Novel humanized antibody against factor XI having antithrombotic and anti-inflammatory effects and its use

Abstract

To provide novel binding molecules that specifically bind to FXI and FXIa and comprise humanized antibodies, fragments, variants, and derivatives thereof; and to provide compositions, methods, and kits for the same.SOLUTION: The binding molecule of the present invention is capable of binding to and forming an immune complex with the factor XI A2 domain, and thereby disrupting the contact activation molecular complex without affecting hemostatic factor XI activity or activation. The binding molecule of this disclosure is useful for safely inhibiting thrombosis and inflammation without compromising hemostasis. The binding molecules, compositions, methods, and kits provided herein are therefore intended to treat, inter alia, thrombosis- and inflammation-related diseases and conditions. Moreover, polynucleotides encoding the binding molecules of this disclosure, vectors comprising the polynucleotides, and host cells for producing the polynucleotides are provided.SELECTED DRAWING: None

Description

Detailed description of the invention

[0001] [Related applications] This application claims priority from U.S. Provisional Patent Application No. 62 / 794,987, filed on 21 January 2019, the disclosures of which are incorporated herein by reference in their entirety.

[0002] [Sequence Listing] This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on January 15, 2020, is named ARB002PCT_SL.txt and has a size of 18,337 bytes.

[0003] [Government support] The inventions described herein were supported, at least in part, by the U.S. Department of Health and Human Services (HHS) and the National Institutes of Health under U.S. government grant numbers R44AI088937, R44HL128016, R43NS077600, and R44HL106919. The U.S. government has certain rights to the inventions arising from this disclosure.

[0004] [Technical Field] This disclosure relates to antibodies in general. More specifically, this disclosure relates to humanized monoclonal antibodies capable of binding to factor XI and methods of using them, including methods of use as antithrombotic and anti-inflammatory agents that do not impair hemostasis.

[0005] 〔background〕 Thromboembolic diseases, including both venous and arterial thrombosis, are major causes of severe chronic morbidity and mortality in developed countries worldwide. These diseases are caused by the formation of abnormal blood clots (thrombi) resulting from the accumulation of fibrin, platelets, and other blood cells within blood vessels, often leading to vascular occlusion, tissue ischemia, and, in some cases, embolus formation due to the detachment and migration of clot fragments from the thrombus.

[0006] Under normal circumstances, blood coagulation results in hemostasis, a crucial mechanism that prevents blood loss from the site of vascular injury by inducing platelet activation and fibrin formation. At the mechanistic level, hemostasis proceeds through two simultaneous processes. During primary hemostasis, blood in contact with the extraluminal environment is activated by the essential hemostatic protein, tissue factor (TF), which triggers the immediate production of the coagulation enzyme, thrombin. Thrombin then activates other coagulation factors (F), such as fibrinogen (FI), FXIII, and FV. Furthermore, platelets adhere to the injury site, are activated primarily by thrombin, and finally aggregate by binding to each other to form a platelet thrombus. The formation of the platelet thrombus is enhanced and stabilized during secondary hemostasis, which results from a series of sequential platelet activation and enzymatic reactions involving coagulation proteins FXI, FIX, FX, FVIII, FV, FXIII, FIII (prothrombin), and FI, ultimately resulting in a more stable hemostatic plug that seals the tear in the vessel wall, preventing bleeding and death.

[0007] Since Macfarlane (Nature. 1964 May 2;202:498-9) introduced the cascade waterfall model for the treatment of blood coagulation in 1964, our understanding of the mechanisms and functions of blood coagulation in vivo has increased. Over the past few decades, the theory of two distinct pathways—the so-called extrinsic pathway and the intrinsic pathway, which initiate coagulation and ultimately converge into a common pathway leading to thrombin production and fibrin deposition—has been partially modified and rigorously investigated.

[0008] In one model generally accepted by many in the relevant medical field, thrombin generation during the hemostatic process is initiated when circulating plasma protease-activated factor VII (FVIIa) comes into contact with cofactor TF and thereby forms a complex with cofactor TF. This TF-FVIIa complex converts zymogen FIX and FX into their active (a) forms, FIXa and FXa. FIXa can then further activate FX in the presence of cofactor FVIIIa, and FVIIIa and FXa, respectively, can activate prothrombin (FII) in the presence of cofactor FVa to form thrombin (FIIa). Thrombin, a key player in coagulation, then catalyzes the conversion of fibrinogen to fibrin and can cleave protease-activated receptors (PARs) 1 and 4 on platelets, causing platelet activation. Activated platelets combined with fibrin are essential for hemostatic clotting and are therefore fundamental players in normal hemostasis.

[0009] FXI has been well-established as a plasma serine protease zymogen that plays a supporting role in bridging the contact and amplification phases of thrombinogenesis both in vitro and in vivo (Davie EW et al., Biochemistry. 1991 Oct 29;30(43):10363-70, Gailani D and Broze GJ Jr, Science. 1991 Aug 23;253(5022):909-12; Kravtsov DV et al., Blood. 2009 Jul 9;114(2):452-8). Both physiological hemostasis and pathological prothrombinogenesis involve the activation of FXI and FXIa.

[0010] However, data demonstrating that hereditary human or other mammalian FXI deficiency does not typically cause spontaneous bleeding suggest that FXI is not an essential contributing factor to hemostatic thrombin generation. FXI deficiency and hemophilia C are associated with an increased risk of bleeding due to certain hemostatic difficulties, such as certain surgical procedures and trauma, although the severity of bleeding does not correlate well with plasma FXI levels or activity. On the other hand, severe FXI deficiency in humans has been reported to have some protective effects against thrombotic diseases, including ischemic stroke and deep vein thrombosis (DVT) (Salomon O et al., Thromb Haemost 2011 Feb;105(2):269-73, Salomon O et al., Blood. 2008 Apr 15;111(8):4113-7). Furthermore, high levels of fxiphoblastic thrombotic events have been reported to be associated with a higher risk of deep vein thrombosis (DVT), myocardial infarction (MI), and stroke (Meijers JC et al., N Engl J Med. 2000 Mar 9;342(10):696-701, Berliner JI et al., Thromb Res. 2002 Jul 15;107(1-2):55-60, Yang DT et al., Am J Clin Pathol. 2006 Sep;126(3):411-5). Therefore, it has been suggested that pharmacological targeting of FXI may be safer than conventional anticoagulants, and primate studies have provided evidence for this hypothesis (Gruber A and Hanson SR, Blood. 2003 Aug 1;102(3):953-955, Tucker EI et al., Blood. 2009 Jan 22;113(4):936-944).

[0011] In summary, theoretical considerations and previous studies suggest that while FXI plays an adjunctive role in maintaining hemostasis, it is a significant contributing factor to the pathogenesis of thrombosis, making it a promising target for safe antithrombotic therapy. Sufficient nonclinical and clinical evidence now supports this view. Currently available antithrombotic drugs either target the building blocks of thrombus (fibrin and platelets) or inhibit molecules (coagulation factors) and cells (platelets) involved in both the thrombus formation and hemostatic processes. Antiplatelet agents, fibrinolytic agents, and anticoagulants have been the mainstays of the treatment and prevention of thromboembolism for decades and are among the most common prescription drugs in clinical practice. However, most of these drugs have dose-limiting antihemostatic toxicity because, when administered at effective doses, they can completely prevent both thrombosis and hemostasis. As a result, current antithrombotic drugs are administered by healthcare professionals in carefully planned doses lower than the sufficiently effective dose, in order to balance the antithrombotic effect with the potential for severe and fatal bleeding.

[0012] To date, one of the few anti-FXI antibodies showing therapeutic potential is the mouse antibody 1A6 (also known as aximab), published by Tucker et al. (Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI. Erik I. Tucker, Ulla M. Marzec, Tara C. White, Sawan Hurst, Sandra Rugonyi, Owen J. M. McCarty, David Gailani, Andras Gruber, and Stephen R. Hanson. Blood. 2009 Jan 22;113(4):936-944). Antibody 1A6 is also disclosed in U.S. Patent No. 9,125,895, which is incorporated herein by reference in its entirety. However, because antibody 1A6 is a mouse antibody, it is unsuitable for chronic applications in human therapy, particularly in antithrombotic therapy. Similarly, another example of an anti-FXI antibody showing therapeutic potential is the mouse antibody 14E11 (also known as xisomab) published by Cheng et al. (A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo, Cheng Q1, Tucker EI, Pine MS, Sisler I, Matafonov A, Sun MF, White-Adams TC, Smith SA, Hanson SR, McCarty OJ, Renne T, Gruber A, Gailani D. Blood. 2010 Nov 11;116(19):3981-3989; Luo, D. et al. (2012) Infect Immun. 80(1):9109; Tucker, E., et al. (2012) Blood. 119(20):4762-8). Antibody 14E11 is also disclosed in U.S. Patent No. 9,637,550, No. 8,940,883 and No. 8,388,959 ("14E11 Patents").

[0013] One method for converting mouse antibodies into tolerable therapeutic antibodies is humanization. (References: O'Brien S. and Jones T. (2001. Humanizing Antibodies by CDR Grafting. In: Kontermann R. Dubel S. (Eds) Antibody Engineering. Pp. 567-590. Springer Lab Manuals. Springer, Berlin, Heidelberg), Hwang, Almagro, Buss, Tan, and) Foote(2005) Use of human germline genes Standard techniques, such as those described in "A CDR homology-based approach to antibody humanization" (Methods, 36(1):35-42) and the references cited therein, are available to those skilled in the art. Further sequence optimization and germlineization are needed to further reduce the potential for intrinsic immunogenicity of humanized antibodies.

[0014] The recombinant humanized antibody (hereinafter referred to as AB023) obtained by applying these standard methods to the humanization and optimization of the mouse 14E11 antibody showed equivalent binding activity to the mouse precursor in biochemical assays. The introduction of sequence modifications produced humanized variants of the mouse 14E11 antibody (e.g., AB023 antibody), which exhibited both equivalent biochemical profiles and antithrombotic activity in vivo.

