Molecular Cloning; scientific treatise, Methods in Enzymology (Academic Press, Inc., New York); Miller and Kalos, eds., (1987), Gene Transfer Vectors for Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods in Enzymology, vol. 154 and 155; Mayer and Walker, eds., (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Wire and Blackwell, eds., (1986), Handbook of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986); and Ausubel et al., (1989), Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, MD). Although any methods, devices and materials similar or equivalent to those described herein may be used in the practice or testing of the disclosed antibodies, fragments, variants and derivatives thereof, preferred methods, devices and materials will be described below.

Inhibition of coagulation factor XI (also called FXI), which has been attributed to a role in the development of pathological thrombus formation with limited impact on physiological hemostasis, represents an innovative and promising approach to the development of new antithrombotic agents to obtain an improved benefit-to-risk ratio. The present invention, among other things, provides a new binding molecule, AB023, which is capable of specifically binding to FXI, forming the FXI-AB023 immune complex, and thereby inhibiting the proper molecular assembly of the normally functioning contact activation complex comprising FX11, FXI, prokallikrein human (PK) and high molecular weight kininogen (HMWK). As a result, the molecular interaction between FXI-AB023, FXII, PK and HMWK is limited and therefore the conversion of FXI-AB023 to its activated form FXIa-AB023 by FXIIa and the conversion of FXII to its activated form from FXIIa by FXIa-AB023 will also be limited . Binding molecule AB023 is a novel anticoagulant recombinant monoclonal antibody targeting FXI. Moreover, the ability of the binding molecule to bind to FXIa has also been demonstrated. Thus, the binding molecule presented herein blocks the reciprocal activation of elements involved in the pathological formation of thrombin and thrombosis, including the formation of kallikrein and bradykinin, which play an active role in the regulation of blood pressure and inflammation (reviewed by Weidmann, G. et al., ( 2017), Journal Biochim Biophys Acta Mol Cell Res.1864, (11 ch. B):2118-2127, Björkvist et al., (2014), Journal of Thrombosis and Hemostasis 112(5): 868. -75; Blood Advances 2019, 3:658-669). In particular, a humanized version of the mouse monoconal antibody 14E11 is provided that preferentially binds to FXI with high binding affinity compared to 14E11. In addition, the formation of an immune complex between the binding molecule and FXI significantly reduces blood clotting in vitro, as indicated by prolongation of activated partial thromboplastin time (aPTT) in the presence of complexes that form at low concentrations of the binding molecule. Thus, the binding molecule of the present invention represents a new substance for the effective treatment and/or prevention of disorders where activation of the contact system plays a pathogenic role, especially inflammatory, thrombotic or thromboembolic disorders and/or complications, and is also considered to be effective without significant disruption of hemostasis, which minimizes the risk of bleeding.

Thanks to the antibody of the present invention, a molecule with therapeutic effect has been obtained that reduces the risk of immunogenicity and slows down the development of blood clot in the body after complexing with circulating FXI. The formation of this immune complex between the antibody and the free FXI antigen in the body effectively blocks the spread of the blood clot, but without harming hemostasis. Moreover, the formation of an immune complex between the humanized antibody 14E11, AB023 and FXI does not prevent the hemostatic feedback activation of the FX1-AB023 immune complex by thrombin. Moreover, the immune complex FXIa-AB023 retains enzymatic activity that promotes hemostatic release of thrombin through activation of FIX and other coagulation factor FXIa, thereby making antithrombotic therapy with antibody binding fragments, variants and derivatives thereof of the present invention more hemostatically safe versus direct inhibition of enzymatic activity or hemostatic activation of FXI, and therefore expanding the range of clinical indications and scenarios in which this type of antithrombotic therapy can be used. It is important to note that in the absence of a circulating immune complex, the antibody itself has no anticoagulant or antithrombotic activity. Moreover, in the absence of free, available and circulating FXI in the circulation, it does not itself have anticoagulant or antithrombotic or other activity. Therefore, in the absence of circulating FXI-AB023 immune complexes, the antibody of the present invention may not have anticoagulant activity and may not have antithrombotic activity in FXI-deficient entities.

Binding molecule

The binding molecule of the present invention is a novel anticoagulant recombinant monoclonal antibody against coagulation factor XI (FXI). It was produced by humanization using the hypervariable region (CDR) grafting of the mouse monoclonal antibody 14E11, disclosed in US patents 8,388,959, 8,940,883 and 9,637,550 entitled: “Anti-FXI Antibodies and Methods of Use.” Surprisingly, the binding molecule of the present invention does not include a number of amino acid substitutions in the CDR regions compared to the 14E11 CDR, but still exhibits advantageous properties. Mouse monoclonal antibody. The characteristics of 14E11 and the humanized antibody, AB023, were determined. Their anticoagulating properties were assessed in vitro and in the body for comparison purposes. The binding molecule of the present invention has the ability to bind to FXI with a binding affinity compared to 14E11, and FXI in the immune complex is not efficiently converted to FXIa by FXIIa, but is efficiently converted to FXIa by thrombin (FIG. 4). It is interesting to note that AB023 is more potent in inhibiting this activation than

14E11 (FIG. 10C). The FXIa-AB023 complex decreased the catalytic activity to convert FXII to its active form, FXIIa, in contrast to 14E11 (FIG. 10D). When converted by thrombin to FXIa-AB023, FXI-AB023 retains enzymatic activity toward FIX (data not available) and other macromolecular and small molecule substrates. As a result, contact activation-mediated states are reduced, and hemostatic thrombin-mediated activation of FXI-AB023 maintains the hemostatic activity of circulating FXI.

The binding molecule of the present invention is a humanized monoclonal antibody, antigen binding fragment, variant or derivative, and preferably AB023, an anti-FXI monoclonal therapeutic antibody. This could be IgG4 with a possible modification at the S241P hinge region to prevent antibody arm exchange. The light chain (LC) amino acid sequence is shown in SEQ ID No: 10 and the DNA coding sequence for LC is shown in SEQ ID No: 12. The heavy chain (HC) amino acid sequence is shown in SEQ ID No: 11 and the DNA coding sequence for HC shown in SEQ ID No: 13. AB023 was produced by CDR grafting and contains a kappa (k) light chain and a heavy chain of the IgG4 isotype. Variable sequences (VH and VL) from the mouse monoclonal antibody precursor, 14E11, were cloned into the human IgG4 heavy chain gene (modified SP241 hinge region of Cabot nomenclature) and the kappa light chain gene. The four chains were held together through a combination of covalent (disulfide) and non-covalent bonds. There are 16 cysteine residues and therefore [16/2] potential disulfide bonds per molecule. The heavy chain subunit includes one consensus sequence (N-X-S/T) for potential N-linked glycosylation in the heavy chain.

The antithrombotic effects observed with 14E11 were maintained after humanization, and AB023 prevented venous and arterial thrombosis. According to a first aspect, the present invention provides a binding molecule that is capable of specifically binding to factor XI, wherein the binding molecule includes the following hypervariable regions (CDRs): a light chain CDR1 comprising the sequence KASQDVSTAVA (SEQ ID No: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2); Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3); Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4); Heavy chain CDR2 comprising the sequence MIW GDGRTDYN SALKS (SEQ ID NO: 5); Heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6). The binding molecule may further include the S241P modification.

During the humanization process, the 14E1 CDR regions were determined, and the VH and VL variable regions were added to the modeling program to determine which amino acid residues in the framework were beneficial for the binding properties of the antibody. The CDR regions were grafted into the human scaffold with the highest degree of homology to the 14E11 scaffold. If necessary, back mutation into a specific mouse scaffold is used for binding. Through this process, 3VH and 3VL were obtained. In some embodiments, antibody AB023 is a combination of VH3 (SEQ. ID No. 8) and VL3 (SEQ. ID No. 9).

The term "amino acid" or "amino acid residue" refers to an amino acid as defined in the art, for example, an amino acid selected from the group consisting of: alanine (Ala or A); agrinin (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (GIu or E); glycine (Gly or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y) and valine (Val or V), although modified, synthetic or unique amino acids may be used. In general, amino acids can be classified as having a non-polar side chain (eg, Ala, Cys, He, Leu, Met, Phe, Pro, Val); negatively charged side chain (eg Asp, Glu); a positively charged side chain (eg Arg, His, Lys) or an uncharged side chain (eg Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp and Tyr).

Number of amino acid substitutions and their distribution

Amino acid substitutions can generally be distributed across the CDR in any manner, i.e. one CDR may, for example, include one or more exchanges, and a second CDR one or more replacements. Or, two CDRs may include one or more amino acid substitutions, or six CDRs may include one or more amino acid substitutions, for example, one or more substitutions on a CDR and, preferably, a binding molecule comprising one or more substitutions on CDR1, CDR2, and/or CDR3 of the lung chain, or one or more amino acid substitutions in CDR1, CDR2 and/or CDR3 of the heavy chain, which retains the CDR of the non-humanized molecule to the maximum extent possible without compromising functionality. In general, amino acid substitutions can be distributed in virtually any manner as long as the number of total amino acid substitutions relative to the CDR 14E11 amino acid does not impair the ability of the binding molecule to bind FXI.

Type of substitutions

In general, any combination of amino acid substitutions in the CDR relative to 14E11 is possible as long as the advantageous properties of the binding molecules of the present invention are not impaired.

Amino acid exchanges can be conservative (ie, the exchange of an amino acid of one class or group with an amino acid of a similar class or group, as defined above) or non-conservative (ie, the exchange of an amino acid of one class/group with an amino acid of another class/group).

Preferred substitutions result in the production of binding molecules of the present invention, which leads to prolongation of aPTT, as described herein.

The binding molecule of the present invention may include one or more of the previously mentioned CDRs, in certain cases in combination. Preferred substitutions result in the production of binding molecules of the present invention, resulting in a 1.5- to 2-fold prolongation of aPTT as described herein.

A preferred embodiment of the binding molecule of the present invention, which may be a monoclonal antibody, an antigen binding fragment, variant or derivative thereof, includes the following CDRs: light chain CDR1 comprising the sequence KASQDVSTAVA (SEQ ID No: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2); Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3); Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4); Heavy chain CDR2 comprising the sequence M1W GDGRTDYNS ALKS (SEQ ID NO: 5); and a heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6). A preferred embodiment of the binding molecule may additionally and in some cases include modification at the S241P hinge region.

Moreover, it is contemplated that the binding molecules of the present invention include a light chain variable region (V _H or V _H ), as set forth in SEQ ID No: 8, and/or a heavy chain variable region (V _L or V _L ), as set forth in SEQ ID No: 9. However, other combinations of the VL and _VH regions are possible. Accordingly, a preferred embodiment is the monoclonal antibody AB023 with the sequences as described herein and SEQ ID Nos: 1, 2, 3, 4, 5, 6, 8 and 9.

Factor XI

As stated herein, the binding molecule of the present invention is preferably capable of binding to two identical exosites on an FXI homodimer that participate in recognition reactions of selected macromolecular substrates. Human "factor XI", which may also be referred to herein as "plasma thromboplastin precursor", "PTA", "Rosenthal factor", "clotting factor XI", "FXI", "Fll" or "fXI", circulates in the blood as a double-chain glycoprotein homodimer with a total molecular weight of approximately 160 kilodaltons (kDa). The two monomers that form the homodimer are identical disulfide-linked polypeptides with a molecular weight of approximately 80,000 daltons each. Each FXI monomer contains 4 "apple domains" (A1 - A4 from the N-terminus, the heavy chain of the monomer) and a C-terminal catalytic domain (the light chain of the monomer). Without wishing to be bound by any particular theory, some experts in the field believe that the 4 apple domains contain FXI binding sites for other proteins, e.g. A1 for thrombin: A2 for high molecular weight kininogen (HK, HMWK), A3 for FIX, a glycoprotein lb (GPIb), as well as heparin and A4 for dimerization and possibly for FXIIa. FXI can be converted to its active form, FXIa, by FXIIa, thrombin, FXIa, and possibly other proteases. The serine protease FXIa can cleave a number of macromolecular substrates, including FXII, FX, FV, TFPI, FIX, and possibly others. One of the best described reactions is the conventional aPTT assay, which is sensitive to the conversion of FIX to FIXa, a value that can be easily measured in plasma or blood in routine clinical laboratories. FXIa subsequently activates coagulation factor IX (IXa), which can activate coagulation factor X (FXa), which can then mediate the activation of coagulation FII (prothrombin) into thrombin. Thrombin can then activate additional FXI molecules, thereby enhancing the enzymatic process through a positive feedback reaction, which then leads to the production of more thrombin and the subsequent clotting of recalcified citrated blood or plasma in the aPTT assay, usually in less than 40 seconds from the start of the reaction with negatively charged surfaces and phospholipids.

The term "factor XI" refers to human coagulation factor XI (FI 1, FXI) with acc. Uniprot No. P03951 Initial Release 194 dated October 14, 2015 (SEQ ID No.: 7). As stated elsewhere herein, it is believed that the binding molecule of the present invention binds to a domain in the amino acid sequence corresponding to amino acids 91-175 of SEQ ID No: 7. The amino acid numbering of human FXI includes the signal sequence starting with methionine at position -18 to -1, and then with glutamine at position 1.

The term "position" as used herein means the position of an amino acid in the amino acid sequence presented herein or the position of a nucleotide in the nucleotide sequence presented herein. The term “corresponding” as used herein also includes that the position is not solely determined by the number of preceding nucleotides/amino acids, but rather must be considered in the context of the adjacent portion of the sequence. Accordingly, the position of a particular amino acid or nucleotide of the present invention may change due to the deletion or addition of amino acids or nucleotides elsewhere in the sequence. Thus, when a position is referred to as the “corresponding position” of the present invention, it is obvious that the nucleotides/amino acids may differ in terms of the number indicated, but there may still be similar adjacent nucleotides/amino acids. To determine whether an amino acid (or nucleotide) residue in a given sequence corresponds to a specific position in the amino acid sequence (or polynucleotide sequence) of the “original” amino acid (or polynucleotide sequence) (for example, human FXI, as shown in SEQ ID No: 7), one skilled in the art may use tools and methods well known in the art, for example, sequence alignment manually or using computer programs in accordance with the examples given in this document.

The term "epitope" generally refers to a region of an antigen, i.e. a (poly-)peptide that recognizes a binding domain and may also be referred to as an "antigenic structure" or an "antigenic determinant". The term "binding domain" refers to the "antigen binding site", i.e. characterizes the domain of a binding molecule that binds/interacts with a given target epitope on an antigen or group of antigens, for example, an identical antigen in different species. The target antigen may contain one epitope and preferably contains at least two epitopes and may include any number of epitopes, depending on the size, conformation and type of the antigen. In addition, it should be noted that the "epitope" of a target antigen may be a (poly-)target peptide, but may also be or may include non-polypeptide elements, for example, the epitope may include a carbohydrate side chain. The term "epitope" generally covers linear and conformational epitopes. Linear epitopes are contiguous epitopes included in the amino acid primary sequence, and may, for example, include at least 2 amino acids or more. Conformational epitopes are formed by non-contiguous amino acids superimposed on each other by folding of the target antigen and preferably the (poly-)target peptide.

The binding molecules of the present invention are expected to recognize a structurally conserved epitope located on the factor XI heavy chain that includes a sequence of at least 2, at least 3, at least 4, at least 5, at least 6, at least of 7, at least 8, at least 9, at least 10 contiguous or non-contiguous factor XI amino acids (SEQ ID No: 7).

The binding molecules provided herein bind and form an immune complex with the A2 domain of human factor XI, which includes amino acids 91-175 of SEQ ID No: 7. However, the binding molecule is also contemplated to be capable of binding to human FXI variants as defined herein. since the parent molecule AB023, 14E11 forms an immune complex or complexes with FXI in the plasma of several, but not all, mammalian species. If the binding molecule is in its native (IgG4) form, it can bind one or two FXI homodimers, and multimers or aggregates can also form. The term "variant" when used in relation to FXI refers to a polypeptide with substitutions, deletions and/or additions of one or more amino acid sequences compared to the "original" FXI sequence and performs the same biological function, i.e. can be converted to the active form FXIa with protease activity and catalyzes the activation of FIX and/or the activation/inactivation of other macromolecular substrates such as TFPI, SERPIN, protein S, FV, FX and FXII. Amino acid substitutions may be conservative, as defined herein, or non-conservative, or any combination thereof. FXI variants may include the addition of amino acid residues at the carboxyl terminus or amino terminus (the amino terminus may or may not include a leader sequence). The term “variant” when used with respect to FXI includes isoforms, allelic variants, splice variants or post-translationally modified variants (eg, glycosylation variants) of known FXI polypeptides, for example, an FXI polypeptide with the sequence as set forth in SEQ ID No: 7. Easy to understand that the binding molecules of the present invention can exhibit binding affinity for FXI variants with the amino acid sequence corresponding to amino acids 91-175 of SEQ ID No: 7. In accordance with the above, it is assumed that the binding molecule is also capable of binding and forming various complexes with FXIa molecules and variants thereof, provided that they include the above amino acid region or corresponding amino acid positions.