[0015] Cardiovascular disease and venous thromboembolism (VTE) remain leading causes of death. Primary prevention, acute treatment, and secondary prevention strategies (such as anticoagulant and antiplatelet therapy) are effective, but these generally increase the risk of bleeding. Therefore, this disclosure addresses an urgent medical need for safe and effective agents for antithrombotic and anti-inflammatory therapy that do not impair hemostasis.

[0016] [Summary of the Invention] The present invention, as described in this disclosure, overcomes existing shortcomings and prior art by providing binding molecules, compositions, methods, and kits for inhibiting thrombosis without impairing hemostasis. The compositions of this disclosure include recombinants, humanized anti-FXI apple 2 domain binding molecules, binding fragments thereof, variants thereof, derivatives thereof, cell lines, and nucleic acid molecules encoding the amino acid sequence of the binding molecule. The disclosure further includes pharmaceutical compositions comprising a therapeutically effective amount of the binding molecule, binding fragment, variant, or derivative thereof in a pharmaceutically acceptable carrier, and methods of using the same. Methods of this disclosure may include administering the compositions of this disclosure to a subject in need, for example, to inhibit thrombosis, prevent thrombosis, or treat inflammation through antithrombotic and anti-inflammatory activity by interfering with factor XIIa-mediated FXI activation without inhibiting thrombin-mediated FXI activation or the procoagulant function of FXIa. Methods for constructing binding molecules, binding fragments, variants, or derivatives thereof are also provided.

[0017] In a preferred embodiment, this disclosure provides a binding molecule comprising: CDR1 light chain containing sequence KASQDVSTAVA (sequence number 1); CDR2 of the light chain containing the sequence LTSYRNT (SEQ ID NO: 2); CDR3 light chain containing sequence QQHYKTPYS (sequence number 3); CDR1 of the heavy chain containing sequence GYGIY (sequence number 4); Heavy chain CDR2 containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); and, A heavy-chain CDR3 containing the sequence DYYGSKDY (sequence number 6).

[0018] The binding molecule is V, as shown in Sequence ID No. 8. H Region, and / or V shown in Sequence ID 9 L It may include a region.

[0019] The binding molecule may include the light chain shown in SEQ ID NO: 10, or the light chain encoded by SEQ ID NO: 12, and / or the heavy chain shown in SEQ ID NO: 11, or the heavy chain encoded by SEQ ID NO: 13.

[0020] The binding molecules of this disclosure can bind to mammalian FXI and / or FXIa, including human or non-human primate FXI, or human or non-human primate FXIa.

[0021] In particular, the binding molecule can bind to the amino acid sequence corresponding to the A2 domain of FXI, including amino acids 91-175 of SEQ ID NO: 7, and form a therapeutic immune complex. Here, the amino acid numbering of human FXI includes a signal sequence starting from methionine at positions -18 to -1, and then from glutamine at position 1. The binding molecule is intended to be an antibody, antigen-binding fragment, variant, or derivative thereof, and in particular may be a humanized monoclonal antibody, antigen-binding fragment, variant, or derivative thereof (e.g., an IgG antibody).

[0022] In further embodiments, the Disclosure provides polynucleotides encoding binding molecules as defined herein, and vectors (e.g., expression vectors containing such polynucleotides). The Disclosure also relates to host cells containing such vectors or polynucleotides.

[0023] In a further embodiment, a process for producing the binding molecules described herein is provided, the process comprising culturing host cells as defined herein under conditions that enable the expression of the binding molecules, and optionally, recovering the produced binding molecules from the culture.

[0024] Furthermore, this disclosure relates to a pharmaceutical composition comprising a binding molecule, polynucleotide, vector and / or host cell, and optionally one or more pharmaceutically acceptable excipients, as defined herein. The pharmaceutical composition may also contain one or more additional active substances, such as antithrombotic and / or anticoagulant agents, or may be administered as part of a combination therapy with additional active substances.

[0025] According to this disclosure, binding molecules, polynucleotides, vectors, host cells, or pharmaceutical compositions can be used in methods to inhibit contact activation, blood coagulation, platelet aggregation, and / or thrombosis in a subject. Therefore, they are useful for the treatment and / or prevention of disorders (e.g., cardiovascular disorders, infectious disorders, or inflammatory disorders, preferably thrombotic disorders or thromboembolic disorders, and / or thrombotic complications or thromboembolic complications).

[0026] Furthermore, the use of binding molecules as anticoagulants in blood samples, blood preservatives, plasma products, biological samples, or pharmaceutical additives, or for coatings on medical devices, is provided herein.

[0027] Furthermore, this disclosure relates to a kit comprising a binding molecule, polynucleotide, vector, host cell, or pharmaceutical composition as described herein.

[0028] [Brief explanation of the drawing] The accompanying drawings are incorporated herein and form part of this specification for illustrative purposes. These drawings, together with this specification, illustrate the principles of this disclosure. The drawings illustrate preferred and alternative embodiments, and this disclosure should not be construed as limiting to the illustrated and described embodiments only. Further features and advantages will become apparent from the various aspects, embodiments, and configurations of this disclosure, as illustrated in detail by the drawings referenced below.

[0029] Figure 1 shows the relationship between activated partial thromboplastin time (aPTT) in mouse plasma (white circles) and human plasma (black circles), and 14E11(10 -5 -10 0 This graph shows the concentration-dependent effect (in μM).

[0030] Figures 2A–F are blots and graphs illustrating the binding properties of 14E11. Figure 2A shows Coomassie blue-stained 10% polyacrylamide gels of human (H) and mouse (M) recombinant FXI; Figures 2B and C show Western blots of unreduced 10% polyacrylamide gels of mouse (B) and human (C) normal (N) and FXI-deficient (XI- / -) plasma using biotinylated-14E11 for detection. rXI in panel B represents the recombinant mouse FXI control; Figure 2D shows the binding of biotinylated 14E11 to immobilized mouse FXI (white circles), human FXI (black circles), or human FXIa (white squares); Figure 2E shows Western blots of unreduced 10% polyacrylamide gels of human FXI (hXI), human prekallikrein (PK), and human FXI in which the A1, A2, A3, or A4 domains are substituted with corresponding domains derived from PK. The position of the FXI dimer is indicated by "D" and the monomer PK by "M," shown on the right (note that FXI with the PK A4 domain is a monomer, as A4 mediates FXI dimer formation); Figure 2F shows Western blots (left panel) of unreduced 10% polyacrylamide gels of individual human FXI apple domains bound to human FXI (hXI) and tissue plasminogen activator (t-PA). The right panel of Figure 2F is a stained gel showing recombinant apple domain-t-PA chimeras (note that the A4 chimera forms a dimer). For panels AC and E, the position of the molecular weight standards, expressed in kDa, is on the left of the figure, and for panel F, it is on the right.

[0031] Figures 3A and 3B are graphs showing the effects of 14E11 (black circles) and its humanized version, AB023 (white circles), on in vitro aPTT. Figure 3A shows the effects of 14E11 and AB023 in pooled human plasma, and Figure 3B shows the effects of 14E11 and AB023 in pooled baboon plasma. Both 14E11 and AB023 similarly prolong aPTT in human and baboon plasma.

[0032] Figures 4A–F are blots and graphs illustrating the binding properties of AB023. Figure 4A shows Western blots of unreduced 10% polyacrylamide gels of human FXI (hXI), human prekallikrein (PK), and human FXI with the A1, A2, A3, or A4 domains substituted with the corresponding domains derived from PK, with biotinylated AB023 used for detection; Figure 4B shows Western blots of unreduced 10% polyacrylamide gels of individual human FXI apple domains (A1–A4) ligated to recombinant t-PA, with AB023 binding to human FXI. Figure 4C shows the binding of AB023 to human FXI (black circles), human FXIa (white squares), and mouse FXI (black triangles); Figure 4D shows that AB023 inhibits FXI-FXIIa activation in a concentration-dependent manner; Figure 4E shows that AB023 does not interfere with thrombin-mediated activation of FXI; Figure 4F shows that AB023 prolongs aPTT in a concentration-dependent manner in plasma derived from humans (black circles), baboons (white squares), cynomolgus monkeys (white circles), and rats (white diamonds). * FXI / PKA4 chimeric protein (A4 * ) is a dimeric molecule created by substituting Cys326 with alanine in the PK A4 domain.

[0033] Figure 5 shows the effect of AB023-FXI complex formation on in vitro aPTT. These experiments were performed sequentially using FXI-deficient plasma (George King, Product #1100, Lot #6538). The baseline aPTT in FXI-deficient plasma was determined to be 118.5 s (circle). Recombinant human FXI (Enzyme Research Labs, catalog number HFXI1111) was added to FXI-deficient plasma at a final concentration of 10 μg / mL, and the aPTT was measured. Addition of FXI to FXI-deficient plasma shortened the aPTT to 32.3 s (square). Subsequently, AB023 was added to the FXI-deficient plasma + 10 μg / mL FXI mixture at a final antibody concentration of 100 μg / mL, and the aPTT was extended to 66.6 seconds (triangle). In the second experiment, AB023 was added to FXI-deficient plasma at a final antibody concentration of 100 μg / mL, and aPTT was measured. Addition of AB023 to FXI-deficient plasma did not alter the aPTT from baseline (117.7 s, inverted triangle). Addition of recombinant human FXI (final concentration 10 μg / mL) to this mixture shortened the aPTT to 59.1 s (diamond), similar to what was observed in previous experiments. These data demonstrate that the anticoagulant effect of AB023 occurs only when a complex is formed between FXI and AB023.

[0034] Figure 6 shows the effects of 14E11 and AB023 in a mouse model of experimental arterial thrombosis. C57Bl / 6 mice [mice treated with or untreated with 14E11 (1.0 mg / kg, iv), or mice treated with or untreated with AB023 (1.0 mg / kg, iv)] or FXI- / - mice were used to test the FeCl3 carotid artery thrombosis model. FeCl3 concentrations of 2.5%–10% were applied to the carotid artery, and the time to occlusion was measured. The height of the bar indicates the percentage of mice with patent arteries 30 minutes after FeCl3 application. Intravenous injection of 1.0 mg / kg AB023 into wild-type mice (bar with circle) protected mice from carotid artery occlusion induced by 3.5%, 5.0%, and 7.5% FeCl3 compared to untreated wild-type mice (bar with dot). These results are comparable to those obtained with FXI- / - mice (white, unmarked lines) and mice treated with 14E11 (grid-patterned lines) (n=10 / group).