The binding molecules of the present invention can also bind to FXI from a variety of other mammalian species, with or without preference for any species. These non-human FXI polypeptides are preferably encoded by the FXI gene or an ortholog or paralog thereof and exhibit the same biological function as human FXI even though they are not present as homodimers. Potential non-human primate protein targets for the binding molecules of the present invention include acc polypeptides. No. Uniprot H2QQJ4 (Pan troglodytes, initial version 26 dated November 11, 2015), acc. No. Uniprot H2PEX7 (Pongoabelii, initial version 27 dated November 11, 2015), acc. Uniprot No. A0A0D9S2M6 (Chlorocebussabaeus, initial version 6 dated November 11, 2015), acc. No. UniProt G3R2X1 (Gorilla gorilla gorilla, initial version 27 dated October 14, 2015), acc. No. Uniprot 20 A0A096NC95 (Papio anubis, initial version 11 dated November 11, 2015), acc. No. Uniprot G1RLE8 (Nomascusleucogenys, initial version 28 dated November 11, 2015), acc. No. Uniprot G7PKF5 (Macaca fascicularis, initial version 13 dated October 14, 2015), acc. UniProt No. G7MSF8 (Macaca mulatto, initial version 12 dated October 14, 2015). Other species include a number of mammalian FXI variants that share conserved antigenic regions in the A2 domain as in humans. Variants of the above polypeptides are also contemplated as targets for the binding molecule of the present invention. The contemplated non-human primate target polypeptides recognized by the binding molecule of the present invention are believed to include a sequence corresponding to amino acids 91-175 of SEQ ID No: 7 or a sequence with at least 95%, 96%, 97%, 98% or 99% identity . Thus, this document also provides cross-species specific binding molecules targeting FXI, for example in non-human primates. The term “cross-species recognition” or “cross-species specificity” as used herein means the binding of a binding molecule described herein to the same target polypeptide in humans and non-human species, for example, non-human primates. 14E11 is a generalist antibody, meaning that it appears to form complexes with a wide variety and range of unrelated mammalian species. This aspect suggests binding to a highly conserved or identical sequence in the A2 domain of FXI. Because AB023 has been shown to be equivalent to 14E11, the versatility of AB023 allows the development of therapeutic antibodies with few species restrictions.

As stated herein, it is believed that the binding molecules described herein are also capable of binding to human or non-human mammal FXIa. Thus, what is disclosed in the context of the binding characteristics of a binding molecule for FXI preferably applies equally to its binding characteristics for FXIa, mutatis mutandis.

Antibody

It is assumed that the binding molecule of the present invention is an antibody. As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target epitope through at least one epitope recognition site located in the variable region of the immunoglobulin molecule. The terms “antibody,” “antibody molecule,” and “immunoglobulin” are used interchangeably and in the broadest sense herein and may include native antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), (natural or synthetic) derivatives, fragments or variants of antibodies, fusion proteins containing an antigen-binding fragment of the required specificity, as well as any other modified antibody configuration that includes an antigen-binding site of the required specificity. The antibodies of the present invention are believed to be capable of binding to mammalian FXI as described herein and preferably exhibit the advantageous characteristics of antibody AB023 as described herein.

Native antibody

A "native antibody" is a tetrameric glycoprotein. In a natural native antibody, each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal part of each chain includes a "(hyper)variable" region of approximately 100-110 amino acids or more, which is primarily responsible for antigen recognition. The hypervariable region includes amino acid residues from the "hypervariable region" or CDR or "CDR regions". "Framework" or FR residues are variable domain residues other than hypervariable region residues.

Both the light and heavy chains are divided into regions of structural and functional homology called the "constant region" and the "variable region". The terms "constant" and "variable region" are used functionally. In this regard, it should be understood that the variable regions of both the light (V _L ) and heavy (V _H ) chains determine antigen recognition and specificity. The terms " _VL ", " _VL region" and " _VL domain" are used interchangeably herein to refer to the light chain variable region. Likewise, the terms " _VH ", " _VH region" and " _VH domain" are used interchangeably herein to refer to the heavy chain variable region.

The terms “ _CL ”, “ _CL region” and “ _CL domain” are used interchangeably herein to refer to the light chain constant region. The terms “ _CH ”, “CH region” and “ _CH domain” are used interchangeably herein to refer to the heavy chain constant region and include the “ _CH1 ”, “ _CH2 ” and “ _CH3 ” regions or domains. At the same time, the light chain ( _CL ) and heavy chain ( _CH1 , _CH2 or _CH3 ) constant domains provide biological properties such as secretion, transplacental motility, Fc receptor binding, complement fixation, etc. Conventionally, the numbering of constant region domains increases as they become more distant from the antigen binding site or the amino terminus of the antibody. The N-terminal region is the variable region, and the C-terminal region is the constant region; the C _H3 and C _L regions actually contain the carboxyl terminus of the heavy and light chain, respectively.

The variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the V _L and V _H regions or a subset of hypervariable regions (CDRs) in these antibody variable domains combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary structure of the antibody forms an antigen-binding site, which is present at the end of each Y arm. More specifically, the antigen-binding site is defined by three CDRs (CDR1, CDR2, CDR3, defined according to Cabot nomenclature) along each of the V _H and V _L regions. The three light chain CDRs are also referred to herein as CDR1 LC or CDRLI, CDR2 LC or CDR _L2 , and CDR3 LC or CDR _L3 . The three heavy chain CDRs are designated CDR1 HC or CDRHI, CDR2 HC or CDR _h, and CDRS HC or CDR _L3 . In native antibodies, the six "hypervariable regions" or "CDRs" or "CDR regions" present in each antigen-binding domain are typically short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment .

It is believed that the binding molecules and, for example, antibodies of the present invention contain a light chain CDR1 comprising the sequence KASQDVSTAVA (SEQ ID No: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2); Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3); Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4); Heavy chain CDR2 comprising the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); Heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6); and, in some cases, a modification of S241P. One skilled in the art will readily understand that the CDRs are located in the light and heavy chain variable regions, respectively. A monoclonal antibody that contains the above CDRs was evaluated as disclosed and is designated herein as "AB023".

The binding molecules and preferred monoclonal antibodies, antigen binding fragments, variants and derivatives thereof of the present invention are contemplated to contain a VL region as set forth in SEQ ID No.: 8 and/or a V _H region as set forth in SEQ ID No.: 9. However, Other combinations of the VL and _VH regions are also possible. The binding molecules and preferred monoclonal antibodies, antigen binding fragments, variants and derivatives thereof of the present invention are contemplated to contain a light chain as set forth in SEQ ID No.: 10 or SEQ ID No.: 12 and/or a heavy chain as set forth in SEQ ID No.: 12 No.: 11 or SEQ ID No.: 13. However, other combinations of light and heavy chains are possible.

The carboxy-terminal portion of each light and heavy chain defines a constant region that is primarily responsible for effector function. Immunoglobulins can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains are classified into mu (m), delta (D), gamma (g), alpha (a), and epsilon (e) and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Some of them can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Different isotypes have different effector functions; for example, the IgG1 and IgG3 isotypes often have antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Light chains are classified as kappa or lambda (k, l). Each heavy chain class can be coupled to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. All types, classes and subclasses of immunoglobulins are included within the scope of the present invention. The antibodies of the present invention may be IgG antibodies and, in particular, IgG1 monoclonal antibodies.

Monoclonal antibodies

The present invention provides monoclonal antibodies and antigen binding fragments, variants and derivatives thereof. As used herein, the term “monoclonal antibody” refers to an antibody derived from a population of substantially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical except for possible natural mutations that may be present in small quantities. Unlike conventional types of (polyclonal) antibody production, which may include different antibodies directed to different epitopes, monoclonal antibodies contain substantially similar epitope binding sites and therefore can be directed to the same epitope on an antigen. The term "monoclonal antibody" thus includes recombinant, chimeric, humanized, human or Human Engineered™ monoclonal antibodies.

Various methods for producing monoclonal antibodies are known in the art and are described, for example, by Goding, Monoclonal Antibodies: Principles and Practice, pp. 116-227 (Academic Press, 1996). Suitable methods include the hybridoma method first described by Kohler et al., Nature 256: 495 (1975), recombinant DNA methods that involve isolation and sequencing of DNA encoding monoclonal antibodies and subsequent administration and expression in suitable host cells, and also the isolation of antibodies from phage antibody libraries prepared using methods first described by McCafferty et al., Nature, 348: 552-554 (1990).

Chimeric antibody

As set forth herein, the term "antibody" also includes chimeric antibodies. As used herein, the phrase “chimeric antibody” refers to an antibody containing a sequence derived from two different antibodies, which may be derived from different species. In particular, the term refers to an antibody in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from or belonging to a class or subclass of antibodies, while the remaining portion of the chain(s) is identical or homologous corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies.

In other words, the term “chimeric antibody” will mean any antibody in which the antigen binding region is derived from a first species and the constant region (which may be intact, partial, or modified in accordance with the present invention) is derived from a second species. For example, the antigen binding site may be derived from a non-human source (eg, mouse or primate) and the constant region from a human. For example, chimeric antibodies may contain fragments of human and murine antibodies, for example, human constant and murine variable regions.

Humanized Antibody

As stated herein, the present invention relates to a (monoclonal) humanized antibody and antigen binding fragments, variants and derivatives thereof derived from the murine anti-FXI antibody 14E11 (made by Abzena (formerly known as Antitope Limited, Cambridge, UK) using both methods well known and used in this field of technology, as well as the author’s methodology).

A “humanized antibody” is generally defined as an antibody that (i) is derived from a non-human source (eg, a transgenic mouse with a heterologous immune system), the antibody being based on a human germline sequence; or (II) is an antibody grafted with CDRs, wherein the variable region CDRs are of non-human origin, while one or more framework regions and/or a portion of the variable region CDR sequence are of human origin and, for example, the constant region (if any) is of human origin.

The term "humanized antibody" thus includes antibodies in which the variable region on the heavy chain, light chain, or both chains of a human antibody is altered by at least partial replacement of one or more CDRs from a non-human antibody of known specificity and, in some cases, , by partially replacing the framework region and changing the sequence. In other words, an antibody in which one or more “donor” CDRs from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate antibody) with known specificity is grafted into a human heavy or light chain framework region is referred to herein as "humanized antibody". Replacing all CDRs with complete CDRs from the donor variable domain to transfer the antigen-binding ability of one variable domain to another may not be practical. More precisely, only those residues that are useful for maintaining the activity of the target binding site can be transferred.

In the present invention, the murine 14E11 precursor antibody was humanized by identifying the 14E11 CDR residues and selecting from a database the human germline sequence with the best overall homology to the mouse _VH and VL sequences as the human germline acceptor scaffold for grafting VH and VL CDR3, respectively. as detailed herein. Briefly, structural models of the V regions of the chimeric anti-FXI antibody were obtained using Swiss PDB and analyzed to identify amino acids in the V region scaffolds that could support the binding properties of the antibody. These amino acids have been flagged for inclusion in one or more CDR-grafted variant antibodies. Both the VH and Vk binding molecule sequences contain typical scaffold residues, and CDR 1, 2 and 3 motifs are comparable to many murine antibodies.

The heavy and light chain V region amino acid sequences were compared to a database of human V region germline sequences to identify human heavy and light chain sequences with the highest degree of homology for use as human V region scaffolds. A series of humanized heavy and light chain V regions were then designed by grafting CDRs onto the scaffolds and, if necessary, by backmutating into the mouse sequence-specific residues identified previously, which can restore antibody binding efficiency. Variant sequences with the lowest frequency of potential T cell epitopes were then selected, as determined using Abzena's proprietary ex vivo technology, EpiScreen™ (Jones T.D., Hanlon M., Smith B.J., Heise K.T., Nighy P.D., Sanders D.A., Hamilton A., Sweet K., Unitt E., Alexander G., Law K.M., Gillies S.D., Carr F.J. and Baker M.P. The development of a modified human IFN-alpha2b linked to the Fc portion of human IgG1 as a novel potential therapeutic for the treatment of hepatitis C virus infection. a new potential therapeutic agent for the treatment of hepatitis C virus infection") J Interferon Cytokine Res 2004 24(9):560-72; Jones T.D., Phillips W.J., Smith B.J., Bamford K.A. ., Nighy P.D., Baglin T.P., Gaston J.S. and Baker M.P. Identification and removal of a promiscuous CD4+ T cell epitope from the Cl domain of factor VIII T cells from factor VIII Cl domain") J Thromb Haemost. 2005 3(5):991-1000). In the context of the present invention, humanized antibodies that have been CDR optimized (“modified at the germline gene level”) are included within the term “humanized” antibodies.

Framework regions (FRs) in the variable region on the heavy chain, light chain, or both chains of a humanized antibody may consist of substantially all or all residues of human origin, in which case these humanized antibody framework regions are referred to as “fully human framework regions.” A human scaffold region that includes a mixture of human and donor scaffold remnants is referred to herein as a “partially human scaffold region.” In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the donor antibody. These modifications are intended to further improve the characteristics of the antibodies (eg, to obtain the desired affinity).

In general, a humanized antibody will thus include substantially all, or at least one, and in some cases two, variable regions in which all or a portion of the CDRs correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are FR sequences of human immunoglobulin. The humanized antibody, in some cases, will also include at least a portion of an immunoglobulin constant region (Fc), for example, human immunoglobulin.

Human antibody

A “human” antibody is hereby defined as an antibody that is not chimeric or “humanized” and is not derived (in whole or in part) from a non-human species. The human antibody or functional antibody fragment may be derived from a human or may be a synthetic human antibody. A “synthetic human antibody” is defined herein as an antibody having a sequence derived in whole or in part from synthetic sequences that are based on analysis of known human antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody sequences or antibody fragments and designing an amino acid sequence using the data obtained thereby. Another example of a human antibody or functional antibody fragment is encoded by a nucleic acid isolated from a library of antibody sequences of human origin (ie, such a library is based on antibodies taken from a natural source of human origin).

Fragments, variants and derivatives

As described herein, the present invention discloses full-length antibodies, as well as antigen-binding fragments, variants and derivatives thereof.

Fragments

The term "antibody fragment" refers to a polypeptide derived from the "parent" antibody and retaining its essential structure and function. Thus, the antibody fragment is preferably capable of binding to its specific antigen, ie, FXI. In addition, the antibody fragment of the present invention includes the minimum structural requirements for the antibody that allow binding to the antigen. This minimum requirement may, for example, be defined by the presence of at least three light chain CDRs (i.e., VL region CDR1, CDR2, and CDR3, i.e., CDR _L1 , CDR _L2 , and CDR _L3 ) and/or three heavy chain CDRs (ie, CDR1, CDR2 and CDR3 of the _VH region, ie, CDR _H1 , CDR _H2 and CDR _H3 ). Thus, the term “antibody fragment” refers to a “functional” or “antigen-binding” polypeptide that retains the antigen-binding site (ie, the CDR and, in some cases, the FR) of the “original” antibody. The antibody fragments of the present invention can be obtained, for example, from monoclonal, recombinant, chimeric, humanized and human parent antibodies.

Preferred antigen binding antibody fragments include at least one of preferably all light chain CDR1s comprising the sequence KASQDVSTAVA (SEQ ID NO: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2); Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3); Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4); Heavy chain CDR2 comprising the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); Heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6).

In accordance with the above, the term "antigen-binding antibody fragments" may refer, for example, to fragments of full-length antibodies such as (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or "rIgG"("half-antibody"). The antibody fragments of the present invention may also be modified antibody fragments such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab ), tandem di-scFv, tandem tri-scFv, "mini-antibodies" represented by a structure that looks like this: (V _H -VL-CH3)2, (SCFV-CH ₃ ) ₂ or (scFv-CH3-scFv)2 , multi-bodies, such as tri-bodies or tetra-bodies. In addition, the definition of the term “antibody fragments” includes constructs containing fragments, i.e., monovalent, bivalent and polyvalent/multivalent constructs, and thus monospecific constructs that specifically bind to only one target antigen, as well as bispecific and polyspecific/multispecific constructs that specifically bind to multiple target antigens, eg, two, three, or more, through separate antigen binding sites. Moreover, the definition of the term “antibody fragments” includes molecules consisting of only one polypeptide chain, as well as molecules consisting of several polypeptide chains, which may be identical (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heterooligomers).

Antibody fragments can be obtained by recombinant DNA methods, enzymatic or chemical cleavage of intact antibodies. Methods for preparing such fragments are well known in the art.

Options

The term “variant” refers to polypeptides comprising the amino acid sequence of the “original” binding molecule, such as an antibody or antibody fragment, but containing at least one amino acid modification (e.g., substitution, deletion, or inclusion) compared to the “original” amino acid sequence, when provided that the variants are still capable of (in a certain manner) binding to FXI, preferably the A2 domain of human FXI, as presented in SEQ ID No: 7, and preferably exhibits similar or even improved characteristics compared to antibody AB023. Variants of the binding molecules of the present invention, for example, antibodies and antibody fragments, can be obtained by introducing appropriate nucleotide changes into the nucleic acids encoding the antibody or antibody fragment, or by peptide synthesis. In general, the above amino acid modifications may be introduced or may be present in the variable or constant region, provided that the two or more CDR variants collectively contain 10, 11, 12, 13 or 14 amino acid substitutions compared to CDR AB023, as presented in SEQ ID NO: 1, 2, 3, 4, 5 and 6. For example, amino acid modifications may be introduced to modulate properties of an antibody, such as thermodynamic stability, solubility or viscosity, which affect pharmaceutical development (“sequence optimization”).

As defined herein, amino acid modifications include, for example, deletions and/or inclusions and/or substitutions of residues in the amino acid sequences of the binding molecules described herein, preferably antibodies or antigen binding fragments of an antibody. Any combination of deletion, inclusion and substitution may be introduced into the "original" amino acid sequence to produce the final product, provided it has the required characteristics as defined herein. Amino acid modifications can also alter post-translational processes of binding molecules, such as changing the number or position of glycosylation sites.

For example, variants may contain 1, 2, 3, 4, 5, or 6 amino acids that may be included or deleted in each of the CDRs (depending on their length, of course), while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be included or deleted in each of the FRs. Amino acid sequence inclusions provided herein contain, for example, amino- and/or carboxy-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues with polypeptides containing a hundred residues or more , as well as inclusions within the sequence of one or more amino acid residues. An insertion variant of a binding molecule, e.g., an antibody or antibody fragment of the present invention may include a fusion product of an antibody or antibody fragment and an enzyme or other functional polypeptide (e.g., that can increase the serum half-life of the binding molecule, e.g., an antibody or antibody fragment) .

Amino acid substitutions can be introduced into the CDRs of the heavy and/or light chain, for example, hypervariable regions or FR regions in the heavy and/or light chain. Conservative amino acid substitutions are provided herein and may be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues involved.

Alternative variants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids substituted in the CDR, compared to the CDR as set forth in SEQ ID NO: 1-6, while 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be substituted in framework regions (FR) in depending on the length of the CDR or FR.

In general, if amino acids are replaced in one or more or all of the heavy and/or light chain CDRs, it is preferred that the resulting “variant” sequence be at least 80%, more preferably at least 90%, and most preferably both a minimum of 95%, 96%, 97%, 98%, or 99% identity to the “original” CDR sequence. Thus, the length of the CDR affects the number of possible amino acid substitutions, so that the variant sequence is still included in the present invention. For example, a CDR having 5 amino acids is preferably 80% identical to its substituted sequence to ensure at least one amino acid substitution. Accordingly, the CDRs of antibody-based constructs may have different degrees of identity to their substituted sequences, for example, the _L1 CDR may have 80% identity, while _{the L3} CDR may have 90% identity.

Preferred substitutions are conservative. However, any substitution (including a non-conservative substitution or one or more exemplary substitutions) is contemplated as long as the antibody construct retains its ability to bind FXI and/or the CDRs are at least 80% identical to the sequence so substituted, more preferably at least by 90%, and most preferably by at least 95%, 96%, 97%, 98% or 99%.

As used herein, the term "sequence identity" indicates the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment and is often expressed as a percentage. Preferably, identity is determined over the entire length of the compared sequences. Thus, two copies of the same sequence have 100% identity, but sequences that are less highly conserved and involve deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will appreciate that several algorithms are available for determining sequence identity using standard parameters, Blast (Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. ( 1990), Journal of Molecular Biology 215:403410), Smith-Waterman (Smith et al. (1981), Journal of Molecular Biology 147:195-197) and Klustal U.

The term "sequence homology" indicates the similarity of two (nucleotide or amino acid) sequences attributed to origin from a common predecessor. Homologous biological components (genes, proteins, structures) are called homologs and include orthologs and paralogs.

Preferred variants of the binding molecule of the present invention have sequence identity or homology at the CDR regions of at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99%, or almost 100% and exhibit comparable or improved binding affinity to FXI and/or comparable or improved biological activity compared to the binding molecules comprising the “original” CDRs, preferably SEQ ID NO: 1, 2, 3, 4, 5 and 6.

Moreover, the homology or nucleic acid sequence similarity between the nucleotide sequences encoding the individual variant CDRs and the nucleotide sequences provided herein is at least 80% and preferably with increasing homology or identity of at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and almost 100 %.

In addition to the CDR and FR, amino acid modifications can also be introduced into the Fc portion of the binding molecule, which is preferably a monoclonal antibody or an antigen binding fragment thereof. Such modifications can be used to modulate the functional properties of the antibody, for example, interaction with complement proteins such as Clq and/or Fc receptors on other immune cells or to modulate serum half-life or antigen-dependent cell-mediated cytotoxicity (ADCC). Thus, mutations to modify effector functions can be introduced into Fc domains using conventional methods known in the art. Examples of modifications include Asn297®Ala297 and Asn297®Gln297, leading to glycosylation of IgG1, or Lys322®Ala322 and, in some cases, Leu234®Ala234 and Leu235®Ala234, which have been reported to reduce or abolish antibody-derived cell-mediated cytotoxicity (ADCC) ), and/or complement-derived cytotoxicity (CDC).

Derivatives

The term "linking molecule" also includes derivatives. Derivatives of antibodies or antibody fragments are provided herein as specified elsewhere herein. The term "derivative" generally refers to a binding molecule that has been covalently modified to introduce additional functionality. Covalent modifications of binding molecules are usually, but not always, carried out post-translationally and can be introduced into the binding molecule by reacting certain amino acid residues of the molecule with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues. Derivatization of binding molecules can be used to attach therapeutic or diagnostic agents, labels, groups that increase the serum half-life of the molecule, or the incorporation of unnatural amino acids. Possible chemical modifications of the binding molecules of the present invention include, for example, acylation or acetylation of the N-terminal region or amidation or esterification of the C-terminal region or, alternatively, both regions. Chemical modifications such as alkylation (eg, methylation, propylation, butylation), arylation, and esterification are also contemplated.

Increased serum half-life

Examples of agents for increasing the serum half-life of binding molecules and preferably antibodies and antigen-binding fragments thereof of the present invention include the attachment of peptides or protein domains that bind to other proteins in the human body (for example, serum albumin, immunoglobulin Fc region or neonatal Fc -receptor (FcRn) Additional possible modifications to increase serum half-life include increasing the amino group using polypeptide chains of varying lengths (e.g., CTEN or PASylation® technology), conjugating non-protein polymers including, but not limited to, various polyols such as polyethylene glycol. (PEGylation), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol or carbohydrates, for example, hydroxyethyl starch (eg HESylation®) or polysialic acid (eg PolyXen® technology), amino acid substitutions can also be made as is known in the prior art. made at different positions in the binding molecule to facilitate the addition of polymers.

Glycosylation

Another type of covalent modification of binding molecules, and also, preferably, antibodies and antigen-binding fragments of the present invention, involves changing the glycosylation profile. As is known in the art, glycosylation profiles can depend on either the amino acid sequence of the molecule (eg, the presence or absence of glycosylation amino acid residues) or the host cell or organism where the protein is produced. Glycosylation of polypeptides can be N-linked or O-linked. N-linked glycosylation involves the addition of a carbohydrate moiety to the side chain of an aspartic residue. The addition of N-linked glycosylation sites to the binding molecule is most conveniently accomplished by altering the amino acid sequence so that it contains one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites can be introduced by adding or substituting serine or threonine residues to the initial sequence.

Another method of glycolysis of the binding molecule is the chemical or enzymatic combination of glycosides with protein. The advantages of these procedures are that they do not require protein production in a host cell that has the appropriate capabilities for N- and O-linked glycosylation. Depending on the coupling conditions contemplated, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine groups, (d) free hydroxyl groups such as serine groups, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan residues, or (f) a glutamine amide group.

Likewise, deglycosylation (ie, removal of carbohydrate moieties present in the binding molecule) can be accomplished chemically, such as by exposing the binding molecule to trifluoromethanesulfonic acid, or enzymatically, using endo- and exoglycosidases.

Entering marks

Additional potential covalent modifications of the binding molecules of the present invention include the addition of one or more tags. A group of tags can be introduced in combination with a binding molecule through spacers of varying lengths to limit potential steric isolation. Various methods for introducing tags into proteins are known in the art and can be used in implementing the present invention. The term "mark" or "group of marks" means any detectable mark. In general, tags are divided into many classes, depending on the analysis that is intended to detect them. Examples of tags include, but are not limited to: isotopic tags, which may be radioactive or heavy isotopes, such as radioisotopes or radionuclides (eg, 3H, 14C, 15N, 35S, 89Zr, 90Y, 99Tc, ll lln, 1251, 1311); magnetic tags (eg magnetic particles); redox-active fragments; optical dyes (including, but not limited to, chromophores, luminophores and fluorophores), such as fluorescent groups (e.g. FUC, rhodamine, lanthanide complex phosphors), chemiluminescent groups and fluorophores, which may be “low molecular weight” fluorophores or protein fluorophores; enzyme groups (for example, horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase); biotinylated groups; or predefined polypeptide epitopes that are recognized by secondary reporters (eg, paired leucine zipper sequences, secondary antibody binding sites, metal binding domains, epitope markers, etc.).

ADC

Among other things, it is possible to add a drug, for example, a composition having a small molecule, to the binding molecules, and, preferably, antibodies or antigen binding fragments thereof of the present invention. Antibody-drug conjugates (ADCs) are antibodies or antigen-binding fragments thereof coupled to a drug or substance. The connection can be established through covalent bonds or non-covalent interactions, such as electrostatic forces. To form an ADC, according to standard practice, various linkers known in the art can be used.

Affine markers

The binding molecule, and preferably the antibody or antigen binding fragments thereof, of the present invention may include additional domains that can aid in purification and isolation of the molecule (affinity markers). Non-limiting examples of such additional domains include peptide motifs known as Myc marker, HAT marker, HA marker, TAP marker, GST marker, chitin binding domain marker (CBD marker), maltose binding protein (MBP marker). ), Flag marker, Strep marker and their variants (for example, StrepII marker), and His marker.

The above fragments, variants and derivatives can be further adapted to improve, for example, their antigen-binding properties. For example, F(ab') ₂ or Fab may be designed to maximally limit or completely remove intermolecular disulfide interactions that form between the C _HI and _CL domains. Fv polypeptides may further include a polypeptide linker between the V _H and V _L domains, allowing the Fv to form the required structure for antigen binding. The Fab fragment also contains a light chain constant domain and a first heavy chain constant region (CH ₁ ). The distinction of Fab fragments from Fab' fragments is determined by adding a number of residues at the carboxyl terminus of the CH ₁ region of the heavy chain, which includes one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab' wherein the constant region cysteine residue(s) bear a free theol group.

F(ab')2 antibody fragments were initially formed as pairs of Fab' fragments containing cysteine residues between them.

The binding molecules of the present invention may be provided in "isolated" or "substantially pure" form. "Isolated" or "substantially pure" form as used herein means that the binding molecule has been identified, separated and/or recovered from a component of its working environment such that the "isolated" binding molecule is free or substantially free from other extraneous components of the work environment that may interfere with the satisfaction of therapeutic or diagnostic goals. Extraneous components may include enzymes, hormones, and other protein or non-protein solutes. Thus, “isolated” binder molecules are obtained by performing at least one purification step to remove or substantially remove these extraneous components. The above definition applies equally to "isolated" polynucleotides, mutatis mutandis.

Specific binding

The binding molecules of the present invention, for example, antibodies and antigen binding fragments thereof, are advantageously capable of binding to various mammalian FXIs, preferably human FXIs, that include or consist of the amino acid sequence as set forth in SEQ ID No: 7. Binding Terms c" and "recognition" in all grammatical forms are used interchangeably in this document. In a preferred embodiment, the binding molecules specifically bind to FXI. The term “specific binding” generally means that a binding molecule, such as an antibody or an antigen-binding fragment thereof, as described herein, binds through its antigen-binding site more readily to a putative target epitope than to a random decoy epitope. The term “specific bond” means that the affinity of the binding molecule will be at least 5 times, preferably 10 times, more preferably 25 times, even more preferably 50 times, most preferably 50 times. 100-fold or more greater for its putative target epitope than the affinity for the decoy target epitope.

Thus, it can be considered that the binding molecule, i.e. An antibody or antigen-binding fragment, variant or derivative thereof has specific binding to a target epitope if it binds the epitope with a decay constant (K _D ) that is less than the K _D of the antibody for the decoy target epitope. The binding molecules of the present invention can also be described by their binding affinity for mammalian FXI, preferably human FXI. The term “affinity” or “binding affinity” refers to the ability of a particular epitope to bind to an antigen binding domain (ie, the CDR of a binding molecule). The binding affinity of a given binding molecule for a particular epitope is typically determined by measuring the equilibrium association constant (ka) and equilibrium dissociation constant (kd), and by calculating the ratio of kd to ka (K _D = kd/ka). Binding affinities can be easily determined using conventional techniques such as equilibrium dialysis; using the BIAcore 2000 device; by radioimmunoassay using a radiolabeled target antigen; or using another method known to those skilled in the art. Affinity data can be analyzed, for example, by the method described by R.J. Kaufman and F.A. Sharpe (1982, Journal of Molecular Biology 159:601-621). Among the preferred binding affinities of innovative binding molecules are those whose decay constant or K _D is less than 5 x 10 ^-6 M, 10 ^-6 M, 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 10 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M, 10 ^-10 M, 5 x 10 ^-11 M, 10 ^-11 M, 5 x 10 ^-12 M, 10 ^{- 12} M, 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M, or 10 ^-15 M.

Cross reactivity

The term “specific binding” does not, however, exclude the possibility that binding molecules having a (specific) binding to human FXI cross-react with another species' FXI protein. Accordingly, the binding molecules of the present invention are also capable of binding to FXI from other mammalian species.

“Interspecies” association or recognition means the association of a binding domain, as described herein, with a similar target antigen in humans and non-human species. Thus, “cross-species specificity” should be understood as cross-species reactivity to FXI in different species, but not to an antigen other than FXI. For example, a binding domain that binds to human FXI, preferably the A2 domain, which includes amino acids 91 to 175 of the amino acid sequence set forth in SEQ ID NO: 7, also binds to other non-human FXI, and in a preferred embodiment, to which has a region characteristic corresponding to or similar to amino acids 91 to 175 of the amino acid sequence presented in SEQ ID No: 7.

Biological activity

It is assumed that the binding molecules proposed herein are biologically active, i.e. bind to mammalian FXIa and/or FXI and block some of the relevant biological functions. In particular, the “biologically active” binding molecules of the present invention form an immune complex with FXI, and the FXI-AB023 immune complex cannot be quickly activated by FXIIa or activated automatically. However, the immune complex can still be activated by thrombin. Once the FXI-AB023 complex is activated relative to FXla-AB023, its ability to convert the zymogen FXII to FXIIa is reduced, but the activated complex can convert FIX to factor IXa without loss of function, resulting in, preferably, complete or partial inhibition of contact activation, while thrombin-mediated activation of blood hemostatic feedback is maintained. Thus, it is expected that the binding of biologically active binding molecules to their FXIa and/or FXI target will cause anticoagulant activity, for example, in an assay involving the initiation of coagulation through a contact activation complex. In other words, it is believed that the binding molecules of the present invention perform their beneficial function by a) binding to FXI, thereby blocking conversion to the active form of FXIa by FXIIa or automatic activation, and/or b) binding to FXIa, thereby limiting binding and activation of FXII. Thus, in a preferred embodiment, the binding molecules of the present invention interfere with the contact activation complex, and thereby limit the pathological processes associated with contact activation, including, for example, inflammation and thrombosis, preferably without interfering with hemostatic processes that are independent of contact activation complex.

The anticoagulant activity of the binding molecule can be determined in vitro as described herein. Briefly, the activated partial thromboplastin time (aPTT) of normal human or other mammalian plasma, which measures thrombin generation as a function of contact system activation, is determined in the presence of varying concentrations of the binding molecule or appropriate solvent using a commercial test kit (SynthASil reagent from Instrumentation Laboratories, Bedford, Massachusetts). The test compositions are incubated with plasma, which typically contains endogenous FXI in the concentration range of 20 to 45 nM, as well as the SynthASil reagent (colloidal silica activator) at 37°C for 3 minutes. Coagulation then begins, which is accomplished by adding 25 nM calcium chloride, and the time at which coagulation occurs is determined, and the concentration of test substance that causes aPTT to prolong by 2.0 times is determined. The binding molecules of the present invention are predicted to cause aPTT to prolong by 1.5-fold, 2.0-fold or more.

Advantageously, the binding molecules, and also preferably the monoclonal antibodies and antigen binding fragments thereof, of the present invention exhibit the above biological properties and therefore represent promising agents for inhibiting thrombosis and inflammation. Since the binding molecules specifically bind to FXI, they are not expected to interfere with hemostasis, and thus preferably do not increase the risk of bleeding.

Polynucleotide

The present invention further discloses a polynucleic acid molecule encoding a binding molecule, or VH or V domain, of the present invention.

As used herein, the term "polynucleotide" means polyribonucleotides and polydeoxyribonucleotides, e.g., modified or unmodified RNA or DNA, each in single-stranded and/or multistranded (e.g., double-stranded) form, linear or circular, or combinations thereof, including hybrids molecules. The polynucleotide may include a conventional phosphodiester linkage or a non-conventional linkage (eg, an amide linkage, as in a peptide nucleic acid (PNA)). The polynucleotides of the present invention may also contain one or more modified bases, such as, for example, tritylated bases, and unusual bases, such as inosine. Other modifications are possible, including chemical, enzymatic or metabolic modifications, if the connecting molecule of the present invention can be obtained from a polynucleotide. The polynucleotide may be presented in isolated form, as defined herein. The polynucleotide may include regulatory sequences, such as transcription control elements (including accelerators, enhancers, operators, repressors, and transcription termination signals), a ribosome binding site, introns, and the like.

The present invention provides a polynucleotide comprising or consisting of a nucleic acid encoding an immunoglobulin heavy chain domain (V _H region), wherein at least one CDR of the V _H region has an amino acid sequence of at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No: 8. A binding molecule comprising encoded CDRs or _VH domains is believed to be capable of bind to FXI and, by forming a complex between FXI and the binding molecule, preferably exhibit the desired biological activity as described herein.

The present invention provides a polynucleotide comprising or consisting of a nucleic acid encoding an immunoglobulin light chain domain ( _VL region), wherein at least one CDR of the VL region has an amino acid sequence of at least 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID No: 9. A binding molecule comprising encoded CDRs or VL regions is believed to be capable of binding to FXI and, by forming a complex between FXI and the binding molecule, preferably exhibit the desired biological activity as described herein.

The polynucleotides that are described herein may or may not include additional nucleotide sequences encoding, for example, a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein . Thus, such polynucleotides may encode fusion polypeptides, fragments, variants, and other derivatives of binding molecules that are described herein.