[0035] Figure 7 shows the relationship between AB023 plasma concentration and aPTT in four baboons. Each graph shows the time course of AB023 plasma concentration (left y axis, black circles) and aPTT (right y axis, white circles) in a single baboon administered 1.0 mg / kg of AB023 intravenously. In each baboon, aPTT was prolonged until the plasma AB023 concentration fell below a detectable level (1000 ng / mL). A partially validated ELISA assay was used to detect free AB023 in baboon plasma. aPTT is shown as a doubling compared to baseline.

[0036] Figures 8A-D show the effect of AB023 in an in vivo baboon thrombosis model (graft + dilation chamber). AB023 reduces platelet-rich thrombus growth in a primate thrombosis model. The effect of AB023 (0.2 mg / kg, iv) on platelet deposition (shown in Figures 8A and 8C) on a collagen-coated (4 mm diameter, 2 cm length) vascular graft (Figure 8A) and a venous dilation chamber (9 mm diameter, 2 cm length) (Figure 8C). Figures 8B and 8D show fibrin deposition in the collagen graft (Figure 8B) and in the venous dilation chamber (Figure 8D) during control treatment (shaded lines) and after AB023 treatment (unmarked white lines). Values ​​are mean ± SEM, n=7 trials / group of 4 animals in the control group (including historical controls from the same experiment using 14E11), and n=2 trials of 2 animals in the AB023 treatment. The values ​​are mean ± SEM.

[0037] Figures 9A-F show the effect of AB023 on platelet-rich thrombus growth in an in vivo baboon thrombosis model (collagen-coated grafts). Figures 9A-9C show the effect of AB023 (1.0 mg / kg, iv) on platelet (4 mm in diameter, 2 cm in length) deposition in collagen-coated (4 mm in diameter, 2 cm in length) vascular grafts (Figure 9A) and 10 cm downstream of the collagen-coated grafts ("tail") (Figure 9B) (Figure 9B) (Figure 9B); Figure 9C shows platelet deposition in both graft + tail combinations; Figures 9D-F show fibrin deposition in collagen grafts (Figure 9D), tails (Figure 9E), and graft + tail combinations (Figure 9F) during control treatment (shaded lines) and after AB023 treatment. Values ​​are mean ± SEM, n=7 trials / group in the control group (including historical controls from the same experiment using 14E11) and n=2 trials in the AB023 treatment in 2 animals. Values ​​are mean ± SEM. Values ​​are mean ± SEM, n=4 trials / group of 4 animals. *p<0.05, **p<0.01, ***p<0.001 vs. control. Each animal was subjected to a control experiment and then to the AB023 experiment.

[0038] Figures 10A-D show a comparison of the activity of AB023 and the activity of the mouse antibody 14E11. Figures 10A-B show the effects of 14E11 (dotted bar) and AB023 (shaded bar) on FXI autoactivation in the presence of dextran sulfate (Figure 10A) and DNA (Figure 10B), as a function of antibody concentration. Humanized antibody AB023 inhibits DNA-induced autoactivation of purified human FXI in a concentration-dependent manner in vitro; Figure 10C shows the effects of 14E11 (white circles) and AB023 (black squares) on inhibiting FXIIa activation of FXI compared to the control (black circles); Figure 10D shows the effects of 14E11 and AB023 on FXIa-mediated FXII activation in vitro. The graph shows that FXIIa activation is shown as a function of antibody concentration. A mixture of purified human FXII and FXIa was incubated with various concentrations of 14E11 (dotted bar) or AB023 (slashed bar), and the FXIIa amid-degradation activity was measured. AB023 inhibited the activation of purified human FXII by purified human FXIa in a concentration-dependent manner in vitro, while 14E11 did not.

[0039] [Detailed explanation] For details, please refer to the representative embodiments of this disclosure. The disclosed antibodies, fragments, variants, or derivatives thereof are described in relation to the listed embodiments, but it will be understood that they are not intended to limit this disclosure to those embodiments. On the contrary, the disclosed antibodies, fragments, variants, or derivatives thereof are intended to encompass all substitutes, variants, and equivalents that may fall within the scope of this disclosure as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which may be used within this disclosure and are within the scope of implementation of this disclosure. This disclosure is by no means limited to the methods and materials described.

[0040] All publications and patents referenced herein are incorporated herein by reference in their entirety for the purposes of description and disclosure. For example, constructs and methodologies described in publications that may be used in connection with this disclosure. Publications discussed throughout this specification are provided for their disclosure prior to the filing date of this application. Nothing herein should be construed as an endorsement.

[0041] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure belongs. The terms “a” or “an” refer to one or more of the real things it refers to, and are therefore understood to mean, for example, “one or more” and “at least one,” which may be used interchangeably.

[0042] The implementation of this disclosure will, unless otherwise indicated, utilize conventional techniques in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, and these conventional techniques are within the scope of prior art. Such techniques can be found in detail in the following literature, for example: 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, NY); DNGlover (eds.) (1985) DNA Cloning, Volumes 1 and II; Gait (eds.) (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Patent No. 4,683,195; Hames and Higgins (eds.) (1984) Nucleic 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; Laboratory); edited by Wu et al., Methods in Enzymology, Vols. 154 and 155; edited by Mayer and Walker (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London; Weir and Blackwell, eds., (1986) Handbook of Experimental Immunology, Volumes I-IV; Manipulating Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold. Spring Harbor, NS, (1986); and Ausubel et al. al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, MD). Any methods, devices, and materials similar to or equivalent to those described herein may be used to test the antibodies, fragments, variants, or derivatives thereof that have been performed or disclosed, but preferred methods, devices, and materials are described below.

[0043] Inhibition of blood coagulation factor XI (also known as FXI) (which has an effect limited to physiological hemostasis but is given a role in the development of pathological thrombus formation) is a promising novel approach in the development of new antithrombotic agents to achieve an improved benefit-risk ratio. This disclosure provides, in particular, a novel binding molecule AB023. AB023 can specifically bind to FXI, forming the immune complex FXI-AB023, thereby inhibiting the proper molecular assembly of a normally functioning contact-activating complex containing FXII, FXI, prekallikrein (PK), and high molecular weight kininogen (HMWK). As a result, molecular interactions between FXI-AB023, FXII, PK, and HMWK are restricted, and therefore, the conversion of FXIa-AB023 to the active form FXIa-AB023 by FXIa-AB023, and the conversion of FXII to the active form FXIIa by FXIa-AB023 are also restricted. The binding molecule AB023 is a novel anticoagulant recombinant monoclonal antibody against FXI. Furthermore, the binding molecule has also been shown to bind to FXIa. Thus, the binding molecule provided herein inhibits the interaction of players involved in pathological thrombinogenesis and thrombosis, including kallikrein and bradykinin production, which are involved in blood pressure regulation and inflammation (as outlined by Weidmann, H. et al. (2017) Biochim Biophys Acta Mol Cell Res. 1864(11 Pt B):2118-2127, Bjorkvist et al. (2014) Thrombosis and Hemostasis. 112(5):868-75; Blood Advances 2019 3:658-669). Specifically, a humanized version of a mouse 14E11 monoclonal antibody is provided that favorably binds to FXI with high binding affinity, comparable to that of 14E11. Furthermore, the formation of immune complexes between the binding molecule and FXI effectively reduces blood coagulation in vitro, as indicated by the prolongation of activated partial thromboplastin time (aPTT) in the presence of complexes formed with low concentrations of the binding molecule.Therefore, the conjugating molecules of this disclosure are promising novel agents for the effective treatment and / or prevention of disorders in which activation of the contact system plays a pathogenic role, particularly inflammatory and thrombotic or thromboembolic diseases, and / or thrombotic or thromboembolic complications. Furthermore, the conjugating molecules of this disclosure are expected to be effective without significantly impairing hemostasis, thereby minimizing the risk of bleeding.

[0044] Using the antibody of this disclosure, a therapeutic molecule was produced that reduces immunogenicity risk and, after forming an immune complex with circulating FXI, reduces thrombosis in vivo. The formation of this immune complex between the antibody and free FXI antigen in vivo effectively inhibits thrombotic proliferation without impairing hemostasis. In fact, the formation of the immune complex between the humanized 14E11 antibody, AB023, and FXI does not interfere with the hemostatic feedback activation of the FXI-AB023 immune complex by thrombin. Furthermore, the FXIa-AB023 immune complex of this disclosure retains enzymatic activity that contributes to hemostatic thrombin generation via the activation of FIX and other coagulation factors by FXIa. Thus, antithrombotic therapy with antibodies, binding fragments, variants, or derivatives thereof is hemostatically safer than directly inhibiting the enzymatic activity or hemostatic activation of FXI, and therefore expands the scope of clinical application and the range of scenarios in which this type of antithrombotic therapy can be applied. It is important to note that in the absence of circulating immune complexes, the antibody alone will not possess anticoagulant or antithrombotic activity. Furthermore, in the absence of free, available, and activatable FXI in circulation, the antibody alone will not possess anticoagulant, antithrombotic, or other activities. Therefore, in the absence of circulating FXI-AB023 immune complexes, the antibodies of this disclosure may lack anticoagulant activity and may not possess antithrombotic activity in FXI-deficient subjects.

[0045] [Binding molecule] The conjugation molecule of this disclosure is a novel recombinant anticoagulant monoclonal antibody against coagulation factor XI (FXI). It was obtained by humanization using the transplantation of the complementarity-determining region (CDR) of the mouse monoclonal antibody 14E11 disclosed in U.S. Patents 8,388,959, 8,940,883, and 9,637,550 (title: Anti-FXI Antibodies and Methods of Use). Surprisingly, the conjugation molecule of this disclosure exhibits advantageous properties compared to the 14E11 CDR, with fewer amino acid substitutions in the CDR region. The mouse monoclonal antibody 14E11 and the humanized antibody AB023 were characterized, and their anticoagulant properties were evaluated both in vitro and in vivo to demonstrate comparability of performance. The conjugation molecule of this disclosure can bind to FXI with a binding affinity equivalent to that of 14E11. Furthermore, FXI in the immune complex is not efficiently converted to FXIa by FXIIa, but is efficiently converted to FXIa by thrombin (Figure 4). Interestingly, AB023 appears to be more potent than 14E11 in inhibiting this activation (Figure 10C). In contrast to 14E11, the FXIa-AB023 complex has reduced catalytic activity for converting FXII to its active form, FXIIa (Figure 10D). When FXI-AB023 is converted to FXIa-AB023 by thrombin, it retains enzymatic activity for FIX (data not shown), as well as other high-molecular-weight and low-molecular-weight substrates. As a result, activation of FXI-AB023 via hemostatic thrombin maintains the hemostatic activity of circulating FXI, while events via contact activation are downregulated.

[0046] The binding molecule of this disclosure is a humanized monoclonal antibody, an antigen-binding fragment, a variant, or a derivative thereof, preferably AB023, a monoclonal therapeutic antibody targeting FXI. It may also be IgG4 and may have an S241P hinge modification to prevent antibody arm exchange. The amino acid sequence of the light chain (LC) is shown in SEQ ID NO: 10, and the coding DNA sequence of LC is shown in SEQ ID NO: 12. The amino acid sequence of the heavy chain (HC) is shown in SEQ ID NO: 11, and the coding DNA sequence of HC is shown in SEQ ID NO: 13. AB023 is generated by CDR transplantation and contains a kappa (κ) light chain and an IgG4 isotype heavy chain. Variable sequences (VH and VL) from the mouse monoclonal precursor antibody 14E11 were cloned into the human IgG4 (SP241 hinge modified using the Kabat numbering system) heavy chain gene and κ light chain gene. The four chains are held together by 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 contains one consensus sequence (NXS / T) for potential N-linked glycosylation located on the heavy chain.

[0047] The antithrombotic effect observed in 14E11 was maintained after humanization, and AB023 prevented venous and arterial thrombosis. In a first embodiment, this disclosure relates to a binding molecule that can specifically bind to factor XI, the binding molecule comprising the following complementarity-determining regions (CDRs): CDR1 of the light chain containing the sequence KASQDVSTAVA (SEQ ID NO: 1); CDR2 of the light chain containing the sequence LTSYRNT (SEQ ID NO: 2); CDR3 of the light chain containing the sequence QQHYKTPYS (SEQ ID NO: 3); CDR1 of the heavy chain containing the sequence GYGIY (SEQ ID NO: 4); CDR2 of the heavy chain containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); and CDR3 of the heavy chain containing the sequence DYYGSKDY (SEQ ID NO: 6). The binding molecule may further include S241P modification.

[0048] During the humanization process, the CDR region of 14E11 was determined, and both the VH and VL variable regions were plugged into a modeling program to identify which amino acid residues in the framework were useful for antibody binding properties. The CDR region was then transplanted onto a human framework having the highest degree of homology to the 14E11 framework. Where necessary, reverse mutations were performed to specific mouse frameworks identified as useful for binding. From this process, 3VH and 3VL were generated. In some embodiments, antibody AB023 is a combination of VH3 (SEQ ID NO: 8) and VL3 (SEQ ID NO: 9).

[0049] The terms “amino acid” or “amino acid residue” refer to amino acids having definitions recognized in the art, such as amino acids selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamic acid (GIu or E); serine (GIy or G); histidine (His or H); isoleucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Ser or S); threonine (Thr or T); tryptophan (Trp or W); and valine (VaI or V), and may be used as desired, even if they are modified, synthetic, or rare amino acids. Generally, amino acids can be classified as having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, VaI); negatively charged side chains (e.g., Asp, GIu); positively charged side chains (e.g., Arg, His, Lys); or uncharged polar side chains (e.g., Asn, Cys, Gin, GIy, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0050] [Number and distribution of substitutions] Amino acid substitutions can generally be distributed throughout the CDRs in any manner. That is, one CDR may contain, for example, one or more exchanges, and a second CDR may contain one or more substitutions. Alternatively, two CDRs may contain one or more amino acid substitutions, or all six CDRs may contain amino acid substitutions. For example, a binding molecule containing one or two substitutions per CDR, and preferably one or more substitutions in CDR1, CDR2 and / or CDR3 of the light chain, or one or more amino acid substitutions in CDR1, CDR2 and / or CDR3 of the heavy chain. The heavy chain retains the CDRs of the non-humanized molecule (to the greatest extent possible without destroying functionality). In general, amino acid substitutions can be distributed in substantially any manner, as long as the number of cumulative amino acid substitutions compared to the 14E11 CDR amino acids does not negate the binding molecule's ability to bind to FXI.

[0051] [Types of substitution] In general, any combination of amino acid substitutions in the CDR compared to 14E11 is possible, as long as it does not negate the advantageous properties of the binding molecule of this disclosure. Amino acid exchanges may be conservative (i.e., exchanging an amino acid of one class or group for another amino acid from the same class or group listed above). Alternatively, amino acid exchanges may be non-conservative (i.e., exchanging an amino acid from one class / group for another amino acid from another class / group).

[0052] Preferred substitutions, as described herein, produce the binding molecules of this disclosure that result in an extension of aPTT.

[0053] The binding molecules according to this disclosure may optionally be combined to comprise one or more of the above-mentioned CDRs. Preferred substitutions, as described herein, produce the binding molecules of this disclosure that result in approximately 1.5, 2, or more extensions of aPTT.

[0054] Preferred binding molecules of the present disclosure can be monoclonal antibodies, antigen-binding fragments, variants, or derivatives thereof, and include the following CDRs: CDR1 of the light chain containing the sequence KASQDVSTAVA (SEQ ID NO: 1); CDR2 of the light chain containing the sequence LTSYRNT (SEQ ID NO: 2); CDR3 of the light chain containing the sequence QQHYKTPYS (SEQ ID NO: 3); CDR1 of the heavy chain containing the sequence GYGIY (SEQ ID NO: 4); CDR2 of the heavy chain containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); and CDR3 of the heavy chain containing the sequence DYYGSKDY (SEQ ID NO: 6). Preferred binding molecules can further and optionally include an S241P hinge modification.

[0055] Furthermore, the binding molecule of the present invention may have a light chain variable region (V H or VH region) shown in SEQ ID NO: 8, and / or a heavy chain variable region (V L or VL region) shown in SEQ ID NO: 9. However, other combinations of V L and V H regions are also conceivable. Therefore, a preferred embodiment is the humanized monoclonal antibody AB023 disclosed herein and having the sequences shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 8 and 9.

[0056] [Factor XI] As described herein, the binding molecules of this disclosure can bind to two identical exocytes on an FXI homodimer that is preferably involved in a selected polymer substrate recognition reaction. Human “Factor XI,” also known as “Plasma Thromboplastin Precursor,” “PTA,” “Rosenthal Factor,” “Coagulation Factor XI,” “FXI,” “F11,” or “FXI,” circulates in the blood as a double-chain glycoprotein homodimer with a binding molecular weight of approximately 160 kilodaltons (kD). The two monomers that form the homodimer are identical disulfide-linked polypeptides, each with a molecular weight of approximately 80,000 daltons. Each FXI monomer has four “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). While we do not wish to be bound to any particular theory, some experts in the field believe that the four apple domains contain FXI binding sites to other proteins. For example, A1 is for thrombin, A2 is for high molecular weight kininogen (HK, HMWK), A3 is for FIX, glycoprotein Ib (GPIb), and heparin, and A4 is for dimerization and possibly FXIIa. FXI can be converted to its active form, coagulation FXIa, by FXIIa, thrombin, FXIa, and possibly other proteases and serine proteases. The serine protease FXIa can cleave many high molecular weight substrates, including FXII, FX, FV, TFPI, FIX, and possibly others. One of the best-described reactions is the common aPTT assay, which is sensitive to the conversion of FIX to FIXa and can be readily measured in plasma or blood in a standard clinical laboratory. FXIa can subsequently activate coagulation factor X (FXa), which in turn activates coagulation factor IX (IXa), which can mediate the activation of coagulation FII (prothrombin) to thrombin. Subsequently, thrombin can activate further FXI molecules, thereby amplifying the enzymatic process through a positive feedback reaction.This, in turn, leads to the generation of more thrombin and, in the aPTT assay, indirect coagulation of recalcified citrated blood or plasma, typically within 40 seconds of the initiation of the reaction with negatively charged surfaces and phospholipids.

[0057] The term "Factor XI" is found in "Uniprot Acc No. P03951, entry This refers to human coagulation factor XI (F11, FXI) having version 194 of 14 October 2015 (SEQ ID NO: 7). As described elsewhere in this specification, the binding molecule of this disclosure is thought to bind to the 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 a signal sequence beginning with methionine at positions -18 to -1, followed by glutamine at position 1.

[0058] The term “position,” as used in accordance with this disclosure, means either the position of an amino acid in an amino acid sequence described herein or the position of a nucleotide in a nucleic acid sequence described herein. The term “corresponding” also includes the fact that a position should be considered not only in terms of the number of preceding nucleotides / amino acids, but rather in terms of the context of the surrounding parts of the sequence. As a result, in this disclosure, the position of a given amino acid or nucleotide may change due to the deletion or addition of an amino acid or nucleotide. Therefore, when a position is referred to as “corresponding position” in accordance with this disclosure, it should be understood that the nucleotide / amino acid may differ in terms of the identified number, but may still have similar adjacent nucleotides / amino acids. To determine whether an amino acid residue (or nucleotide) in a given sequence corresponds to a particular position in the amino acid sequence (or polynucleotide sequence) of a “parent” amino acid (or polynucleotide sequence) (e.g., the amino acid sequence of human FXI shown in SEQ ID NO: 7), a person skilled in the art may use means and methods well known in the art (e.g., sequence alignment manually or using a computer program as illustrated herein).