In addition, the present invention includes compositions consisting of one or more polynucleotides as described above. Further provided herein are compositions comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes the _VH region as described herein and the second polynucleotide encodes the VL region as described herein, particularly a composition that includes or consists of the V _H region presented in SEQ ID No.: 8 and/or the VL region as presented in SEQ ID No.: 9.

Polynucleotide formation

The polynucleotides of the present invention can be prepared using the most common methods known in the art. For example, if the nucleotide sequence of the binding molecule is known, then a polynucleotide encoding the binding molecule can be assembled from chemically synthesized oligonucleotides by annealing and ligating the oligonucleotides and then amplifying the ligated oligonucleotides using PCR. The polynucleotide encoding the binding molecule can be obtained from a suitable source (for example, a cDNA library or a nucleic acid additive such as poly(A)+ mRNA from any tissue or cell from which the binding molecule can be obtained, such as hybridoma cells) by PCR amplification using synthetic hybridizable primers (3' and 5' ends of the sequence), or by cloning using an oligonucleotide probe specific for the nucleotide sequence of the gene to identify, for example, a cDNA derived from a cDNA library encoding a binding molecule.

Once the nucleotide sequence and corresponding amino acid sequence of the binding molecule have been determined, its nudeotide sequence can be modified using methods well known in the art for manipulating nudeotide sequences, for example, recombinant DNA techniques, site-specific mutagenesis, PCR, etc. ..., thereby introducing one or more nucleotide substitutions, additions or deletions into a polynucleotide sequence (see, for example, the methods described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual). (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012)) to create non-natural fragments, variants or derivatives of the binding molecule, i.e. the monoclonal anti-FXI antibody described herein (eg, immunoglobulin heavy chain region or light chain region).

Vectors

Presented hereinafter is a vector comprising a polynucleotide as described herein. The polynucleotide encodes a binding molecule of the present invention, preferably a monoclonal antibody or an antigen binding fragment thereof. A "vector" is a nucleic acid molecule used as a carrier medium for transferring (foreign) genetic material to a host cell, where it can, for example, be replicated and/or expressed.

The term “vector” includes, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids , cosmids and artificial chromosomes (including bacterial artificial chromosomes and yeast artificial chromosomes). The vector itself is generally a nucleotide sequence, usually a DNA sequence, including an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. Designed vectors may include an origin for autonomous replication in host cells (if stable expression of the polynucleotide is desired), selectable markers, as well as restriction enzyme cleavage sites (eg, multiple cloning site, MCS). Vectors may further include accelerators, genetic markers, reporter genes, targeting sequences and/or protein purification tags. Vectors called expression vectors (expression constructs) are specifically designed to express a transgene in a target cell, and usually have control sequences. Those skilled in the art are aware of a large number of suitable vectors available on the market. Examples of suitable vectors are given by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. (2012).

Targeted Vectors

Targeting vectors can be used to integrate a polynucleotide into the host cell chromosome (Sambrook et al., 2012). Briefly, suitable means include, but are not limited to, homologous recombination or the use of a hybrid recombinase that targets sequences to sites of integration. Targeted vectors can be made circular or linear before being used for homologous recombination. Alternatively, the foreign polynucleotides may be DNA fragments combined by PCR fusion or synthetically engineered DNA fragments that are then recombined into the host cell. In addition, the possibility of using heterologous recombination, which causes random or untargeted integration, is envisaged.

Receipt

Expression vectors

"Expression vectors" or "expression constructs" can be used to transcribe heterologous polynucleotide sequences, such as those encoding binding molecules of the present invention, and to translate their mRNA in a suitable host cell. This process is also referred to herein as “expression” of the binding molecules of the present invention. In addition to the origin of replication, selectable markers and restriction enzyme cleavage sites, expression vectors may include one or more regulatory sequences operably linked to the heterologous polynucleotide to be expressed.

The term “regulatory sequence” refers to a nucleic acid sequence necessary for the expression of an operably linked coding sequence of a (heterologous) polynucleotide in a host organism, and thus includes transcriptional and translational regulatory sequences. Regulatory sequences required for the expression of heterologous polynucleotide sequences in prokaryotes include, for example, accelerator(s), in some cases operator sequence(s), and ribosome binding site(s). In eukaryotes, accelerators, polyadenylation signals, amplifiers, and, in some cases, splicing signals may be required. In addition, specific initiation and secretory signals may also be introduced into the vector to allow secretion of the polypeptide of interest into the culture medium.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence, for example, in the same polynucleotide molecule. For example, an accelerator operably binds to the coding sequence of a heterologous gene when it can affect the expression of that coding sequence. The accelerator can be located upstream of the gene encoding the polypeptide of interest and regulate gene expression.

Examples of regulatory sequences for mammalian host cell expression include viral elements that control high levels of expression in mammalian cells, such as accelerators and/or enhancers derived from cytomegalovirus (CMV) (eg, CMV accelerator/enhancer), simian virus 40 ( SV40) (eg, SV40 accelerator/booster), adenovirus (eg, adenovirus major late booster (AdMLP)), and polyoma. For further description of viral regulatory elements, and their sequences, see, for example: US Pat. No. 5,168,062 issued to Stinsky; US Patent No. 4,510,245 to Couzens et al.; and US Patent No. 4,968,615 to Koszynowski et al. Expression vectors may also include origins of replication and selectable markers.

The vectors of the present invention may further include one or more selectable markers. Selectable markers suitable for use with eukaryotic host cells include the herpes simplex virus thymidine kinase (tk), hypoxanthine guanine phosphoribosyltransferase (hgprt), and adenine phosphoribosyltransferase (aprt) genes, among others. Other genes include dhfr (methotrixate resistance), gpt (mycophenolic acid resistance), neo (G-418 resistance) and hygro (hygromycin resistance). Vector amplification can be used to increase expression levels. In general, a gene-selectable marker can be directly linked to the polynucleotide sequences to be expressed, or introduced into a similar host cell through co-transformation.

In view of the above, the present invention further provides one or more polynucleotide sequences described herein that can be introduced into a vector. The present invention provides replicable vectors comprising a nucleotide sequence encoding a binding molecule of the present invention, or a heavy or light chain, or a heavy or light chain variable domain thereof, functionally linked to an accelerator. Such vectors may include a nudeotide sequence encoding the constant region of a binding molecule, and the variable domain of the binding molecule may be cloned into such a vector to express the entire heavy or light chain.

Host cell

In general, a variety of host cells can be used to express the binding molecule of the present invention from an expression vector. As used herein, the term “host cell” refers to a cell that may be or has been the recipient of polynucleotides or vectors, or a cell encoding a binding molecule of the present invention. In particular, the host cell may subsequently be capable of expressing, or in some cases secreting, the binding molecule. In descriptions of processes for obtaining binding molecules from host cells, the terms “cell” and “cell culture” are used interchangeably to identify the source of the binding molecule, unless clearly stated otherwise. The term "host cell" also includes "host cell lines".

In general, the term includes prokaryotic and eukaryotic cells, and also includes, but is not limited to, bacteria, yeast cells, fungal cells, plant cells and animal cells, such as insect cells and mammalian cells, for example, mammalian cells, e.g. mouse, rat, macaque, or human cells. The polynucleotides and/or vectors of the present invention can be introduced into host cells using the most common methods known in the art, for example, by transfection, transformation, and the like.

"Transfection" is the process of deliberately introducing nucleic acid molecules or polynocleotides (including vectors) into target cells. The term is mainly used for non-viral methods in relation to eukaryotic cells. Transduction is generally used to describe the transfer of nucleic acid molecules or polynucleotides mediated by a virus. Transfection of animal cells may involve opening transition pores or “holes” in the cell membrane to allow material to be taken up. Transfection can be done using calcium phosphate, by electrically pulsing open cell pores, compressing cells, or mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo molecules inside. Examples of methods for transfecting eukaryotic host cells include lipid vesicle-mediated uptake; heat shock-mediated capture; calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation); microinjection; as well as electric pulse opening of cell pores.

The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) to bacteria, as well as to non-animal eukaryotic cells, including plant cells. Thus, transformation is a genetic change in a bacterial or non-animal eukaryotic cell resulting from direct uptake through the cell membrane(s) from the environment and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be influenced artificially. For transformation to occur, the cells or bacteria must be in a state of competence, which can occur as a time-limited response to environmental conditions such as growth in minimal media and cell density. For prokaryotic transformation, methods include heat shock-mediated capture; fusion of bacterial protoplasts with intact cells; microinjection; as well as electric pulse opening of cell pores. Among the methods for plant transformation can be highlighted the transfer mediated by Agrobacterium, such as A. tumefaciens, with rapidly moving microparticles of tungsten or gold; electric pulse opening of cell pores; microinjection; and polyethylene glycol-mediated uptake.

In view of the above, the present invention further provides host cells comprising at least one polynucleotide sequence and/or vector as described herein.

For expression of the binding molecule of the present invention, a host cell may be selected that modulates the expression of the introduced polynucleotide sequences, and/or modifies and processes the gene product (ie, RNA and/or protein) as required. Such modifications (eg, glycosylation) and processing (eg, cleavage) of gene products may contribute to the function of the binding molecule. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of gene products. Suitable cell lines or host systems can be selected to ensure proper modification and processing of the product. For this purpose, eukaryotic host cells that possess the cellular machinery to properly process the primary transcript, glycosylate, and phosphorylate the gene product can be used.

Examples of mammalian host cells that can be used to express binding molecules provided herein include Chinese hamster ovary (CHO cells), including DHFR minus CHO cells, such as DG44 and DUXB1 1, and as described in US Pat. No. 4,634,665 (eg, used with a DHFR selectable marker, eg, as described in US Pat. No. 5,179,017), NSO, COS (CVI derivative with SV40 T antigen), HEK293 (human kidney), and SP2 (mouse myeloma). Other examples of host cells include, but are not limited to: HELA (human cervical carcinoma), CVI (monkey kidney line), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells) and RAJI (human lymphocyte). Host cell lines can be obtained from commercial services, the American Tissue Culture Collection, or from the published literature.

Non-mammalian cells, such as bacterial, yeast, insect or plant cells, can also be easily obtained and, in principle, can be used to express the binding molecules of the present invention. Examples of bacterial host cells include enterobacteriaceae such as Escherichia coli, Salmonella; bacilli such as Bacillus subtilis; Pneumococcus; Streptococcus; as well as Haemophilus influenzae.

Other host cells include yeast cells such as Saccharomyces cerevisiae and pichia pastoris. Among insect cells, the most notable are the cells of Spodoptera frugiperda.

In accordance with the above, possible expression systems (i.e., host cells incorporating an expression vector) include microorganisms such as bacteria (eg, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or vectors cosmid DNA expression; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus); plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmids); or mammalian cell systems (eg, COS, CHO, BLK, 293, 3T3 cells) bearing recombinant expression constructs containing accelerators derived from the genome of mammalian cells (eg, metallothionein accelerator) or mammalian viruses (eg, adenovirus late accelerator; virus accelerator vaccinia 7.5K).

For expression of the binding molecules of the present invention, eukaryotic cells are preferably provided. Accordingly, CHO cells comprising a eukaryotic vector with a polynucleotide sequence encoding a binding molecule of the present invention (which may, for example, be operably linked to the human cytomegalovirus (CMV) major enhancer of immediate response gene (MIEP)) are useful expression systems for producing binding molecules of the present invention.

Cultivation

Host cells carrying the expression vector are grown under conditions suitable for producing the binding molecules described herein, eg, light chains and heavy chains as described herein, and assayed for heavy and/or light chain protein synthesis. Thus, the present invention includes host cells comprising a polynucleotide encoding a binding molecule of the present invention, or a heavy or light chain thereof, operably linked to an enhancer. For the expression of double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell to express the entire molecule.

Cleaning

Once the binding molecule of the present invention has been recombinantly expressed, it can be purified by any method known in the art, for example, by chromatography (e.g., ion exchange chromatography (e.g., hydroxylapatite chromatography), affinity chromatography, using protein A, protein G, or by lectin affinity chromatography, size distribution column chromatography), centrifugation, differential dissolution, hydrophobic interaction chromatography, or any other standard protein purification method. One skilled in the art will readily be able to select an appropriate purification method based on the individual characteristics of the binding molecule to be recovered.

In view of the foregoing, the present invention further provides a process for producing the binding molecule of the present invention, comprising culturing a host cell, as defined herein, under conditions allowing expression of the binding molecule and, in some cases, recovery of the resulting molecule from the culture.

Pharmaceutical composition

The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a binding molecule, nucleic acid, vector and/or host cell of the present invention, as well as, in some cases, one or more pharmaceutically acceptable excipients or carriers. A preferred pharmaceutical composition includes an antibody of the present invention and, in some cases, one or more pharmaceutically acceptable excipients.

Thus, in one aspect, the present invention provides a pharmaceutical composition comprising as an active ingredient a binding molecule as described herein, preferably an anti-FXI antibody or an antigen binding fragment thereof. Accordingly, the present invention also provides for the possibility of using a binding molecule for the manufacture of a pharmaceutical composition. The term "pharmaceutical composition", preferably, refers to a composition suitable for administration to a subject, specifically a human. However, compositions suitable for administration to non-human animals are also included within this term.

The pharmaceutical composition and its components (i.e. active ingredients and, in some cases, excipients) are preferably pharmaceutically acceptable, i.e. are capable of causing the required therapeutic effect without causing undesirable local or systemic effects in the recipient. Pharmaceutically acceptable compositions of the present invention may be, for example, sterile and/or pharmaceutically inert. In particular, the term "pharmaceutically acceptable" may mean approval by a regulatory authority or other generally accepted pharmacopoeia for use in animals, and, preferably, in humans.

The binding molecule described herein is preferably present in the pharmaceutical composition in a therapeutically effective amount. By "therapeutically effective amount" is meant the amount or dosage of a binding molecule that produces the desired therapeutic effect. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures, experimental animals, or clinical trials, for example, ED ₅₀ (dose therapeutically effective in 50% of the population) and LD ₅₀ (dose lethal in 50% of the population). The dose ratio between therapeutic and toxic effects represents the therapeutic index and can be expressed as the ratio ED ₅₀ /LD ₅₀ . Pharmaceutical compositions demonstrating high therapeutic indices are preferred.

As indicated herein, the pharmaceutical composition may, in some cases, include one or more excipients and/or additional active ingredients.

Antibodies and fragments thereof are usually administered parenterally, preferably intravenously (injection or infusion) or subcutaneously. Compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be selected from the group consisting of water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffer media such as, but not limited to, sodium phosphate buffer solution. Additional parenteral carrier media include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer, or fixed oils. Other suitable pharmaceutical carriers, diluents and/or excipients are well known in the art. A binding molecule, such as an antibody or fragment thereof, of the present invention may be combined with a pharmaceutically acceptable carrier, diluent and/or excipient, such as those discussed above, to form a pharmaceutical composition. Pharmaceutical compositions may include the binding molecule of the present invention in an aqueous carrier that includes a buffer substance selected from the group consisting of histidine buffer, acetic acid buffer, citric acid buffer and histidine hydrochloride buffer. Additional information on buffers and composition can be found by one skilled in the art in, for example, Wang W. et al., J. Pharmaceutical Sci., January 2007 (1): 1-26. In addition, preservatives may be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, etc.

The pharmaceutical composition may further include protein carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. According to one embodiment, the pharmaceutical composition includes the binding molecule in lyophilized form, and, preferably, is reconstituted in a solution or suspension before administration. In other embodiments, the pharmaceutical composition includes a binding molecule and is in liquid form.

Once the pharmaceutical compositions of the present invention and, in some cases, a suitable excipient have been prepared, they may be appropriately packaged and labeled for the treatment of the specified condition. Such labeling may, for example, include the amount, frequency, and route of administration.