[0059] The term “epitope” generally refers to a site on an antigen, i.e., a site on a (poly)peptide recognized by a binding domain, also called the “antigenic structure” or “antigenic determinant.” The term “binding domain” refers to the antigen-binding site. In other words, it characterizes the domain of a binding molecule that binds to / interacts with a target epitope or group of antigens (e.g., the same antigen of different species) on a given antigen or group of antigens. A target antigen may contain a single epitope, preferably at least two, and may contain any number of epitopes depending on the size, conformation, and type of the antigen. Furthermore, it should be noted that while an “epitope” on a target antigen may be a target (poly)peptide, an “epitope” on a target antigen may also be, for example, a non-polypeptide element, or may contain a non-polypeptide element (e.g., an epitope may contain a carbohydrate side chain).

[0060] The term "epitope" generally encompasses linear epitopes and conformational epitopes. Linear epitopes are consecutive epitopes contained in a primary amino acid sequence, and may, for example, contain at least two or more amino acids. Conformational epitopes are formed by discontinuous amino acids arranged in parallel by the folding of a target antigen, preferably a target (poly)peptide.

[0061] The binding molecule of this disclosure is thought to recognize a structurally conserved epitope located on the heavy chain of factor XI (SEQ ID NO: 7) whose epitope comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, and at least 10 consecutive or discontinuous amino acid sequences of factor XI.

[0062] The binding molecule provided herein binds to the A2 domain of human factor XI, including amino acids 91-175 of SEQ ID NO: 7, to form an immune complex. However, since the parent molecule AB023, 14E11 forms immune complexes or complexes with FXI in plasma from several, but not all, mammalian species, the binding molecule may also have binding ability to the variants of human FXI identified herein. If the binding molecule is in its native (IgG4) form, it can bind to one or two FXI homodimers. Multimers or aggregates can also be formed. When used in relation to FXI, the term “variant” refers to a polypeptide that can be converted to active FXIa, comprising one or more amino acid sequence substitutions, deletions, and / or additions compared to the “parent” FXI sequence, and exhibiting the same biological function as the “parent” FXI sequence, i.e., active FXIa has protease activity and catalyzes the activation of FIX and / or the activation / inactivation of other polymer substrates such as TFPI, SERPIN-s, protein S, FV, FX, and FXII. Amino acid substitutions may be conserved, non-conserved, or any combination thereof, as defined herein. FXI variants may have the addition of an amino acid residue at either the carboxyl terminus or the amino terminus (the amino terminus may or may not contain the leader sequence). When used in relation to FXI, the term “variant” includes isoforms, alleles, or splicing variants of known FXI polypeptides (e.g., FXI polypeptides having the sequence shown in SEQ ID NO: 7), or post-translational modification variants (e.g., glycosylation variants). It is readily apparent that the binding molecules of this disclosure may exhibit binding affinity to FXI variants containing amino acid sequences corresponding to amino acids 91-175 of SEQ ID NO: 7. Accordingly, it is conceivable that such binding molecules may also have the ability to bind to FXIa molecules and their variants and form variable complexes with them, if they contain the aforementioned amino acid extensions or the corresponding amino acid positions.

[0063] The binding molecules of this disclosure may also have binding ability to a number of other mammalian species, with or without preference to any particular species. These non-human FXI polypeptides are preferably encoded by the FXI gene or its orthologs or paralogs and exhibit the same biological function as human FXI, even if they are not present as homodimers. Potential non-human primate protein targets of the binding molecules of this disclosure include polypeptides having the following: Uniprot Acc.No.H2QQJ4 (Pan troglodytes, entry version 26 of 11 November 2015), Uniprot Acc.No.H2PEX7 (Pongoabelii, entry version 27 of 11 November 2015), Uniprot Acc.No.A0A0D9s2M6 (Chlorocebussabaeus, entry version 6 of 11 November 2015), Uniprot Acc.No.G3R2X1 (Gorilla gorilla gorilla, entry version 27 of 14 October 2015), Uniprot Acc.No.20 A0A096NC95 (Papio anubis, entry version 11 of 11 November 2015), Uniprot Acc.No.G1RLE8 (Nomascus leucogenys, entry version 28 of 11 November 2015), Uniprot Acc.No.G7PKF5 (Macaca fascicularis, entry version 13 of 14 October 2015), UniProt Acc.No.G7MSF8 (Macaca mulatta, entry version 12 of 14 October 2015). Other species include a series of mammalian FXI variants that have the same conserved antigenic region in the A2 domain as humans. Variants of the polypeptides described above can also be considered targets for the binding molecules of this disclosure.The non-human primate polypeptide targets expected to be recognized by the binding molecules of this disclosure may include sequences corresponding to amino acids 91-175 of SEQ ID NO: 7, or sequences having at least 95%, 96%, 97%, 98%, or 99% sequence identity to it. Thus, interspecies-specific binding molecules directed to FXI, e.g., in non-human primates, are also provided herein. Therefore, the terms “interspecies recognition” or “interspecies specificity” as used herein mean that the binding molecules described herein bind to the same target polypeptide in humans and non-humans, e.g., non-human primate species. 14E11 is a universal antibody, meaning it appears to form complexes with a wide range of unrelated mammalian species. This view suggests that it binds to highly conserved or identical sequences on the A2 domain of FXI. Since the equivalence of AB023 to 14E11 has been demonstrated, the universality of AB023 enables the development of therapeutic antibodies with virtually no species limitations.

[0064] As described herein, the binding molecules described herein may also bind to human or non-human mammalian FXIa. Therefore, those disclosed in the context of the binding properties of binding molecules to FXI are equally applicable to their binding properties to FXIa, preferably with necessary modifications.

[0065] 〔antibody〕 The binding molecules of this disclosure are presumed to be antibodies. As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target epitope via 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 their broadest sense within this specification. They may also include native antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), (native or synthetic) antibody derivatives, fragments or variants, fusion proteins containing antigen-binding fragments having the required specificity, and any other modified forms of antibodies containing antigen-binding sites having the required specificity. The antibodies according to this disclosure are assumed to have the ability to bind to mammalian FXI as described herein and preferably exhibit the advantageous features of antibody AB023 as described herein.

[0066] [Natural antibodies] "Natural antibodies" are tetrameric glycoproteins. In natural antibodies, each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a "(hyper)variable" region consisting of approximately 100-110 or more amino acids, primarily involved in antigen recognition. The hypervariable region contains amino acid residues from the "complementarity-determining region" or CDR or "CDR region". "Framework" or FR residues are variable domain residues other than those in the hypervariable region.

[0067] Both light and heavy chains are divided into regions of structural and functional homology called the "steady region" and the "variable region." The terms "steady" and "variable" are used functionally. In this regard, naturally, the light chain (V L ) and heavy chain (V H Both variable regions of ) determine antigen recognition and specificity. L "V L "Area" and "V LThe term "domain" is used interchangeably throughout this specification to refer to a variable region of a light chain. Similarly, the term "V" H "V H "Area" and "V H In this specification, the term "domain" is used interchangeably to refer to a variable region of a heavy chain.

[0068] The term “C L "C L "Area" and "C L The term "domain" is used interchangeably here to refer to the steady region of the light chain. H "C H "Area" and "C H The term "domain" is used interchangeably here to indicate the constant region of the heavy chain, and "C H1 "C H2 ", and "C H3 Includes a region or domain. Conversely, light chain (C L ) and heavy chain (C H1 , C H2 , or C H3 The constant region of the antibody confers biological properties such as secretion, transplacental motility, Fc receptor binding, and complement binding. By convention, the numbering of constant region domains increases as they become more distal to the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region. H3 Region and C L The regions actually include the C-terminuses of the heavy and light chains, respectively.

[0069] Variable regions allow antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, the V within these variable domains. L and V H The regions, or subsets of complementarity-determining regions (CDRs) within these variable domains, bind to form variable domains that define a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y-shaped antibody. More specifically, V H Region and V LFor each region, the antigen-binding site is defined by three CDRs (CDR1, CDR2, CDR3, determined according to the Kabat numbering system). The three CDRs of the light chain are referred to herein as CDR1 LC or CDR L1 , CDR2 LC or CDR L2 , and CDR3 LC or CDR L3 It is also designated as follows. The three CDRs of the heavy chain are CDR1 HC or CDR H1 , CDR2 HC or CDR H2 , and CDR3 HC or CDR H3 These are referred to as "complementarity-determining regions" (CDRs) or "CDR regions" in natural antibodies. In each antigen-binding domain, the six "complementarity-determining regions" or "CDRs" are typically short, discontinuous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the antibody takes its three-dimensional configuration in an aqueous environment.

[0070] The binding molecules and, for example, antibodies of this disclosure are thought to include a light chain CDR1 containing the sequence KASQDVSTAVA (SEQ ID NO: 1); a light chain CDR2 containing the sequence LTSYRNT (SEQ ID NO: 2); a light chain CDR3 containing the sequence QQHYKTPYS (SEQ ID NO: 3); a heavy chain CDR1 containing the sequence GYGIY (SEQ ID NO: 4); a heavy chain CDR2 containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); a heavy chain CDR3 containing the sequence DYYGSKDY (SEQ ID NO: 6); and optionally, an S241P modification. Those skilled in the art will readily understand that the CDRs are located in the variable regions of the light and heavy chains, respectively. Monoclonal antibodies containing the aforementioned CDRs are disclosed herein and referred to herein as "AB023".

[0071] The binding molecules and preferred monoclonal antibodies, their antigen-binding fragments, their variants, or derivatives are as shown in SEQ ID NO: 8. L Region, and / or V shown in Sequence ID 9 H It is thought to include the region. However, V L Region and V HOther combinations of regions are also possible. The binding molecules of this disclosure and preferred monoclonal antibodies, antigen-binding fragments, variants thereof, or derivatives thereof are thought to include a light chain as shown in SEQ ID NO: 10 or SEQ ID NO: 12, and / or a heavy chain as shown in SEQ ID NO: 11 or SEQ ID NO: 13. However, other combinations of light and heavy chains are also possible.

[0072] The carboxyl-terminal regions of each light and heavy chain define the constant region primarily involved in effector function. Immunoglobulins can be assigned to various classes depending on the amino acid sequence of the constant domain of their heavy chains. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Some of these can be further classified into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Different isotypes have different effector functions. For example, IgG1 and IgG3 isotypes often possess antibody-dependent cytotoxicity (ADCC) activity. Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. All immunoglobulin types, classes, and subclasses are within the scope of this disclosure. The antibodies according to this disclosure may be IgG antibodies, particularly IgG4 monoclonal antibodies.