Additional active ingredients

The present invention further provides medicinal preparations or pharmaceutical compositions comprising an innovative compound and one or more active ingredients, including those intended for the treatment and/or prevention of the disorders specified herein. Preferred examples of active ingredients suitable for combination include, but are not limited to:

- lipid-lowering substances, especially hydroxymethylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, including, but not limited to, lovastatin (Mevacor), simvastatin (Zocor), pravastatin (Lescol). Leskol)) and atorvastatin (Lipitor (Lipitor));

- coronary therapeutic agents/vasodilators, especially ACE (angiotensin-converting enzyme) inhibitors, including, but not limited to, captopril, lisinopil, enalapril, ramipril, dlazapril, benazepril, fosinopril, quinapril and perindopril; or AII (angiotensin II) receptor blockers, including, but not limited to, embusartan, losartan, valsartan, irbesartan, candesartan, eprosartan and temisartan; or adrenergic blockers including, but not limited to, carvedilol, alprenolol, bisoprolol, acebutolol, atenolol, betaxolol, carteolol, metoprolol, nadolol, penbutolol, pindolol, propanolol and timolol; or alpha-1 adrenergic blockers including, but not limited to, prazosin, bunazosin, doxzosin and terazosin; or diuretics, including, but not limited to, hydrochlorothiazide, furosemide, amiloride and dihydrazaline; or calcium channel blockers including, but not limited to, verapamil and diltiazem; or dihydropyridine derivatives including, but not limited to, nifedipine (Adalat) and netrendipine (Bayotensin); or nitro preparations including, but not limited to, isosorbide 5-mononitrate, isosorbide dinitrate and glycerol trinitrate; or substances that cause an increase in cycloguanosine monophosphate (cGMP), including, but not limited to, riociguat;

- plasminogen activators (thrombolytics/fibrinolytics) and compounds promoting thrombolysis/fibrinolysis, including, but not limited to, plasminogen activator inhibitor inhibitors (PAI inhibitors) or thrombin-activated fibrinolysis inhibitors (TAFI inhibitors), including, but not limited to, tissue plasminogen activator (t-PA), streptokinase, reteplase and urokinase;

- anticoagulants (anticoagulants), including, but not limited to, heparin (NG), low molecular weight heparins (LMWH), which include, but not limited to, tinzaparin, certoparin, pamaparin, nadroparin, ardeparin, enoxaparin, reviparin, dalteparin, danaparoid , semuloparin (AVE 5026), adomiparin (Ml 18) and EP-42675/ORG42675;

- direct thrombin inhibitors (DTI), including, but not limited to, Pradaxa (dabigatran), atecegatran AZD-0837), DP-4088, SSR-182289A, argatroban, bivalirodin and tanogitran (BIBT-986 and the prodrug BIBT- 1011), hirudin;

- direct factor Xa inhibitors, including, but not limited to, rivaroxaban, apixaban, edoxaban (DU-176b), betrixaban (PRT-54021), R-1663, darexaban (YM-150), otamixaban (FXV-673/RPR- 130673), letaxaban (TAK-442), razaxaban (DPC-906), DX-9065a, LY-517717, tanogitran (BIBT-986, prodrug: BIBT-1011), idraparinux and fondaparinux;

- substances that inhibit platelet aggregation (blood cell aggregation inhibitors, platelet aggregation inhibitors), including, but not limited to, acetylsalicylic acid (for example, Aspirin), ticlopidine (Ticlid), clopidogrel (Plavix) , prasugrel, ticagrelor, cangrelor, elinogrel, vorapaxar;

- fibrinogen receptor blockers (glycoprotein-IIb/IIIa), including, but not limited to, abciximab, eptifibatide, tirofiban, lamifiban, lefradafiban and fradafiban;

- antiarrhythmic drugs;

- various antibiotics or antifungal medications either as calculated therapy (before a diagnosis based on microorganisms is available) or as specific therapy;

- vasoconstrictor factors, including, but not limited to, norepinephrine, dopamine and vasopressin;

- inotropic therapy, including, among other things, dobutamine;

- human recombinant activated protein C, for example, Sigris;

- blood products, including, but not limited to, red blood cells, platelets, erythropoietin and fresh frozen plasma;

- platelet adhesion inhibitors, such as GPVI and/or GPIb blockers, including, but not limited to, Revacept or Caplacizumab.

- inhibitors of signal transduction pathways dependent on VEGF and/or PDGF, including, but not limited to, Aflibercept, Ranibizumab, Bevacizumab, KH-902, Pegaptanib, Ramucirumab, Squalaminoder, Bevasiranib, Apatinib, Axitinib, Brivanib, Cediranib, Dovitinib, Lenvatinib, Linifanib, Motesanib, Pazopanib, Regorafenib, Sorafenib, Sunitinib, Tivozanib, Vandetanib, Vatalanib, Vargatef or E-10030;

- signal transduction pathway inhibitors (angiopoietin-TIe), including, but not limited to, AMG386;

- inhibitors of TIe2 receptor tyrosine kinase activity;

- inhibitors of the integrin-dependent signal transduction pathway, including, but not limited to, Volociximab, Cilengitid or ALG1001;

- inhibitors of signal transduction dependent on the PI3K/AKT/mTOR signaling pathway, including, but not limited to, XL-147, Perifosin, MK2206, Sirolimus, Temsirolimus or Everolimus;

- corticosteroids, including, but not limited to, hydrocortisone, fludrocortisone, Anecortave, Betamethason, Dexamethasone, Triamcinolon, Fluocinolon or Fluocinolonacetonid;

- inhibitors of the ALK.l-Smadl/5 dependent signal transduction pathway, including, but not limited to, ACE041;

- cyclooxygenase inhibitors, including, but not limited to, Bromfenac (Bromfenac) or Nepafenac (Nepafenac);

- kallikrein-kinin system inhibitors, including, but not limited to, Safotibant or Ecallantid;

- inhibitors of sphingosine-I-phosphate-dependent signal transduction pathways, including, but not limited to, Sonepcizumab;

- complement receptor C5a inhibitors, including, but not limited to, Eculizumab; - 5HTla receptor inhibitors, including, but not limited to, Tandospiron;

- inhibitors of the Raf-Mek-Erk-dependent signal transduction pathway, inhibitors of the mitogen-activated protein kinase signal transduction pathway; inhibitors of the fibroblast growth factor signal transduction pathway, inhibitors of endothelial cell proliferation and compounds capable of inducing apoptosis; or

- photodynamic types of therapy, involving the use of an active substance and exposure to light, and the active substance can be represented, for example, by Verteporfin.

"Combinations" for the purposes of the present invention mean not only dosage forms containing all components (so-called fixed combinations) and blocks of combinations that contain components separated from each other, but also components administered simultaneously or sequentially, provided that they used for the prevention and/or treatment of the same disease. It is also possible to combine two or more active ingredients with each other, which means that each of them will thus be included in two- or multi-component combinations.

Introduction

There are various routes for administering the pharmaceutical composition of the present invention. Administration can be carried out parenterally. Methods of parenteral delivery include, for example, topical, intra-arterial, intramuscular, subcutaneous, bone marrow, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical compositions of the present invention can be formulated as aqueous solutions, preferably as physiologically compatible buffers such as Hank's solution, Ringer's solution or buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Examples of lipophilic solvents or carrier media include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. In addition to this, the suspension may also contain suitable stabilizers or substances that can increase the solubility of the compounds to enable the preparation of highly concentrated solutions. For topical or nasal administration, the compositions use penetration agents appropriate to the barrier to be overcome. Such penetration agents are well known in the art. Additional information regarding preparation and administration procedures can be found in Remington's Pharmaceutical Sciences, 22nd edition (ed. Maack Publishing Co., Easton, PA, 2012).

Treatment

As used herein, the terms “treat” or “treatment” (in all grammatical forms) imply a therapeutic or prophylactic effect on the diseases described herein. “Therapeutic or prophylactic treatment” includes types of prophylactic treatment aimed at slowing or completely preventing clinical and/or pathological manifestations, or therapeutic treatment aimed at reducing the intensity or remission of clinical and/or pathological manifestations. Thus, the term "treatment" also implies the reduction or prevention of the described diseases. Treatment may also mean increased survival compared to what would be expected without treatment. Persons in need of treatment include those who already have a disorder or disorder, those who are susceptible to such a disorder or disorder, or those who are to be prevented from developing a disorder or disorder.

The terms “patient” or “individual”, “animal” or “patient”, or “mammal” are used interchangeably herein and refer to a patient, preferably a mammalian subject, for whom diagnosis, prognosis or treatment is to be performed. . Mammalian subjects include humans, non-human primates, domestic animals, farm animals, companion animals, zoo, sporting or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle cattle, cows, etc.

As used herein, expressions including "will benefit" and "in need of treatment" refer to patients who will benefit from administration of a humanized monoclonal antibody, binding fragment, variant or derivative thereof.

Dosage

Through the use of known techniques, one skilled in the art will be able to determine the precise dosage (therapeutically effective amount) of binding molecules, polynucleotides, vectors, or host cells. In appropriate dosages, a sufficient amount of binding molecules is provided, preferably being therapeutically effective, i.e., providing the desired therapeutic or prophylactic effect.

As is known from the prior art, it is possible to determine the treatment and adjust according to its purpose (for example, prevention, maintenance of remission, treatment of sudden acute exacerbation of the disease) for the route, time and frequency of administration, time and frequency of preparation of the drug for administration, age, body weight , general health, gender, diet, disease severity, drug combination(s), allergic reactions, and tolerance/response to treatment. Ranges of appropriate therapeutically effective doses can be determined by cell culture and animal studies and may include ED ₅₀ . The amount per dose can vary from 0.1 to 100,000 micrograms, reaching a total dose of about 2 g, depending on the route of administration. Examples of binding molecule dosages may range from 0.01 to 10 mg/kg, 0.1 to 10 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg, 0.01 to 1 mg/kg or from 0.1 to 1 mg/kg. Guidance on dosages and delivery methods is provided in the literature. It is known that treatment may require a single administration of a therapeutically effective dose or multiple administrations of such a dose containing a binding molecule, polynucleotide, vector or host cell of the present invention. For example, depending on the form, half-life, and elimination rate of the composition, some pharmaceutical compositions may be administered once, periodically over a period measured in hours, every 3-4 days, weekly or biweekly, monthly, every other month. Methods for determining each of the applicable variables are well known to those skilled in the art.

Kit

According to the present invention, pharmaceutical kits and kits are further disclosed, including one or more packages or bottles filled with one or more active ingredients included in the above pharmaceutical compositions of the present invention, to which instructions for their administration are attached. Thus, provided herein is a kit comprising a binding molecule, a polynucleotide, a vector, a host cell, and/or a pharmaceutical composition as described herein. The above kits described herein may be used to treat the diseases described herein or for other purposes.

The above packaging may include notices in a form prescribed by the government agency regulating the manufacture, use or sale of pharmaceutical or biological products indicating that agency's approval of the manufacture, use or sale of the product for human consumption.

The kit may include one or more active ingredients (additionally in the form of a pharmaceutical composition with one or more excipients). The corresponding active ingredients have previously been described in the context of a pharmaceutical composition and could potentially form part of an innovative kit. The additional active ingredient may be administered to the patient simultaneously or sequentially in accordance with the binding molecule, nucleotide sequence, vector, host cell and/or pharmaceutical composition. The present invention also discloses the administration of active substances through various routes, for example, orally and intravenously.

Further provided herein are kits comprising polynucleotide sequences encoding the binding molecules of the present invention. The polynucleotides may be presented in a vector, such as a plasmid, suitable for transfection and expression by a host cell. A description of such vectors and host cells is provided herein.

Therapeutic Use

The present invention further discloses binding molecules, preferably antibodies and antigen-binding fragments thereof, for use as medicinal products for the treatment and/or prevention of diseases in humans and/or animals.

The present invention further discloses binding molecules, preferably represented by antibodies and antigen-binding fragments thereof, for use in the treatment and/or prevention of disorders, for example cardiovascular disorders, preferably thrombotic or thromboembolic disorders and/or complications, as well as inflammatory processes, preferably autoimmune inflammation or inflammatory reactions associated with infections.

FXIa is an enzyme that, in the context of coagulation, can be activated by both thrombin and FXIIa, and therefore may be involved in the coagulation process. FXI is a central component of the transition from initiation to enhancement and propagation of coagulation. Moreover, FXIa is a component for initiating and maintaining pathological blood coagulation in blood vessels. The blood contact activation system can be activated on intravascular surfaces with a negative charge, which involves the effects of subendothelial and extravascular contents (including collagen and laminin) not only on the bloodstream, on the surface structures of foreign cells (for example, bacteria), but also on such artificial surfaces such as vascular grafts, catheters, stents, ventricular assist devices, valves and extracorporeal life support systems such as oxygenators, pumps, tubing, dialysis units, etc. At surfaces, factor XII (FXII) is activated by factor Xlla (FXIIa), which subsequently activates FXI, which attaches to FXIa surfaces. FXI can also self-activate on negatively charged surfaces. This activation of FXI results in the release of thrombin downstream, as well as a response upregulation of all contact activation complex enzymes, which include FXI, FXII, and prekallikrein.

In contrast, the hemostatic release of thrombin in extravascular blood that exits the vessel through a wound is not affected by inhibition of contact activation, since the hemostatic release of thrombin occurs under the influence of the TF/FVIIa complex and the response activation of FXI by thrombin, neither of which is affected there is a significant impact upon pharmacological intervention in the functions of the contact activation complex. The lack of apparent bleeding tendency and predisposition to acute inflammation in exposed mammals, which characterizes the complexing effect between FXI and the binding molecule in the body, represents a major advantage over other types of FXI/FXla inhibitors or protease inhibitors for use in humans, for example in patients with an increased risk of bleeding or inflammatory reactions. Accordingly, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are intended for use in the treatment and/or prevention of disorders or complications that may develop due to enzymatic and non-enzymatic activity of the contact activation complex, including, but not limited to, thrombin, initiated by pathological contact in the blood, and the release of bradykinin, leading to thrombosis and inflammation, respectively.

In the context of the present invention, “thrombic or thromboembolic disorders” include, for example, disorders occurring in both the arterial and venous vasculature, which can be affected by the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof; and it is also preferable that the disorders be represented by disorders of the coronary arteries of the heart, such as acute coronary syndrome (ACS), ST-segment elevation myocardial infarction

(STEMI) and without (NSTEMI), stable thoracic angina, unstable thoracic angina, reocclusions and restenoses after interventions in the coronary arteries, such as angioplasty, stenting or coronary artery bypass grafting, as well as thrombus or thromboembolic disorders in vessels located further, which leads to occlusive disorders of the arterial periphery, pulmonary embolism, venous thromboembolism, venous thrombosis, i.e., thrombosis in the deep veins of the legs and kidneys, transient ischemic attacks, as well as thrombotic and thromboembolic strokes.

In the context of the present invention, “inflammatory disorders” include pathological conditions based on or triggered by the activation of the total contact system complex (FXFI/FXJ/PK/HK), including excessive degradation of kininogen (HMWK) and the release of the most potent known pro-inflammatory peptide, bradykinin, with subsequent or associated pathological vasodilation and increased permeability of blood vessels, deregulation of blood pressure, increased immune response to foreign substances, and increased endogenous autoimmune reactions, including those involving antigen-antibody reactions and therefore , contribute to the activation of plasminogen and other resulting conditions, affecting either locally (cells, organs) or the entire body system.

Activation of the contact system can occur for various reasons or as a result of related disorders. In the context of surgery and other tissue trauma, mobility limitations, bed rest, infections, inflammation, cancer, tissue damage and necrosis, autoimmunity, temporary or permanent presence of implanted foreign bodies, and ischemic disease, among others, the contact system can be activated and provoke thrombotic and inflammatory complications. Thus, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are useful for the prevention of thrombosis and inflammation in the context of surgical intervention, for example, in relation to patients undergoing operations on the gastrointestinal tract, lungs, nervous system, urinary tracts, orthopedic and other major surgeries that are known to be associated with thrombus formation and/or inflammation. Therefore, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are also useful for the prevention of thrombosis in patients with activated contact system.

Thus, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are also useful in the treatment and/or prevention of venous and cardiogenic thromboembolism, for example, coronary artery disease, stroke and systemic thromboembolism and ischemic diseases in patients with acute, intermittent or persistent cardiac arrhythmia, such as atrial fibrillation, in patients undergoing electropulse therapy, as well as in patients with heart valve disorders or artificial heart valves. Additionally, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are useful in the treatment and/or prevention of generalized thrombohemorrhagic syndrome (DIC), which can occur, inter alia, due to sepsis, but also due to surgery , disorders due to neoplasms, burns or other injuries and lead to severe organ damage due to microthrombosis.

In addition, thromboembolic and inflammatory complications occur with hemolytic microangiopathic anemia and with contact of blood with foreign surfaces during extracorporeal circulation (for example, during extrapulmonary circulation), when using other life support systems, such as hemodialysis units, extracorporeal membrane oxygenation (ECMO), device left ventricular support (LVS) and similar methods, arteriovenous fistulas, vascular and heart valve prostheses.

Moreover, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are useful in the treatment and/or prevention of disorders involving the formation of microclots or fibrin deposits in cerebral blood vessels, which can lead to disorders associated with dementia, such as vascular dementia or Alzheimer's disease. In this case, the clot can provoke a disorder both through occlusion and through the introduction of additional factors associated with the disease.

Moreover, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, can be used for the prevention and/or treatment of thrombotic and/or thromboembolic complications, for example, venous thromboembolism in patients with cancer, including those who have undergone major surgery intervention or chemotherapy or radiation therapy. In the context of the present invention, the term “pulmonary hypertension” includes pulmonary arterial hypertension, pulmonary hypertension associated with left heart disorders, pulmonary hypertension associated with pulmonary disorders and/or hypoxia, and pulmonary hypertension due to chronic thromboembolism (CTPE).

In addition, the binding molecules of the present invention, preferably represented by antibodies and antigen-binding fragments thereof, are also useful in the treatment and/or prevention of systemic inflammation and generalized thrombohemorrhagic syndrome (DIC) secondary to infectious disease and/or systemic inflammatory response syndrome (SIRS), organ and multiple organ failure due to sepsis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), septic shock and/or organ failure due to sepsis. During the development of infection, generalized activation of the contact and coagulation systems may occur,

which will provoke symptomatic DIC (with or without debilitating coagulopathy) with (micro)thrombosis in various organs, as well as secondary hemorrhagic complications. Moreover, endothelial damage may occur with increased vascular permeability and leakage of fluid and proteins into the extravasal space. As the infection progresses, single organ failure may occur (eg, kidney failure, liver failure, respiratory failure, central nervous system failure and cardiovascular failure, etc.) or multiple organ failure may occur. In the case of DIC, extensive activation of the coagulation system occurs on the surface of damaged endothelial cells, the surfaces of foreign bodies, or intersecting extravascular tissue. As a result, coagulation occurs in small vessels of various organs with the development of hypoxia and subsequent functional failure of the organs. A secondary effect is the depletion of clotting factors (wasting coagulopathy), such as proteins C, S, Z, FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, as well as fibrinogen (FI) and platelets, leading to to a decrease in control over the homeostatic balance of the blood and can provoke severe and even fatal bleeding.

The binding molecules of the present invention, preferably antibodies and antigen-binding fragments thereof, are also useful in the primary prevention of thrombotic or thromboembolic disorders and/or inflammatory disorders and/or disorders associated with increased vascular permeability in patients whose gene mutations lead to increased enzyme activity or increased levels of zymogens, the fact of which is determined by appropriate tests/measurements of enzyme activity or zymogen concentrations.