[0073] [Monoclonal Antibodies] Based on this disclosure, monoclonal antibodies, antigen-binding fragments, variants, and derivatives thereof are envisioned. As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, in other words, the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in small amounts. In contrast to conventional (polyclonal) antibody preparations, which may contain different antibodies against different epitopes, monoclonal antibodies contain substantially similar epitope-binding sites, and therefore monoclonal antibodies may be against the same epitope on an antigen. Accordingly, the term “monoclonal antibody” is equivalent to recombinant antibodies, chimeric antibodies, humanized antibodies, human antibodies, or Human Contains Engineered® monoclonal antibody.

[0074] Various methods for producing monoclonal antibodies are well known in the field and are described, for example, in Goding, Monoclonal Antibodies: Principles and Practice, pp. 116-227 (Academic Press, 1996). Suitable techniques include the hybridoma method, first described by Kohler et al., Nature, 256:495 (1975); the recombinant DNA method, which includes isolation and sequencing of DNA encoding monoclonal antibodies and subsequent introduction and expression in suitable host cells; and the isolation of antibodies from antibody phage libraries prepared using the technique first described by McCafferty et al., Nature, 348:552-554 (1990).

[0075] [Chimera antibody] As used herein, the term “antibody” also encompasses chimeric antibodies. As used herein, “chimeric antibody” means an antibody containing sequences derived from two different antibodies, which may originate from different species. Specifically, the term means an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody belonging to one species or antibody class or subclass. The remainder of the chain is identical or homologous to a corresponding sequence in an antibody belonging to another species or antibody class or subclass, as well as a fragment of such an antibody. In other words, “chimeric antibody” means any antibody in which the antigen-binding site is obtained from or induced from a first species, and the constant region (which may be intact, partially, or modified according to the present invention) is obtained from a second species. For example, the antigen-binding site may originate from a non-human source (e.g., mouse or primate), and the constant region may be human. A chimeric antibody may, for example, include human and mouse antibody fragments (e.g., a human constant region and a mouse variable region).

[0076] [Humanized antibodies] As described herein, this disclosure relates to (monoclonal) humanized antibodies, antigen-binding fragments, variants, and derivatives derived from mouse anti-FXI 14E11 (as carried out by Abzena (aka Antitope Limited, Cambridge, GB) using both methods well known and used in the art and proprietary methodologies).

[0077] A "humanized antibody" is generally defined as (I) derived from a non-human source (e.g., a transgenic mouse with a heterologous immune system), with the antibody based on a human germline sequence, or (II) a CDR-implanted antibody whose variable region CDR is of non-human origin, with one or more framework regions and / or parts of the CDR sequence of the variable region being of human origin, for example, the constant region (if any) being of human origin.

[0078] Therefore, the term “humanized antibody” includes antibodies in which the variable region in the heavy chain, light chain, or both of the human antibody is modified by at least partial substitution of one or more CDRs from a non-human antibody of known specificity, and in some cases, the human antibody is modified by partial framework region substitution and sequence transposition. In other words, an antibody in which one or more “donor” CDRs from a non-human antibody of known specificity (such as a mouse, rat, rabbit, or non-human primate antibody) are transplanted into the human heavy chain or light chain framework region is referred to herein as a “humanized antibody.” It may not be useful to replace the entire CDR with a complete CDR from a donor variable domain in order to transfer the antigen-binding ability of one variable domain to another. Rather, only residues useful in maintaining the activity of the target binding site can be transferred.

[0079] In this disclosure, as detailed herein, the precursor mouse 14E11 antibody determines the 14E11 CDR residue, and V H and V L As an acceptor human germline framework for transplanting each CDR, mouse V H and V LHumanization was achieved by selecting human germline sequences with the best overall homology to the sequence from a database. Briefly, a structural model of the chimeric anti-FXI antibody V region was constructed using Swiss PDB and analyzed to identify amino acids in the V region framework that could support the antibody binding properties. These amino acids were considered for incorporation into one or more mutant CDR-implanted antibodies. Both the VH and Vκ sequences of the binding molecule contained typical framework residues, and the CDR1, 2, and 3 motifs were equivalent to many mouse antibodies. To identify the heavy and light chain human sequences with the greatest degree of homology for use as a human V region framework, the heavy and light chain V region amino acid sequences were compared to a database of human germline V region sequences. Subsequently, a series of humanized heavy and light chain V regions were designed by transplanting CDRs onto the framework and, if necessary, by reverse mutations to specific mouse sequences of previously identified residues that could restore antibody binding efficiency. Next, Abzena's proprietary ex vivo technology, EpiScreen TM (Jones TD, Hanlon M, Smith BJ, Heise CT, Nayee PD, Sanders DA, Hamilton A, Sweet C, Unitt E, Alexander G, Lo KM, Gillies SD, Carr FJ and Baker MP. 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.J Interferon Cytokine Res.2004 24(9):560-72;Jones TD,Phillips WJ,Smith BJ,Bamford CA,Nayee PD,Baglin TP,Gaston JS and Baker MP.Identification and removal of a promiscuous CD4+ t cell epitope from the C1 domain of By applying factor VIII (J Thromb Haemost. 2005 3(5):991-1000), the variant sequence with the lowest incidence of possible T cell epitopes was selected. For the purposes of this disclosure, CDR-optimized ("germline-optimized") humanized antibodies are included within the term "humanized" antibody.

[0080] The framework regions (FRs) within the variable regions of the heavy chain, light chain, or both of a humanized antibody may consist of substantially all or all residues of human origin. In this case, these framework regions of the humanized antibody are referred to as “fully human framework regions.” Human framework regions containing a mixture of human and donor framework residues are referred to herein as “partially human framework regions.” Furthermore, humanized antibodies may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain a desired affinity). Thus, generally, a humanized antibody contains substantially all of at least one, and possibly two, variable regions (in which all or part of the CDRs corresponding to those of non-human immunoglobulins, and all or substantially all of the FRs, are from human immunoglobulin sequences). Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), e.g., that of human immunoglobulin.

[0081] [Human antibodies] In this specification, “human” antibodies are defined as those that are not chimeric or “humanized” and do not originate from a non-human species (in whole or in part). Human antibodies or functional antibody fragments may be of human origin or synthetic human antibodies. In this specification, “synthetic human antibodies” are defined as antibodies having a sequence that is generated in silico from a synthetic sequence based on the analysis of known human antibody sequences, in whole or in part. In silico design of human antibody sequences or fragments can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and using the data obtained therefrom to devise an amino acid sequence. Another example of a human antibody or functional antibody fragment is one encoded by nucleic acids isolated from a library of human-derived antibody sequences (in other words, such a library is based on antibodies taken from natural human sources).

[0082] [Fragments, variants, and derivatives] As described herein, this disclosure encompasses full-length antibodies, as well as their antigen-binding fragments, variants, and derivatives.

[0083] [Fragment] The term “antibody fragment” refers to a polypeptide derived from a “parent” antibody that retains its basic structure and function. Therefore, the antibody fragment preferably has the ability to bind to its specific antigen, i.e., FXI. Furthermore, the antibody fragment according to this disclosure includes the minimum structural requirements of the antibody that enable antigen binding. These minimum requirements include, for example, at least three light chain CDRs (i.e., V L CDR1, CDR2, and CDR3 of the region, in other words, CDR L1 , CDR L2 and CDR L3 ), and / or three heavy chain CDRs (i.e., V H CDR1, CDR2, and CDR3 of the region, i.e., CDR H1 , CDR H2 and CDR H3This is defined by the presence of the antigen-binding site (CDR) of the “parent” antibody. Accordingly, the term “antibody fragment” means a “functional” or “antigen-binding” polypeptide that holds the antigen-binding site (i.e., the CDR and optionally a portion of the FR) of the “parent” antibody. The antibody fragments of this disclosure may be derived, for example, from monoclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, and human “parent” antibodies.

[0084] A preferred antigen-binding antibody fragment comprises at least one, preferably all, of the following: a light chain CDR1 containing the sequence KASQDVSTAVA (SEQ ID NO: 1); a light chain CDR2 containing the sequence LTSYRNT (SEQ ID NO: 2); a light chain CDR3 containing the sequence QQHYKTPYS (SEQ ID NO: 3); a heavy chain CDR1 containing the sequence GYGIY (SEQ ID NO: 4); a heavy chain CDR2 containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5); and a heavy chain CDR3 containing the sequence DYYGSKDY (SEQ ID NO: 6).

[0085] Based on the foregoing, the term “antigen-binding antibody fragment” may refer to a fragment of a full-length antibody such as (s)dAb, Fv, Fab', F(ab')2 or “IgG” (“half-antibody”). Antibody fragments under this disclosure may also be modified antibody fragments such as “mini-bodies,” “triabodies,” or “tetrabodies,” as exemplified by configurations such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, (VH-VL-CH3)2, (scFv-CH3)2, or (scFv-CH3-scFv)2. Furthermore, the definition of the term “antibody fragment” encompasses constructs including monovalent, bivalent, and polyvalent (poly / multi) constructs. Therefore, the term "antibody fragment" encompasses both monospecific constructs that specifically bind to only one target antigen and bispecific / multispecific constructs that specifically bind to one or more antigens (e.g., two, three, or more distinct antigen-binding sites). Furthermore, the definition of "antibody fragment" encompasses molecules consisting of only one polypeptide chain and molecules consisting of multiple polypeptide chains, which may be identical (homodimer, homotrimer, or homooligomer) or different (heterodimer, heterotrimer, or heterooligomer).

[0086] Antibody fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Methods for producing such fragments are well known in the art.