In addition, the binding molecules of the present invention, preferably antibodies and antigen-binding fragments thereof, can also be used to prevent blood clotting in the laboratory, for example to protect organs for transplantation from damage caused by clot formation, as well as to protect the organ recipient against thromboembolism caused by this organ, to preserve blood and plasma products, to clean/pre-treat catheters and other medical ancillary devices and instruments, to cover synthetic surfaces of medical ancillary devices and instruments used indoors or in the laboratory, or to provide biological samples, which may include FXI/FXIa.

The present invention further discloses the use of binding molecules, preferably represented by antibodies and antigen binding fragments thereof, for the treatment and/or prevention of the disorders described above.

The present invention further discloses the use of binding molecules, preferably represented by antibodies and antigen-binding fragments thereof, for the production of a medicinal product for the purpose of treating and/or preventing disorders, especially those described herein, preferably for the production of a medicinal product for the treatment and/or prevention of thrombotic or thromboembolic disorders.

The present invention further discloses a method of treating and/or preventing disorders, particularly those described herein, using a therapeutically effective amount of the binding molecules of the present invention, preferably antibodies and antigen binding fragments thereof.

The present invention further discloses binding molecules, preferably represented by antibodies and antigen-binding fragments thereof, for use in a method of treating and/or preventing disorders, particularly those provided herein, using a therapeutically effective amount of the binding molecules of the present invention, preferably represented by antibodies and their antigen-binding fragments.

The present invention further discloses methods of treating thrombotic or thromboembolic disorders in humans and/or animals by administering a therapeutically effective amount of at least one binding molecule of the present invention, preferably an antibody and an antigen binding fragment thereof, or a pharmaceutical composition of the present invention. The present invention further discloses a method of inhibiting blood coagulation, platelet aggregation and/or thrombosis in patients by administering a therapeutically effective amount of at least one binding molecule of the present invention, preferably an antibody and an antigen binding fragment thereof, or a pharmaceutical composition of the present invention.

Gene therapy

Provided hereinafter is a transfer vector for use in gene therapy for mammals, comprising a polynucleotide as provided herein, and methods for treating or preventing diseases comprising introducing a foreign nucleic acid as described herein to enable expression of that foreign nucleic acid, as well as prevention or treatment of disease. In one embodiment, nucleic acid molecules encoding both the heavy and light chains are administered to a patient. In a preferred embodiment, the nucleic acid molecules are introduced so that they are stably integrated into the chromosomes of B lymphocytes, since these cells specialize in producing antibodies. In one embodiment, B cells are transfected or infected in the laboratory and then re-transplanted into a patient in need. In another embodiment, B lymphocytes are infected within the body with a recombinant virus known to infect the cell type in question.

In a preferred embodiment, a method of gene therapy comprises administering an isolated nucleic acid molecule encoding a heavy chain, or an anti-FXI antibody antigen binding portion thereof, as provided herein, and causing the nucleic acid molecule to be expressed. In another preferred embodiment, a method of gene therapy includes administering an isolated nucleic acid molecule encoding a light chain, or an anti-FXI antibody antigen binding portion thereof, as provided herein, and causing the nucleic acid molecule to be expressed. In another embodiment, a method of gene therapy includes administering an isolated heavy chain encoding nucleic acid molecule or antigen binding portion thereof and an isolated light chain encoding nucleic acid molecule or antigen binding portion thereof to an anti-FXI antibody as provided herein, and ensuring the expression of this nucleic acid molecule.

The specific conditions for uptake of foreign nucleic acid are well known in the art. These include, but are not limited to, retrovirus infection, adenovirus infection, transformation with plasmids, transformation with liposomes containing foreign nucleic acid, biolistic delivery of nucleic acid (i.e., loading the nucleic acid onto gold or other metal particles and launching or injecting them into cells) , infection with adeno-associated virus and Epstein-Barr virus. Collectively, for the purposes of the present invention, these may be referred to as "expression vectors". Expression vectors can be either extrachromosomal vectors or vectors that integrate into the host genome. In general, these expression vectors include a transcriptional and translational regulatory nucleic acid operably linked to a foreign nucleic acid. In general, transcriptional and translational control sequences may include, but are not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer and activator sequences. In a preferred embodiment, the regulatory sequences include a promoter sequence and transcription start and stop sequences. In addition, the expression vector may include additional elements. For example, in the case of integrating expression vectors, the latter contain at least one sequence homologous to the host cell genome, and preferably two homologous sequences flanking the expression construct. The integrating vector can be targeted to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Designs for integrating vectors are well known in the art.

Experiments

Starting with the mouse anti-FXI antibody 14E11, new humanized antibodies have been developed. The potential antibodies were tested for their FXI-binding activity, as determined by a competitive ELISA assay, as well as their ability to inhibit the conversion of FXI to its active form.

The characteristics of an example antibody represented by AB023 were established. Studies were conducted using AB023 to confirm that binding and anticoagulant properties were maintained and comparable after humanization of 14E11 by CDR grafting. In vivo binding studies showed that AB023 binds with high affinity to both murine and human FXI (0.16 and 3.2 nm, respectively), but with lower affinity than the murine monoclonal antibody 14E11. In vivo aPTT assays confirmed that the anticoagulant effect was maintained after humanization as AB023 prolonged aPTT in human, baboon, cynomolgus and rat plasma in a concentration-dependent manner.

Notably, several in-body tests revealed some differences between 14E11 and AB023. First, AB023 was able to inhibit FXI self-activation in the presence of both dextran sulfate and DNA in a concentration-dependent manner, whereas 14E11 failed to do so at all concentrations tested. Second, AB023 inhibited the activation of FXII by FXIa in a concentration-dependent manner, unlike 14E11. Finally, AB023 was effective in inhibiting the activation of human FXIIa, related to human FXI, in the presence of HK and dextran sulfate.

After humanization (AB023), the antithrombotic effects observed in 14E11 were maintained (Cheng K., Tucker E.I., Pine M.S., Sisler I., Matafonov A., Sun M.F., White-Adams T.K. , Smith SA, Henson SR, McCarthy OJ, Renne T, Gruber A, Gailani D. J Blood Nov 11 2010 116(19):3981-3989; .I., Verbout NG, Leung PI, Hirst S, McCarthy OJ, Gailani D, Gruber A. Blood 17 May 2012 119(20):4762-8) , which was demonstrated both in a mouse model of arterial thrombosis and in a baboon model of arterial and venous thrombosis. In a mouse model of arterial thrombosis, AB023 prevented FeCl ₃ -induced carotid artery occlusion comparable to general FXI deficiency, but the effect was not as potent as that obtained with the same dose of mouse monoclonal antibody (14E11). Relative to the established baboon model of thrombosis, using thrombus grafting and an expansion chamber to simulate arterial or venous flow, respectively, administration of a low dose of AB023 (0.2 mg/kg IV) slightly reduced the rate of platelet accumulation within vascular grafts compared with control groups, while how almost complete inhibition of platelet accumulation was achieved in the expansion chamber. Fibrin deposition was also lower in both arterial and venous thrombosis. It is noteworthy that when using a thrombus-forming graft without an expansion chamber, the administration of 1.0 mg/kg AB023 intravenously led to both a decrease in the amount of platelet deposits and fibrin in the vascular graft segment itself, as well as to the prevention of the formation of a thrombus “tail” downstream, which demonstrated that AB023, like its murine precursor, 14E11, prevented venous thrombosis at all doses tested. In addition, the study suggests that, apparently, AB023 at higher doses also led to attenuation of arterial thrombosis, as evidenced by a reduction in platelet and fibrin counts in the thrombus-forming collagen-coated graft.

The anti-inflammatory effects of AB023 were tested in a baboon model of sepsis and, as in a previous trial in a mouse infection model with 14E11 (Silasi R. et al., article Inhibition of contact-mediated activation of factor XI protects baboons against S aureus-induced organ damage and death (“Inhibition of contact-mediated factor XI activation protects baboons from organ damage and death by Staphylococcus aureus.” Journal of Blood Advances, 2019; 3:658-669, data not shown; , article Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis ("Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving survival in mouse polymicrobial sepsis"). Journal of Blood. May 17, 2012. (20):4762-8). All tests conducted to compare the activity of AB023 and 14E11 showed identical effects. The anticoagulant effect of AB023 was also evaluated in healthy human subjects (K. U. Lorenz et al., Contact Activation Inhibitor and Factor XI Antibody, AB023, Produces Safe, Dose-Dependent Anticoagulation in a Phase 1 First-In-Human Trial contact activation and factor XI antibody, AB023, provides safe, dose-dependent anticoagulation in Phase 1 of first use in humans." Journal Arterioscler Thromb Vase Biol Apr 2019 39(4):799-809), and data (not presented) showed that AB023 was safe and also provided a dose-dependent anticoagulant effect.

Possible cross-reactivity of AB023 was assessed using frozen sections of human tissue and was not detected.

Taken together, the results of the studies conducted on AB023 demonstrate the anticoagulating and antithrombotic properties of AB023. Additional studies have shown a low risk of haemostatic impairment, low potential for cross-reactivity and adverse effects, low potential for immune activation and immunogenicity, and low potential for neuro- and cardiotoxic effects (data not shown).

Characterization of mouse antibody 14E11

As described in the 14E11 patents and other publications cited herein, studies have determined the binding and anticoagulant properties of the mouse monoclonal anti-FXI antibody 14E11. Briefly, binding and anticoagulant properties were tested in the laboratory. The results of the aPTT assay showed that 14E11 prolonged the aPTT of murine (O) or human (∙) plasma (FIG. 1), with maximum inhibition occurring at 25-50 nM, which is within the concentration range of FXI in human plasma (2,045 nM). The prolongation of clotting time was approximately half that of human and murine FXI-deficient plasma. 14E11 had no negative effect on human plasma clotting time (data not shown).

14E11 was found to bind and recognize one colored band of the predicted size of the FXI homodimer (-160 kDa) in normal mouse and human plasma, as well as recombinant mouse FXI plasma (FIG. 2-A-C), but binding in FXI-deficient plasma was not observed. Solid-phase binding assays revealed the binding affinity of 14E11 relative to murine and human FXI, as well as FXIa (observed K _d ~2-3 pM, FIG. 2D). For most mammals, FXI is a homodimer of two 80 kDa subunits, each of which contains four apple domains (A1-A4) and a protease domain. Prekallikrein (PK), a monomeric homologue of FXI, has a structure almost identical to the FXI monomer. Human FXI/PK fusion proteins were used to demonstrate the requirement of the A2 domain for 14E11 binding to FXI (FIG. 2E). Western blots using individual tPA-bound FXI apple domains show that the 14E11 binding site is likely located entirely within the A2 domain (FIG. 2F).

As shown, mouse monoclonal antibody 14E11 binds to the apple domain 2 (A2) of human and mouse FXI and inhibits activation of FXI by FXIIa and downstream thrombin generation, as well as self-activation of FXI. 14E11 binds to FXI in many different species and can prolong aPTT in mouse, human, baboon, rabbit, rat, pig, and rhesus monkey plasma (data not shown).

Humanization of mouse antibody 14E11

Humanizing the mouse monoclonal antibody 14E11 was carried out at Abzena (formerly known as Antitope Limited, Cambridge, UK) using hypervariable region grafting (CDR) technology (O'Brien S. and Jones T., 2001. Humanizing Antibodies by CDR Grafting. Humanization of Antibodies by CDR Grafting" References: Kontermann R., Diebel S. (eds.) Antibody Engineering (Springer Lab Manuals, Berlin, Heidelberg). In detail, to select germline acceptor family species, mouse antibody CDR residues were identified and annotated according to Cabot nomenclature (for details, see http://www.bioinf.org.Uk/abs/#kabatnum). Canonical heavy and light chain CDR structures were determined using human germline genes according to the CDR homology approach to antibody humanization, and human germline acceptor scaffolds with similar canonical structures were selected (O'Brien and Jones, 2001; Hwang , 2005). Human germline acceptor scaffolds with similar canonical structures were selected.

The 14-E11 variable region (V) genes were sequenced from RNA isolated from the 14E11 hybridoma line, and these sequences were used to generate a chimeric antibody and to develop a number of germline humanized antibody variants. Amino acids in the 14E11 variable region scaffolds that can support antibody binding properties were identified in silico by generating chimeric antibody structural models consisting of 14E11 variable domains and human IgG4 immunoglobulin and kappa light chains using the Swiss PDB, and they were marked for inclusion in one or more CDR-grafted variant antibodies. Humanized region V genes were designed from human germline sequences with homology closest to murine sequences and were also constructed through gene synthesis. The humanized heavy chain variable region (VH) variant antibodies were then cloned into a first vector containing the human immunoglobulin heavy chain common region (CH) 1-3 genes of the IgG4 subclass with a modified S241P hinge region using the restriction enzymes Hind PI and MluI. The humanized light chain variable region (Vk) variant antibodies were cloned into a second vector containing the human common kappa gene (Ck) using the restriction enzymes BssH I and BamHI. Also, by cloning mouse variable regions into similar vectors, a chimeric antibody was obtained. The humanized and chimeric antibodies were stably expressed in NSO cells and tested for binding to the target antigen (recombinant human FXI) in a competitive ELISA against 14E11 (data not shown). In addition, the anticoagulant properties of the humanized antibodies were tested using aPTT assay (FIG. 3). The results of these assays indicated that the preferred AB023 antibody candidate was the VH3/Vk3 variant (clone 3G3), and a stable production cell line was established at Abzena using CHO™ technology.

Cells from a stable cell line can be used for introduction into a production bioreactor. For example, cells can be cultured using a fed-batch process and harvested, for example, after 14 days. The cells can then be purified through one or more steps of column chromatography, virus removal, concentration and diafiltration. Once finalized in buffer, the antibody, ie AB023, can be filtered and stored frozen as a lyophilized product (eg 15 mg/ml after reconstitution), but this is not necessary.

Determination of characteristics of AB023

The characteristics of AB023 were determined using a number of analytical methods. These methods have been used to understand the primary, partial conformational structures, binding, and post-translational changes of the protein. Analysis techniques used include peptide mapping, mass spectrometry (MS), capillary on-chip electrophoresis, capillary isoelectric focusing (cIEF), size exclusion chromatography (SEC), oligosaccharide mapping, fluorescence, circular dichroism (CD), and differential scanning calorimetry ( DSC), each of which is well known in the art.

The molecular mass of AB023, 146,560 Da, was determined by MS to determine the structure and post-translational changes, as well as to determine the integrity of the molecule. The molecular weight of the heavy and light chains (49.890 and 23.401 Da, respectively) was determined after reduction of disulfide bonds in the molecule. The differences compared to the theoretical molecular weight (144.020 Da) were due to post-translational changes, especially glycolysis.

Binding affinity

The binding affinity of the humanized monoclonal antibody AB023 for human and murine factor XI (FXI) was determined using solid-phase binding assays. In addition, the binding affinity of AB023 relative to activated human FXI (FXIa) was assessed for both AB023 and 14E11. Briefly, for this solid-phase binding assay, both 14E11 and AB023 were biotinylated using the Sulfo-NHS EZ-Link™ Biotinylation Kit (ThermoFisher Scientific, Waltham, MA) according to the instructions. Microtiter plates were coated with FXI or FXIa (2 mg/ml, 100 ml/well) in 50 mmol/l Na ₂ CO ₃ , pH 9.6 and incubated overnight at 4°C on Immulon 2HB microtiter plates (Thermo Scientific). The wells were blocked with 150 pl of sodium phosphate buffer solution (PBS) with bovine serum albumin (BSA) 2% for 1 hour at room temperature. Then one hundred milliliters of biotinylated 14E11 or AB023 (0.7-6.7 pmol/l) was added ) in HEPES 90 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.2, NaCl 100 mmol/L, BSA 0.1% with Tween 20 (HBS [HEPES buffered solution]) and performed incubation for 90 minutes at room temperature. After washing with PBS 0.1% Tween 20 (PBS-T), 100 pL of ThermoFisher Scientific streptavidin-horseradish peroxidase, diluted 1:8000 in HBS) was added and incubated at room temperature for 90 minutes. After washing with PBS-T, 100 pl of substrate solution (12 ml citric acid 30 mmol/l; Na ₂ HPO ₄ 100 mmol/l, pH 5.0; and 1 tablet of o-phenylenediamine dihydrochloride, 12 pl H ₂ O ₂ 30%) was added. Reactions were stopped after 10 min with 50 pl H ₂ SO ₄ 2.5 M. Optical density at 495 nm was measured using a SpectroMax 340 microplate reader (Molecular Devices, San Jose, CA). Data analysis was performed by linear regression and the observed _Kd value (equilibrium dissociation constant) was calculated at the concentration of AB023 required to achieve half-maximal binding at equilibrium using GraphPad Prism (ver. 5.0).

The binding affinity of 14E11 and AB023 for murine and human FXI was determined using solid-phase binding assays. Binding of 14E11 to murine FXI had lower affinity (as previously found) (observed _Kd value ~0.07 nM versus ~2-3 pM). 14E11 also showed lower binding affinity for human FXI and FXla than for mouse FXI (observed K _d value ~0.39 nM and 0.20 nM, respectively) (Table 1). AB023 also demonstrated nanomolar binding affinities for murine and human FXI (observed mFXI ~0.16 nM, observed _hFXI Kd ~3.2 nM, and _hFXIa observed Kd ~1.3 nM), although the affinity binding for both human and mouse FXI was lower than that of 14E11 (FIG. 4C, Table 1). Taken together, these results confirm that the 14E11 antibody retained high binding affinity to the same domain after humanization.