[0087] [Mutant] The term “mutant” refers to a polypeptide containing the amino acid sequence of a “parent” binding molecule, such as an antibody or antibody fragment, but the amino acid sequence contains at least one amino acid modification (e.g., substitution, deletion, or insertion) compared to the “parent” amino acid sequence, such that the mutant can still (specifically) bind to FXI, preferably the A2 domain of human FXI as shown in SEQ ID NO: 7, and they preferably exhibit similar or even improved properties compared to the antibody AB023. Mutants of the binding molecules of this disclosure (e.g., antibodies and antibody fragments) can be prepared by introducing appropriate nucleotide conversions into the nucleic acid encoding the antibody or antibody fragment, or by peptide synthesis. Generally, the above amino acid modifications can be introduced into or present in the variable or constant region, under the premise that the mutant CDR contains two or more cumulatively 10, 11, 12, 13, or 14 amino acid substitutions compared to the AB023 CDR as shown in SEQ ID NOs: 1, 2, 3, 4, 5, and 6. Amino acid modifications can be introduced to modulate antibody properties such as thermodynamic stability, solubility, or viscosity, which can affect drug development ("sequence optimization").

[0088] As described herein, amino acid modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of a binding molecule (preferably an antibody or antigen-binding antibody fragment) as described herein. Any combination of deletions, insertions, and substitutions can be introduced into the “parent” amino acid sequence to reach the final product, insofar as it has the desired characteristics as described herein. Amino acid modifications may also alter the post-translational processes of the binding molecule, such as changing the number or location of glycosylation sites.

[0089] For example, a variant may contain 1, 2, 3, 4, 5, or 6 amino acids (naturally, depending on the length of the CDR) inserted or deleted in each CDR. On the other hand, 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 inserted or deleted in each FR. Amino acid sequence insertions as conceivable herein include, for example, (1) intrasequential insertions of single or multiple amino acid residues, and (2) fusions to the amino-terminus / carboxyl-terminus of a length range from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues to polypeptides containing 100 or more residues. Insertion variants of the binding molecule of this disclosure (e.g., an antibody or antibody fragment) may include fusion products of the antibody or antibody fragment with an enzyme or another functional polypeptide (e.g., a polypeptide that can increase the serum half-life of the binding molecule (e.g., an antibody or antibody fragment)).

[0090] Amino acid substitutions can be introduced into the CDR (e.g., hypervariable region) of the heavy chain and / or light chain, or into the FR region of the heavy chain and / or light chain. For example, conservative amino acid substitutions that can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the relevant residues are intended herein.

[0091] Another variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids substituted in the CDR compared to the CDRs shown in SEQ ID NOs. On the other hand, 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 the framework region (FR), depending on the length of the CDR or FR.

[0092] Generally, when an amino acid is substituted in one or more or all of the heavy chain and / or light chain CDRs, the resulting “mutant” sequence is preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, or 99% identical to the “parent” CDR sequence. Therefore, the length of the CDR affects the number of possible amino acid substitutions, and as a result, mutant sequences are still included in this disclosure. For example, a CDR with five amino acids is preferably 80% identical to the substituted sequence in order to have at least one substituted amino acid. Thus, the CDRs of an antibody construct may have varying degrees of identity with respect to their substituted sequences, for example, CDR L1 It can have 80% identity, while CDR L3 It can have 90% identity.

[0093] The preferred substitution (or exchange) is a conservative substitution. However, any substitution (including one or more non-conservative or exemplary substitutions) is conceivable as long as the antibody construct retains its ability to bind to FXI and / or its CDR, having at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to the substituted sequence.

[0094] As used herein, the term “sequence identity” refers to the range over which two (nucleotide or amino acid) sequences have identical residues at the same position in their alignment, and is often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence are 100% identical, but sequences that are not so highly conserved and have deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several algorithms are available for determining sequence identity using standard parameters. For example, Blast (Altschul et al. (1997) Nucleic Acids Res). 25:3389-3402), Blast2 (Altschul et al. (1990) J.Mol.Biol. 215:403-410), Smith-Waterman (Smith et al. (1981) J.Mol.Biol. 147:195-197), and Clustal W.

[0095] The term "sequence homology" refers to the similarity between two (nucleotide or amino acid) sequences of offspring that originate from a common ancestor. Homologous biological constructs (genes, proteins, structures) are called homologs, and include orthologues and paralogs.

[0096] Preferred binding molecule variants of this disclosure have at least 80%, more preferably at least 90%, most preferably at least 95%, 96%, 97%, 98%, 99%, or nearly 100% sequence identity or homology in the CDR region and exhibit equivalent or improved binding affinity and / or equivalent or improved biological activity to FXI compared to binding molecules containing the “parent” CDR, preferably SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

[0097] Furthermore, the nucleic acid sequence homology or identity between the nucleotide sequences encoding the CDRs of individual variants and the nucleotide sequences shown herein is at least 80%, and preferably increases to at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and nearly 100%.

[0098] In addition to CDRs and FRs, amino acid modifications can also be introduced into the Fc portion of the binding molecule, preferably a monoclonal antibody or its antigen-binding fragment. Such modifications can be used to modulate the functional properties of the antibody, such as interaction with complement proteins like C1q and / or Fc receptors on other immune cells, or regulation of serum half-life or antigen-dependent cell-mediated cytotoxicity (ADCC). Thus, mutations for altering effector function can be introduced into the Fc domain using conventional methods known in the art. Exemplary modifications include Asn297 to Ala297 and Asn297 to Gln297, or Lys3220 to Ala322, resulting in glycosylation of IgG1, and optionally, Leu234 to Ala234 and Leu235 to Ala234, which have been reported to reduce or eliminate antibody-derived cell-mediated cytotoxicity (ADCC) and / or complement-derived cytotoxicity (CDC).

[0099] [Derivative] The term “binding molecule” also encompasses derivatives. Those intended herein are derivatives of antibodies or antibody fragments, as disclosed elsewhere herein. The term “derivative” generally refers to a binding molecule that has been covalently modified to introduce additional functionality. Covalent modification of a binding molecule is generally, but not always, performed post-translation and can be introduced into the binding molecule by reacting specific amino acid residues of the molecule with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues. Derivatization of binding molecules may be used for therapeutic or diagnostic agents, labeling, addition of groups to extend the serum half-life of the molecule, or insertion of non-natural amino acids. Possible chemical modifications of binding molecules in this disclosure include, for example, N-terminal acylation or acetylation, or C-terminal amidation or esterification, or both. Chemical modifications such as alkylation (e.g., methylation, propylation, butylation), arylation, and etherification are also possible.

[0100] [Prolongation of serum half-life] Examples of means for extending the serum half-life of a binding molecule, and preferably of the antibody and its antigen-binding fragment, include the addition of peptide or protein domains that bind to other proteins in the human body (such as serum albumin, immunoglobulin Fc region, or neonatal Fc receptor (FcRn)). Further possible modifications for extending the serum half-life include the extension of amino groups having polypeptide chains of varying lengths (e.g., XTEN technology or PASylation®), and the binding of non-proteinoid polymers. Polymers include, but are not limited to, various polyols such as polyethylene glycol (PEGylated), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol, or carbohydrates such as hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology). In addition, as is known in the art, amino acid substitutions can be made at various positions within the binding molecule to facilitate the addition of polymers.

[0101] [Glycosylation] Another type of covalent modification of a binding molecule, and preferably the antibody and its antigen-binding fragment of this disclosure, involves modifying its glycosylation pattern. As is known, the glycosylation pattern may depend on both the amino acid sequence of the molecule (e.g., the presence or absence of glycosylated amino acid residues) or the host cell or organism from which the protein is produced. Glycosylation of a polypeptide may be either N-linked or O-linked. N-linking refers to the attachment of an asparagine residue of the carbohydrate moiety to a side chain. The addition of an N-linked glycosylation site to a binding molecule is conveniently achieved by altering the amino acid sequence to include 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 may be introduced by the addition or substitution of one or more serine or threonine residues to the starting sequence. Another means of glycosylation of a binding molecule is by the chemical or enzymatic binding of a glycoside to a protein. These methods are advantageous in that they do not require the production of proteins in host cells that have glycosylation capacity for N- and O-linked glycosylation. Depending on the coupling mode used, sugars can be coupled to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine, or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine, or tryptophan, or (f) amide groups of glutamine.

[0102] Similarly, deglycosylation (i.e., removal of the carbohydrate portion present on the binding molecule) can be achieved chemically, for example, by exposing the binding molecule to trifluoromethanesulfonic acid, or enzymatically by using endoglycosidases and exoglycosidases.

[0103] [Labeling] Further potential covalent modifications of the binding molecules of this disclosure include the addition of one or more labels. Labeling groups may be attached to the binding molecules via spacers of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used when carrying out this disclosure. The terms “label” or “labeling group” refer to any detectable label. Generally, labels are classified into various classes depending on the assay in which they are detected. Exemplary labels include, but are not limited to, isotopic labels such as radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 89Zr, 90Y, 99Tc, 111In, 125I, 131I); magnetic labels (e.g., magnetic particles); redox active moieties; fluorescent groups (e.g., FITC, rhodamine, lanthanidrine), chemiluminescent groups, and fluorophores (these may be either “small molecule” fluorophores or protein fluorophores); enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); biotinylating groups; or optical dyes such as predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags, etc.) (including, but not limited to, chromophores, phosphors, and fluorophores).

[0104] [ADC] An antibody-drug conjugate ("ADC"), which may also involve adding a drug such as a small molecule compound to the conjugating molecule, preferably an antibody or its antigen-binding fragment, is an antibody or its antigen-binding fragment linked to a drug or pharmaceutical agent. Binding can be established by covalent bonding or by non-covalent interactions such as electrostatic forces. As is known in the art, various linkers known in the art can be used to form an ADC.

[0105] [Affinity Tag] The binding molecules of this disclosure, and preferably antibodies or their antigen-binding fragments, may also include further domains that can assist in the purification and isolation of molecules (affinity tags). Non-limiting examples of such additional domains include Myc-tags, HAT-tags, HA-tags, TAP-tags, GST-tags, chitin-binding domains (CBD-tags), maltose-binding proteins (MBP-tags), Flag-tags, Strep-tags and their variants (e.g., StrepII-tags), and peptide motifs known as His-tags.