Table 1: Observed _Kd values for 14E11 and AB023

Observed _Kd value (nM) Human FXI Human FXla Myshin. FXI AB023 3.2 1.3 0.16 14E11 0.39 0.20 0.07

Confirmation of A2 domain binding

To confirm the binding of AB023 to apple domain 2 (A2) of FXI, immunoblots were performed using standard techniques (Cheng et al., 2010). In FIG. 4A shows immunoblots of human recombinant FXI (track 1) and human FXI in which the A1, A2, A3 or A4 domain was replaced with the corresponding prekallikrein (PK) domain, separated by electrophoresis and immunoblotted with AB023, and also confirms that AB023 , like 14E11, binds apple domain 2 (A2) of FXI. Fusion proteins were also produced in which each individual apple domain from FXI was fused to tissue plasma activator (tPA). The fusion proteins were then separated by electrophoresis and immunoblotted with AB023. In FIG. 4B shows the binding of AB023 to the A2 domain of FXI.

Inhibition of FXIIa activation by FXI

A laboratory assay was performed to determine whether AB023 retained its ability to inhibit FXIIa activation within FXI after humanization of 14E11. Briefly, FXI (30 nmol/L) was incubated with 0.5 nmol/L a-FXIIa and dextran sulfate (0.1 mg/mL) at 37°C in 25 mmol/L HEPES, pH 7.4, 150 mmol/L. l NaCl and BSA 0.1% with or without AB023 (0-300 nmol/l). After incubation for 30 min, samples were removed and screened with polybrene (6 mg/ml) to neutralize dextran sulfate and CTI (50 mg/ml) to

inactivation of FXIIa, after which FXIa generation was calculated according to measurements of the hydrolysis rate of S-2366 at 405 nm, and the hydrolysis rate of S-2366 was converted to FXIa concentration using a calibration curve. In FIG. 4D shows that AB023 inhibits the activation of FXlIa in FXI in a concentration-dependent manner. Like 14E11, AB023 did not inhibit thrombin-mediated activation of FXI (FIG. 4E). To determine this fact, an in vivo assay was performed in which FXI (30 nM) was incubated with 2.5 nM α-thrombin and dextran sulfate (0.1 mg/ml) at 37°C in 25 mM HEPES, pH 7.4 , 150 mM NaCl and 0.1% BSA with or without AB023 (30 nM). After 0, 5, 15, 30 or 60 minutes of incubation, samples were removed and screened with polybrene (6 mg/ml) to neutralize dextran sulfate and hirudin (10 U/ml) to inactivate thrombin, after which FXIa generation was calculated according to measurements of the hydrolysis rate of S-2366 at a wavelength of 405 nm. The hydrolysis rate of S-2366 was converted to FXIa concentration using a calibration curve.

Prolongation of activated partial thromboplastin time (aPTT)

The ability of AB023 to prolong aPTT in the plasma of certain mammals was tested. Mixed human, baboon, rat and cynomolgus plasma (90 pL from three separate subjects), anticoagulated with sodium citrate 0.38%, was mixed with 10 pL AB023 (0.183-1,500 mg/L) or control group ( PBS) and left to incubate at room temperature for 5 minutes. Forty microliters of plasma/antibody mixture was then incubated with 40 pl of aPTT reagent (SynthASil, #0020006800, Instrumentation Laboratory, Bedford, MA) for 3 minutes at 37°C. After incubation, 40 ml CaCl ₂ were added and clotting times were determined using a KC4™ analyzer (TCoag, Bree, Ireland). Each sample was subjected to two separate analyses. For each of the species tested, AB023 prolonged aPTT in a concentration-dependent manner in all of them (FIG. 4F). aPTT data were expressed as fold change from baseline plotted as a function of Boolean AB023 concentration.

Taken together, these data confirm the fact that the properties of 14E11 are retained after humanization.

Formation of an anticoagulant complex

These experiments examined the presence of a complex formed between a monoclonal antibody or any binding fragment, variant or derivative thereof and the FXI represented by the anticoagulant using activated partial thromboplastin time (aPTT). In other examples (FIG. 4), it is well established that FXI instantly forms a stable immune complex with the high affinity A2 domain anti-FXI antibody AB023. In this case, two separate experiments were conducted using FXI-deficient plasma obtained from FXI-deficient human subjects (George King Bio-medical Inc., Overland Park, Kansas), the results of which are presented in FIG. 5.

In the first experiment, the initial aPTT value was measured in FXI-deficient plasma. Forty microliters of plasma were incubated with 40 pL of aPTT reagent (SynthASil, #0020006800, Instrumentation Laboratory, Bedford, MA) for 3 minutes at 37°C. After incubation, 40 pl CaCl ₂ was added and clotting time was determined using a KC4™ analyzer (TCoag, Bree, Ireland). Each sample was analyzed eight times. The mean baseline aPTT for FXI-deficient plasma was 118.5 seconds. After initial measurements, FXI (Enzyme Research Laboratories, no. HCFXI-1111) was added to 400 pL of FXI-deficient plasma to obtain a final concentration of 10 mg/ml. FXI/FXI-deficient plasma was incubated at room temperature for 5 minutes and the aPTT value was measured on 200 pL of the mixture as presented above. In FIG. 5 shows that addition of purified FXI (molecular weight 160 kDa) to FXI-deficient plasma predictably reduces clotting time to 32.3 seconds, since FXI is a zymogen of the procoagulant enzyme, FXIa. Finally, AB023 was added to the remaining 200 pL of FXI-deficient plasma + 10 mg/mL FXI to obtain a final concentration of 100 mg/mL (10-fold excess antibody). This mixture was incubated at room temperature for 5 min, and the aPTT value was measured as presented above. Addition of antibody AB023 (molecular weight 146 kDa) with a 10-fold excess molecular weight of added FXI relative to FXI-deficient plasma resulted in an approximately 2-fold prolongation of aPTT to an average of 66.6 seconds, which is consistent with laboratory experiments (FIG. 4) and about a coagulant in the body (Table 2), during which the concentration of AB023 was excessive compared to FXI. Addition of AB023 to FXI-containing normal plasma did not prolong aPTT to the same extent as observed in FXI-deficient plasma. This is most likely due to the fact that the procoagulant activity of FXIa obtained from the aPTT assay is not inhibited by AB023 (FIG. 4E).

In the second experiment, AB023 was added to 400 pL of FXI-deficient plasma to obtain a final concentration of 100 mg/ml, incubation was performed at room temperature for 5 min, and the aPTT value was measured on 200 pL of the mixture as presented above. Addition of AB023 did not result in a change in clotting time (mean aPTT = 117.7 seconds compared with mean aPTT of FXI-deficient plasma of 118.5 seconds), indicating that AB023 itself is not an anticoagulant without complexation with FXI. Finally, FXI was added to the FXI-deficient plasma/AB023 mixture (100 mg/ml) with a final FXI concentration of 10 mg/ml (10-fold excess of antibody relative to FXI to ensure transfer of FXI to the immune complex). The mixture was incubated at room temperature for 5 minutes and the aPTT value was measured as presented above. The addition of FXI reduced aPTT from 117.7 seconds to an average of 59.1 seconds (FIG. 5).

These experiments indicate that 1) addition of FXI to FXI-deficient plasma provides a procoagulant effect, 2) AB023 by itself does not provide an anticoagulant effect, and 3) the immune complex formed between FXI and AB023 is an anticoagulant but does not provide an equivalent anticoagulant effect. effect similar to FXI deficiency.

Determination of characteristics of the anticoagulating effect

Mouse model of arterial thrombosis

Using an established experimental mouse model of arterial thrombosis, the antithrombotic properties of AB023 were determined as previously done for 14E11 (Cheng et al., 2010). Briefly, mice were anesthetized by IP injection of 50 mg/kg pentobarbital. The right common carotid artery was exposed and a Doppler flow probe was placed. AB023 (1.0 mg/kg, intravenous) was injected into the internal jugular vein over 15 min. to the lesion and induced thrombus formation by placing two sheets of 1 x 1.5 mm filter paper saturated with FeCl ₃ (2.5% to 10% solution) on opposite sides of the artery for 3 min. After removal of the pads, the area was rinsed with sodium phosphate buffer and flow was monitored for 30 min.

Intravenous infusion of 1.0 mg/kg AB023 into wild mice protected mice from carotid artery occlusion induced by 3.5%, 5.0% and 7.5% FeCl ₃ (FIG. 6). These results are comparable to those from FXI-/- mice. The results are consistent with the proposal that FXI is activated by FXIIa in this thrombosis model and demonstrate that the antithrombotic effect was maintained even in mice after humanization of the 14E11 antibody.

PK/PD in baboon model

This study assessed plasma concentrations of AB023 over time and correlated the effects of AB023 on aPTT. Six male baboons were administered 1.0 mg/kg AB023 on day 1 via a single intravenous bolus injection or a single subcutaneous injection. A blood sample was collected at several time points after administration and anticoagulated with sodium citrate 0.32% (1/10 volume). One aliquot was used to determine the plasma concentration of AB023, and the other aliquot was used to measure aPTT. To measure aPTT, plasma (40 pL) was incubated with 40 pL aPTT reagent for 3 minutes at 37°C. After a 3-min incubation at 37°C, 40 pL CaCl2 was added and clotting time was determined using a KC4 Delta™ analyzer (Tcoag Ireland Ltd, Wicklow, Ireland). AB023 plasma concentrations were determined using a partially validated enzyme-linked immunosorbent assay (ELISA) to detect free AB023 in plasma.

Results showed a rapid and immediate prolongation of aPTT and approximately 2-fold greater than baseline aPTT prolongation for at least 1 week (168 h) in all animals. In FIG. 7 shows the relationship between plasma concentrations of AB023 and aPTT. Similarly, administration of 1.0 mg/kg AB023 subcutaneously also resulted in a rapid and immediate prolongation of aPTT. However, after subcutaneous administration, aPTT remained prolonged, approximately 2-fold above baseline, for at least 2 weeks (336 hours) (not shown). These studies indicate that prolongation of aPTT is closely associated with exposure to AB023.

Prophylactic treatment in baboon models of arterial and venous thrombosis

These studies determined the antithrombotic effect of inhibition of FXI activation by FXIIa using the humanized monoclonal antibody AB023 in an established experimental primate model of arterial and venous thrombosis.

Non-terminal studies were performed on young male baboons (Papio anubis) weighing 9-13 kg. All studies were approved by the Institutional Animal Care and Use Committee. Each baboon had a closed, surgically placed, permanent exteriorized arteriovenous (AV) shunt connecting the femoral artery and vein, as described elsewhere (Henson SR, Griffin JH, Harker LA, et al. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates (J Clin Invest, October 1993:92(4):2003-12). Experiments were carried out on awake animals without anticoagulation, which were kept in a sitting position. Anxiety was controlled with a low dose of ketamine, not exceeding 2 mg/kg intramuscularly, up to once an hour. Experimental treatments were performed and blood samples were collected through a silicone rubber extension tube inserted into the AV shunt during the experiments. The red blood cell count and hematocrit were measured daily in each animal, including before and after the experiments, and the estimated blood loss did not exceed 4% of the total blood volume on any day of the experiments. Multiple experiments were carried out on the same animals on different days with and without treatment. Only animals with shunts that had good unrestricted initial flow (>250 ml/min) were used in replicate experiments.

In this experimental thrombosis model, acute local thrombosis was initiated by inserting a short prosthetic modified segment of a thrombus graft within the AV shunt as previously described (Henson SR, Griffin JH, Harker LA, et al Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates J Clin Invest October 1993:92(4):2003-12; .B., Marzek U.M., Krupsky V. et al. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons (“Prevention of heparin-resistant arterial thrombus formation with hirudin in baboons”) Journal Blood, March 1 1991:77(5):1006-12). Because vascular injury exposes the bloodstream to an extracellular matrix that contains structural proteins such as collagen that trigger platelet activation and FXI1, we made the graft segments thrombus-forming with an immobilized collagen coating. The lumens of 20 mm long clinical vascular grafts (expanded polytetrafluoroethylene, ePTFE, Gore-Tex; W. L. Gore and Associates, Flagstaff, AZ) with an internal diameter (i.d.) of 4 mm were covered with equine type I collagen (CHRONO-LOG Corporation, Havertown , Pennsylvania) for 15 min. and dried overnight under sterile air flow. This method ensures uniform collagen coverage within the graft lumen as determined by scanning electron microscopy (data not shown). Segments of collagen-coated (platelet-forming) grafts were embedded in silicone rubber tubing and placed in AV shunts in baboons throughout the 60-minute acute thrombosis experiments.

Shunt flow in unanticoagulated baboons consistently causes acute thrombosis in segments of collagen-coated ePTFE grafts. During each experiment, the maximum blood flow rate through the grafts (about 250 ml/min) was limited by the distal clamp to 100 ml/min, providing an average initial wall shear rate of 265 ^s-1 in 4-mm grafts. Flow rate was continuously monitored using an ultrasonic graft flow meter (Transonics Systems, Ithaca, NY). These 4-mm diameter grafts did not occlude, and the pulsatile flow rate remained at 100 mL/min during thrombus formation. The graft segment (and thrombus) was removed from the shunt after 60 or 90 min, and the permanent shunt was restored after each experiment. Because thrombus formation was found to propagate downstream from the collagen surface over time, platelet accumulation was also measured within a 10-cm arteriovenous shunt area immediately distal to the graft. This model of thrombus growth on the proximal collagen surface (at the “head” of the thrombus) with the thrombus spreading distally to the collagen segment (forming the “tail” of the thrombus).

Thrombus formation was assessed during trials lasting 60–90 minutes using a gamma quantitation camera imaging radiolabeled platelets in the graft segment and further assessed by determining radiolabeled fibrin deposition at the end point after completion of each trial as described ( 1). To quantify platelet storage, autologous baboon platelets were labeled with 1 mCi of ¹¹¹ In, then reintroduced to the animals and allowed to circulate for at least 1 hour and up to 4 days before testing. The accumulation of platelet-associated radioactivity on the grafts was determined at 5-min intervals. using a GE-400A-61 gamma scintillation camera coupled to the NuQuest InteCam computer system, as described for other devices in the permanent AV shunt model (Gruber and Henson, Blood 2003; 102:953-955; Gruber et al ., Thromb Res 2007; 119: 121-127; J Clin Invest 1993; Tucker et al 2009; 944). After 60-90 minutes, the graft was removed, washed, dried, and kept refrigerated for subsequent assessment of ^125I -fibrin content as described previously (Gruber and Henson, Blood 2003; 102:953-955; Henson et al., J Clin Invest 1993;92:2003-2012). Briefly, homologous ¹²⁵ I-labeled baboon fibrinogen (5–25 mg, 4 mCi, coagulability >90%) was administered intravenously over 10 min. before each study. Incorporation of labeled fibrinogen/fibrin into the thrombus was assessed using a gamma counter (Wizard-3, PerkinElmer, Shelton, CT) at least 30 days after removal of the graft from the AV shunt to ensure degradation of ¹¹¹ In attached to the platelets. ¹²⁵ I-radioactivity deposited in the graft was measured and compared with the radioactivity of clotting fibrin (fibrinogen) in plasma samples collected during the primary study.

In the first experiment (FIG. 8), a 4 mm diameter collagen-coated vascular graft segment with a 9 mm diameter silicon chamber to simulate venous blood flow was positioned 20 mm downstream and used to test the antithrombotic effect of AB023 (0.2 mg/kg, administered intravenously 1 hour before the experiment). A total of 4 non-terminal studies were conducted using 2 young male baboons and the data were compared with data from control groups used in the identical trial 14E11 (n = 7 trials/group with 4 animals for the control group).

The results of this study are shown in FIG. 8A-D. The panels to the left of the figures represent platelet deposition, and the panels to the right represent terminal fibrin deposition. The results of this study showed that the rate of platelet accumulation in vascular grafts was slightly lower in animals that received AB023 than in control groups that did not receive AB023 (FIG. 8A), while almost complete inhibition of platelet accumulation was observed. achieved in the expansion chamber of baboons injected with AB023 (FIG. 8C). These data are similar to those observed after administration of 14E11 and demonstrate that the antithrombotic effect of AB023 is comparable to the murine precursor 14E11. Fibrin deposition was also lower in both arterial and venous thrombosis (FIG. 8B and D).

In the second set of trials, acute local thrombosis was initiated by prosthetic segments of collagen-coated vascular grafts placed in permanent AV shunts for 60 minutes in 8 nonterminal trials on 4 young male baboons. Baboons were administered either AB023 (1.0 mg/kg intravenous injection) or saline (control group, intravenous). The antithrombotic effects of AB023 were assessed 24 hours after intravenous administration.

Platelet accumulation in collagen-coated vascular grafts was lower in animals treated with AB023 (FIG. 9A), and in the region 10 cm downstream of the graft (the “tail”), almost complete inhibition of platelet accumulation was achieved by AB023 (FIG. .9B). Overall, after pre-administration of AB023 (1.0 mg/kg IV), total platelet accumulation (platelet accumulation in the thrombus-forming tail graft) was reduced (FIG. 9C). These studies showed that AB023, like its murine precursor, provides antithrombotic effects in primate models of thrombosis. In addition, at an increased dose of 1.0 mg/kg, AB023 was effective in attenuating the rate of arterial thrombus formation in the thrombotic graft as well as in the tail. Fibrin deposition in both the graft and the tail decreased after the administration of AB023.

During the study, aPTT was monitored, which was prolonged after administration of AB023 by infusion and remained elevated 24 hours after administration in the AB023 treatment group (30 and 60 min of experiment, Table 2), while in the control group there were no changes aPTT were not observed. PT measurements were also taken throughout the study. Compared with control groups, AB023 did not lead to a change in PT (Table 2).

Table 2: aPTT and PT from a baboon model of vascular thrombosis

aPTT (c) thirty 60 Prev. research min min 33.2 33.4 32.7 Counter. group ±3.2 ±3.2 ±1.8 78.4 78.5 78.7 AB023 ±11.0 ±12.4 ±12.9 PT (c) 60 24 hours Prev. research min 8.9 8.8 - Counter. group ±0.1 ±0.2 9.2 9.0 9.1 AB023 ±0.1 ±0.4 ±0.4

Data are expressed as mean ± standard deviation

The present data indicate that the humanized antibody AB023 retained its anticoagulant properties and resulted in a reduction in experimental thrombosis events in both murine and non-human primate models of thrombosis.

Comparison of 14E11 and AB023

After humanization, the anticoagulant effect of AB023 was compared with 14E11 using the aPTT assay as presented above, which showed that, compared with 14E11, AB023 resulted in prolongation of aPTT in human and baboon plasma to a similar extent (FIG. 3).

The effects of 14E11 or AB023 on contact pathway activation, self-activation of FXI, and reciprocal activation of FXII by FXIa were also examined. Briefly, to compare the effects of 14E11 and AB023 on contact pathway activation, incubation of FXI (80 nM) in HEPES (20 mM HEPES, pH 7.4), 100 mM NaCl, PEG-8000 0.1% at 37°C for 15 min with or without 1,280 nM 14E11 or 800 nM AB023. At time zero, FXIIa, HK, and dextran sulfate (all in HEPES) were added to obtain final concentrations of FXI (30 nM), FXIIa (5 nM), HK (30 nM), dextran sulfate (0.1 mg/ ml), as well as 14E11 at a concentration of 480 nM or AB023 at a concentration of 300 nM. At various time points, corn trypsin inhibitor (CTI) (500 nM final concentration) and 20 mg/mL polybrene (final) were added to 5 pL aliquots, and the optical density ΔOD ₄₀₅ nm was monitored on a microplate reader in the presence of S-2366 250 mM.

To compare the effects of 14E11 and AB023 on FXIa self-activation, purified human FXI was mixed with DXS (0.1 pM) in the presence of 25 and 100 nM 14E11 or AB023; or purified human FXI was mixed with purified leukocyte-derived DNA in the presence of various concentrations of 14E11 (0-100 nM) or AB023 (0-100 nM) in 20 mM HEPES, pH 7.4, 100 mM NaCl, PEG-8000 0.1 %, ZnCl ₂ (10 pM) at 37°C for 60 min. FXI activation was stopped by mixing aliquots with polybrene (0.2 mg/ml), and the amidolytic activity of FXIa was measured using the chromogenic substrate S-2366 (1 mM), as well as the V _max value. measured as ΔOD 405 nm/min.

Finally, to compare the effects of 14E11 and AB023 on the reciprocal activation of FXII by FXIa, purified human FXII (200 nM) and FXIa (10 nM) were incubated with 14E11 or AB023 (0-200 nM) in 20 mM HEPES, pH 7.4, 100 mM NaCl, PEG-8000 0.1%, ZnCl ₂ (10 pM) at 37°C for 60 min. Activation of FXII was stopped by aprotinin (10 mM), and the amidolytic activity of FXIIa was measured using the chromogenic substrate S-2302 (1 mM), as well as the V _{max value.} measured as ΔOD 405 nm/min.

Despite the lower observed affinity for FXI, AB023 was more effective in inhibiting FXIIa activation within FXI (FIG. 10C). Additionally, the attenuation of FXI self-activation in the presence of a negatively charged surface (dextran sulfate or DNA) by AB023 was greater compared to 14E11 (FIG. 10A-B).

The humanization of 14E11 into AB023 resulted in an innovative binding molecule that has, for example, an unpredicted function, i.e., bidirectional inhibitory activity between FXIl and FXI (FIG. 10C, D). In FIG. 10C shows activation of FXI by FXIIa. In FIG. 10D shows the corresponding inhibitory effects of anti-FXl IgG on FXII activation by FXIa when human FXII (200 nM) was activated by 10 nM human FXIa in the presence of 14E11 or AB023. AB023 is not only more effective than its murine precursor in inhibiting FXIIa activation within FXI (FIG. 10C), but also inhibits the cross-activation process between FXII and FXI, whereas 14E11 does not provide this effect. The unexpected acquisition of inhibitory capacity may be due to sequence similarity between molecules. Alignment using the Basic Local Alignment Finder showed that the sequence similarity between 14E11 and AB023 was about 60–70% (data not shown).

SEQUENCES INCLUDED IN THE LIST

The nucleotide sequences and amino acid sequences included in the accompanying sequence listing are represented by standard alphabetic abbreviations for nucleic acid bases and a three-letter code for amino acids as defined in paragraph 37 of section 1.822 of the CFP. Only one strand of each nucleic acid sequence is presented, however, the complementary strand is considered to be part of the presented strand, incorporated by any reference.

CDR 1LC SEQ ID NO: 1 KASQDVSTAVA CDR 2LC SEQ ID NO: 2 LTSYPNT CDR 3LC SEQ ID NO: 3 QQHYKTPUS CDR HC SEQ ID NO: 4 GYGIY

CDR 2 NS SEQ ID NO: 5 MIWGDGRTDYNSALKS CDR 3 NS SEQ ID NO: 6 DYYGSKDY FXI with A2 domain in bold SEQ ID NO: 7 MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPPCLFTFTAESPSEDPT
RWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTD
DVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFENTVFADS
NIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCR
HSIPVFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEGKCYLKLSSNGS
PTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGNQWI
LTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIIIHDQYKMAESGYDIALLKLETTVNYJDSQR
PICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGGK
DACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILEKTCAV Domain VH AB023 SEQ ID NO: 8 QVQLQESGPGLVKPSETLSITCTVSGFSLTGYGIYWVRQPPGKGLEWLGMIWGDGRTDYNSALKSRVTISK DNSKSQVSLKLSSVTAADTARYYGSKDYWGQGTTVTVSS Domain VL AB023 SEQ ID NO: 9 DIQMIQSPSSLSSASVGDRVTITCKASQDVSTAVANYQQKPGKAPKLLIYLTSYRNTGVPDRFSGSGSGTDF TFTISSLQPEDIAVYYCQQHYKTPYSFGGGTKVEIK LC AB023 SEQ ID NO: 10 DIQMTQSPSSLSSASVGDRVTITCKASQDVSTAVANYQQKPGKAPKLLIYLTSYRNTGVPDRFSGSGSGTDF
TFTISSLQPEDIAVYYCQQHYKTPYSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLAATLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

HC AB023 SEQ ID NO: 11 QVQLQESGPLVKPSETLSITCTVSGFSLTGYGIYWVRQPPGKGLEWLGMIWGDGRTDYNSALKSRVTISK
DNSKSQVSLKLSSVTAADTARYYCARDYYGSKDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALG
CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYICNVDHKPSNTKVDKR
VESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
ALHNHYTQKSLSLSSLGK LC domain of DNA AB023 encoding SEQ ID No: 10) SEQ ID NO: 12 ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGCGCGCGATGTGACATCCAGAT
GACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGG
ATGTGAGTACTGCTGTTGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACTTGACA
TCCTACCGGAACACTGGGGTCCCAGATAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCAT
CAGCAGCCTGCAGCCTGAAGATATTGCAGTTTATTACTGTCAGCAACATTATAAAACTCCGTATTCGTTCG
GCGGAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT
ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG
ACAGCACCTACAGCCTCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC
TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCAAAGAGCTTCAACAGGGGAGAGTGTTAG HC domain of DNA AB023 encoding SEQ ID No: 11) SEQ ID NO: 13 ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCACTCCCAGGTGCAGCTGCA
GGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCATCACCTGCACTGTCTCIGGGTTCTCAT
TAACOGGCTATGGTATATACTGGGTGCGGCAGCCCCCAGGGAAGGGACTGGAGTGGCTGGGGATGATATGG
GGTGATGGAAGAACAGACTATAATTCAGCTTCAAATCCCGAGTCACCATATCAAAGGACAACTCCAAGAG
CCAGGTGTCCCTGAAGCTGAGCTCTGTGACCGCGCGGACACGGCCAGGTATTACTGTGCGAGAGATTACT
ACGGTAGTAAGGACTCTGGGGCCAGGGCACCACGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCC
GTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACG
AAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAAGGTGGACAAGAGAGTTGAGTCCAAATA
TGGTOCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAA AACCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGACGTGAGCCAGGAA
GACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA
GGAGCAGTTCAACAGCACGTACCGTGTGGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCA
AGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATTGACCAAGAACCAGGTCAG
CCTGACCTGCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACC
GTGGACAAGAGCAGGTGGCAGGAGGGGATGTCTTCTCATGCTCCGTGATGCATGAGGCTCGCACAACCA
CTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

The description of the invention above has been presented for purposes of illustration and explanation. It does not limit the scope of the invention to the shapes or forms that are described herein. For example, various features of the invention are grouped into one or more aspects, embodiments, or configurations to organize the description and aid understanding. Features of the aspects, embodiments and configurations of the invention may be combined into alternative aspects, embodiments and configurations different from those described. Therefore, the present invention should not be construed to require more features than those clearly stated in each claim. Rather, as reflected in the following claims, aspects of the invention are not embodied in all features of the above-described aspect, embodiment and configuration. Accordingly, the claims are incorporated into the present detailed description, with each claim representing a separate preferred embodiment of the invention. In addition, although the description of the invention has been included in the description of one or more aspects, embodiments or configurations, as well as certain variations and modifications, other variations, combinations and modifications are within the scope of the invention, for example, within the capabilities and knowledge of specialists in the art after becoming familiar with the present invention. This document is intended to grant rights that cover alternative aspects, embodiments and configuration to the extent permitted, including designs, functions, ranges or steps that are alternative, interchangeable and/or similar to those set forth in the claims, whether such alternative, interchangeable and/or similar designs, functions, ranges or steps are described herein, there is no intent to disclose to the public any patentable subject matter.

--->

LIST OF SEQUENCES

<110> ARONORA INC.

OREGON HEALTH AND SCIENCE UNIVERSITY

<120> NEW HUMANIZED ANTIBODIES TO FACTOR XI WITH ANTITHROMBOTIC AND

ANTI-INFLAMMATORY ACTION AND THEIR APPLICATION

<130>ARB002PCT

<140>

<141>

<150> 62/794,987

<151> 2019-01-21

<160> 13

<170> Patent In version 3.5

<210> 1

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 1

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 2

<211> 7

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 2

Leu Thr Ser Tyr Arg Asn Thr

15

<210> 3

<211> 9

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 3

Gln Gln His Tyr Lys Thr Pro Tyr Ser

15

<210> 4

<211> 5

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 4

Gly Tyr Gly Ile Tyr

15

<210> 5

<211> 16

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 5

Met Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 6

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic peptide

<400> 6

Asp Tyr Tyr Gly Ser Lys Asp Tyr

15

<210> 7

<211> 625

<212>PRT

<213> Homo sapiens

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Val

625

<210> 8

<211> 116

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 8

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

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Ile Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Tyr Gly Ser Lys Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 9

<211> 107

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 9

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Thr Ser Tyr Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln His Tyr Lys Thr Pro Tyr

85 90 95

Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 10

<211> 214

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 10

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Leu Thr Ser Tyr Arg Asn Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln His Tyr Lys Thr Pro Tyr

85 90 95

Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 11

<211> 443

<212>PRT

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 11

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

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Ile Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Tyr Gly Ser Lys Asp Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

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

115 120 125

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 12

<211> 705

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polynucleotide

<400> 12

atgaggtcc ccgctcagct cctggggctc ctgctgctct ggctcccagg cgcgcgatgt 60

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120

atcacttgca aggccagtca ggatgtgagt actgctgttg cctggtatca gcagaaacca 180

gggaaagccc ctaagctcct gatctacttg acatcctacc ggaacactgg ggtcccagat 240

aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300

gaagatattg cagtttatta ctgtcagcaa cattataaaa ctccgtattc gttcggcgga 360

gggaccaagg tggaaatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 420

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 480

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 540

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 600

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 705

<210> 13

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polynucleotide

<400> 13

atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt ccactcccag 60

gtgcagctgc aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccatcacc 120

tgcactgtct ctgggttctc attaaccggc tatggtatat actgggtgcg gcagccccca 180

gggaagggac tggagtggct ggggatgata tggggtgatg gaagaacaga ctataattca 240

gctctcaaat cccgagtcac catatcaaag gacaactcca agagccaggt gtccctgaag 300

ctgagctctg tgaccgctgc ggacacggcc aggtattact gtgcgagaga ttactacggt 360

agtaaggact actggggcca gggcaccacg gtcaccgtct cctcagcttc caccaagggc 420

ccatccgtct tccccctggc gccctgctcc aggagcacct ccgagagcac agccgccctg 480

ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540

ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600

agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga agacctacac ctgcaatgta 660

gatcacaagc ccagcaacac caaggtggac aagagagttg agtccaaata tggtccccca 720

tgcccaccat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 780

aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 840

gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 900

aatgccaaga caaagccgcg ggagggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 960

ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 1020

aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1080

ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1140

acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1200

cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1260

ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 1320

tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 1380

ggtaaatga 1389

<---

Claims (27)

1. A monoclonal antibody or an antigen-binding fragment thereof that specifically binds human factor XI or human factor XIa, and also includes: Light chain CDR1 comprising the sequence KASQDVSTAVA (SEQ ID NO: 1); Light chain CDR2 comprising the sequence LTSYRNT (SEQ ID NO: 2); Light chain CDR3 comprising the sequence QQHYKTPYS (SEQ ID NO: 3); Heavy chain CDR1 including the sequence GYGIY (SEQ ID NO: 4); Heavy chain CDR2 comprising the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); Heavy chain CDR3 comprising the sequence DYYGSKDY (SEQ ID NO: 6). 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, including the V _H region as presented in SEQ ID No: 8. 3. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, characterized in that it includes the V _L region as presented in SEQ ID No.: 9. 4. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, characterized in that it includes a V _H region as presented in SEQ ID No.: 8, and a V _L region as presented in SEQ ID No.: 9. 5. The monoclonal antibody or antigen binding fragment thereof according to claim 1, characterized in that it includes a light chain as set forth in SEQ ID No.: 10, or may be encoded by SEQ ID No.: 12. 6. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that it includes a heavy chain as set forth in SEQ ID No.: 11, or may be encoded by SEQ ID No.: 13. 7. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, characterized in that it includes a light chain as set forth in SEQ ID No.: 10 or may be encoded by SEQ ID No.: 12 and a heavy chain as set forth in SEQ ID No.: 11 , or may be coded SEQ ID No: 13. 8. A monoclonal antibody or an antigen-binding fragment thereof according to claim 1, characterized in that it is a humanized monoclonal antibody or a chimeric monoclonal antibody. 9. Monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that it is IgG. 10. Monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that it is IgG4. 11. Polynucleotide encoding a monoclonal antibody or antigen-binding fragment according to claim 1. 12. A host cell used to inhibit contact activation of blood coagulation, platelet aggregation and/or thrombosis in a patient, which is the recipient of the polynucleotide of claim 11 or vectors including the polynucleotide of claim 11, wherein the host cell is prokaryotic or eukaryotic cells, or a line of said cells. 13. A vector for transferring genetic material to a host cell, including a polynucleotide according to claim 11. 14. The vector according to claim 13, characterized in that it is an expression vector. 15. A method for producing a monoclonal antibody or an antigen-binding fragment thereof according to claim 1, including cultivating a host cell according to claim 12 under conditions that allow the expression of the monoclonal antibody or its antigen-binding fragment. 16. The method according to claim 15, further comprising extracting the resulting antibody, its antigen-binding fragment, from the culture. 17. A pharmaceutical composition for use in the treatment or prevention of thrombotic or thromboembolic disorders, comprising a monoclonal antibody, or an antigen-binding fragment thereof, obtained in accordance with the method of claim 15, as well as a pharmaceutically acceptable excipient. 18. A pharmaceutical composition for use in the treatment or prevention of thrombotic or thromboembolic disorders, comprising a therapeutically effective amount of a monoclonal antibody or an antigen-binding fragment thereof according to claim 1, as well as a pharmaceutically acceptable excipient. 19. The pharmaceutical composition according to claim 18, further comprising one or more active ingredients, which is selected from the group including plasminogen activators, thrombolytics/fibrinolytics; plasminogen activator inhibitors; thrombin-activated fibrinolysis inhibitors (TAFIs), including tissue plasminogen activator (t-PA), streptokinase, reteplase and urokinase; unfractionated heparins; low molecular weight heparins; heparinoid; hirudin; bivalirudin; as well as argatroban. 20. A method of using a monoclonal antibody or an antigen-binding fragment thereof according to claim 1 as a medicinal product, comprising administering a therapeutically effective amount of the monoclonal antibody or an antigen-binding fragment thereof to a subject in need of a medicinal product for use in the treatment or prevention of thrombotic or thromboembolic disorders in humans. 21. The method according to claim 20, characterized in that the subject requires treatment and/or prevention of thrombotic or thromboembolic disorders and/or complications. 22. The method of claim 20, wherein the subject requires treatment and/or prophylaxis to inhibit blood clotting, platelet aggregation and/or thrombosis. 23. A method of using a monoclonal antibody or its antigen-binding fragment according to claim 1 as an anticoagulant in blood samples, during blood conservation, in plasma preparations, biological samples, medicinal supplements or in medical products, including the addition of a monoclonal antibody or its antigen-binding fragment into a sample that requires an anticoagulant. 24. A kit for use in the treatment or prevention of thrombotic or thromboembolic disorders in humans, including a monoclonal antibody or its antigen-binding fragment according to claim 1 and instructions for its use. 25. The kit according to claim 24, characterized in that the monoclonal antibody or antigen-binding fragment thereof is in lyophilized form, and further comprising a pharmaceutically acceptable carrier for resuspending the monoclonal antibody or antigen-binding fragment thereof.