[0106] The above fragments, variants, and derivatives can be further adapted, for example, to improve their antigen-binding properties. For example, F(ab')2 or Fab is C H1 Region and C L The Fv polypeptide may be designed to minimize or completely eliminate intermolecular disulfide interactions that occur with the region. The Fv polypeptide allows Fv to form the desired structure for antigen binding. H Domain and V L The Fab fragment may further include a polypeptide linker between the domains. The Fab fragment also includes a constant region of the light chain and a first constant region (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has several residues added to the carboxyl terminus of the heavy chain CH1 region, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein-name for Fab' fragments in which the cysteine ​​residue in the constant region has a free thiol group. F(ab')2 antibody fragments were initially produced as pairs of Fab' fragments having a hinged cysteine ​​residue between them.

[0107] The conjugated molecules of the present invention may be provided in an “isolated” or “substantially pure” form. Where “isolated” or “substantially pure” is used herein, it means that the conjugated molecule has been identified, separated, and / or recovered from the components of its production environment. Such “isolated” conjugated molecules do not contain, or substantially contain, other contaminants from the production environment of the conjugated molecule that could interfere with its therapeutic or diagnostic use. Contaminants may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Accordingly, “isolated” conjugated molecules are prepared by at least one purification step that removes or substantially removes these contaminants. The above provisions are also applicable to “isolated” polynucleotides with necessary modifications.

[0108] [Specific binding] The binding molecules of this disclosure (e.g., antibodies and their antigen-binding fragments) can advantageously bind to various mammalian FXI (preferably human FXI) and contain or consist of the amino acid sequence shown in SEQ ID NO: 7. In all grammatical forms, the terms “binding” and “recognizing” are used interchangeably herein. Preferably, the binding molecule binds specifically to FXI. The term “specifically binding” generally indicates that the binding molecule, for example, an antibody or its antigen-binding fragment as described herein, binds more readily to its intended target epitope via its antigen-binding site than to random, unrelated non-target epitopes. The term “specifically binding” indicates that the affinity of the binding molecule to its target epitope is at least about 5 times, preferably 10 times, more preferably 25 times, even more preferably 50 times, and most preferably 100 times or more than its affinity to non-target epitopes. Thus, the binding molecule, i.e., an antibody, or its antigen-binding fragment, variant, or derivative, is more readily bound to the antibody’s K against non-target epitopes. D A dissociation constant (K) smaller than DWhen binding to a target epitope, it can be considered to specifically bind to that target epitope. The binding molecules of the present disclosure can also be described in terms of their binding affinity for mammalian FXI (preferably human FXI). The terms "affinity" or "binding affinity" refer to the strength of the binding between an individual epitope and an antigen-binding domain (i.e., the CDR of the binding molecule). The affinity of a particular binding molecule for its specific epitope is often determined by measurement of the equilibrium association constant (ka) and the equilibrium dissociation constant (kd), and calculation of the quotient of kd to ka (K D =kd / ka). Binding affinity can be readily determined using conventional techniques such as equilibrium dialysis; using a BIAcore 2000 instrument; radioimmunoassay using a radiolabeled target antigen; and other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method described in "Kaufman RJ and Sharp PA. (1982) J Mol Biol. 159:601-621". The preferred binding affinity of the binding molecules of the invention is a dissociation constant or K D of 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 [[ID=z8]]M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or less than 10 -15 M are included.

[0109] 〔Cross-reactivity〕 However, the term "specifically binds" does not preclude the possibility that a binding molecule that (specifically) binds to human FXI may cross-react with FXI proteins from different species. Therefore, the binding molecules of this disclosure may also bind to FXI from other mammalian species.

[0110] "Interspecies" binding or recognition means the binding of the binding domain described herein to the same target antigen in human and non-human species. Therefore, "interspecies specificity" should be understood as interspecies responsiveness to FXI expressed in different species, rather than to antigens other than FXI. For example, a binding domain that binds to human FXI (preferably the A2 domain containing amino acids 91-175 of the amino acid sequence shown in SEQ ID NO: 7) also binds to other non-human FXI and, preferably, to a corresponding or similar region characteristic of amino acids 91-175 of the amino acid sequence shown in SEQ ID NO: 7.

[0111] [Biological activity] The binding molecules provided herein are biologically active, i.e., they bind to mammalian FXI and / or FXIa and are thought to inhibit some of their respective biological functions. Specifically, the "biologically active" binding molecules form an immune complex with FXI based on this disclosure, and the FXI-AB023 immune complex cannot be efficiently activated by FXIIa or self-activated. However, the immune complex can be activated by thrombin. Once the FXI-AB023 complex is activated to FXIa-AB023, its activity in converting FXII zymogen to FXIIa is reduced. Furthermore, the activated complex can carry out the conversion of FIX to factor IXa without loss of function, preferably resulting in complete or partial inhibition of contact activation while preserving thrombin-dependent hemostatic feedback activation of the blood. Thus, the binding of biologically active binding molecules to their targets, FXI and / or FXIa, is thought to result in anticoagulant activity, for example, in assays that initiate coagulation via a contact-activated complex. In other words, the binding molecules of this disclosure are thought to exert their beneficial functions by a) binding to FXI, thereby preventing its conversion to its active form FXIa by FXIIa or inhibiting its self-activation, and / or b) binding to FXIa, thereby reducing its binding and the activation of FXII. Thereafter, the binding molecules of this disclosure preferably interfere with the contact activation complex, thereby advantageously reducing pathological processes associated with contact activation, including inflammation and thrombosis, without impairing hemostatic processes independent of the contact activation complex.

[0112] The anticoagulant activity of the binding molecule can be determined in vitro as described herein. Briefly, activated partial thromboplastin time (aPTT) in normal human or other mammalian plasma, which measures contact activation-dependent thrombinogenesis, was measured using a commercially available test kit (SynthASil reagent from Instrumentation Laboratories, Bedford, MA) in the presence of various concentrations of the binding molecule or the corresponding solvent. The test compound was incubated at 37°C for approximately 3 minutes with plasma containing endogenous FXI and SynthASil reagent (colloidal silica activator) typically in the concentration range of 20–45 nM. Coagulation was then initiated by the addition of 25 mM calcium chloride, and the time it took for coagulation to occur was measured to determine the concentration of the test substance that showed an aPTT prolongation effect of approximately 2.0 times. The binding molecules of this disclosure may result in a 1.5-fold, 2.0-fold, or greater aPTT prolongation.

[0113] Advantageously, the binding molecules provided in this disclosure, preferably monoclonal antibodies and their antigen-binding fragments, exhibit the above-described biological properties, and therefore, they are promising new agents for the suppression of thrombosis and inflammation. This is because, since the binding molecules are thought to bind specifically to FXI, they are thought not to impair or severely impair hemostasis, and thereby preferably not to increase the risk of bleeding.

[0114] [Polynucleotides] This disclosure further relates to the binding molecules or V of this disclosure. H or V L It provides polynucleotide / nucleic acid molecules that encode a region.

[0115] As used herein, the term "polynucleotide" includes polyribonucleotides and polydeoxyribonucleotides. For example, each modified or unmodified RNA or DNA in single-stranded and / or multi-stranded (e.g., double-stranded) form, linear or circular, or a mixture thereof (including hybrid molecules). Polynucleotides can include conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNA)). The polynucleotides of the present disclosure can also include one or more modified bases (e.g., tritylated bases) and unusual bases (e.g., inosine). Other modifications, including chemical, enzymatic, or metabolic modifications, are contemplated as long as the binding molecules of the present disclosure can be expressed from the polynucleotides. The polynucleotides can be provided in isolated form as defined herein. Polynucleotides can include regulatory sequences such as transcriptional control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

[0116] The present invention relates to an immunoglobulin heavy chain region (V H region) having at least one CDR in the V region with an amino acid sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 8, or a polynucleotide comprising or consisting of a nucleic acid encoding such an immunoglobulin heavy chain region (V H region). A binding molecule comprising the encoded CDR or V H domain is preferably considered to be able to bind to FXI and, upon complex formation between FXI and the binding molecule, exhibit the desired biological activity described herein.

[0117] The present invention relates to a nucleic acid comprising or consisting of a nucleic acid encoding an immunoglobulin light chain domain (V L region), and the V LThe CDR of the region provides a polynucleotide having an amino acid sequence that is at least approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or 100% identical to SEQ ID NO: 9. The encoded CDR or V L The binding molecule containing the region can bind to FXI, and it is believed that the formation of a complex between FXI and the binding molecule preferably exhibits the desired biological activity described herein.

[0118] The polynucleotides described herein may or may not include additional nucleotide sequences encoding, for example, a signal peptide that directs the secretion of the encoded polypeptide, an antibody constant region described herein, or other heterologous polypeptides described herein. Such polynucleotides may therefore encode fusion polypeptides, fragments, variants, and other derivatives of the binding molecules described herein.

[0119] Furthermore, this disclosure includes compositions comprising one or more of the above-described polynucleotides. Compositions comprising a first polynucleotide and a second polynucleotide are also provided herein. The first polynucleotide is V as described herein. H The region is coded. And the second polyphonic octet is V as described herein. L The region, specifically V shown in Sequence ID 8 H The region and / or V shown in Sequence ID 9 L This code codes for a composition that includes or consists of regions.

[0120] [Production of polynucleotides] The polynucleotides of this disclosure may be produced by conventional methods known in the art. For example, if the nucleotide sequence of the binding molecule is known, the polynucleotide encoding the binding molecule may be assembled from chemically synthesized oligonucleotides, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. The polynucleotide encoding the binding molecule may be obtained from nucleic acids (e.g., poly(A)+mRNA) isolated from any tissue or cells expressing the binding molecule (e.g., hybridoma cells) by a suitable source (e.g., a cDNA library, or by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using a gene sequence-specific oligonucleotide probe for identification (e.g., a cDNA clone from a cDNA library encoding the binding molecule)).

[0121] Once the nucleotide sequence of the binding molecule and the corresponding amino acid sequence are determined, the nucleotide sequence may be modified using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc., thereby introducing one or more nucleotide substitutions, additions, or deletions into the polynucleotide sequence to create non-naturally occurring fragments, variants, or derivatives of the binding molecule, i.e., monoclonal anti-FXI antibodies (e.g., immunoglobulin heavy chain or light chain regions) as described herein (see, for example, the techniques described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012)).

[0122] 〔vector〕 Vectors comprising polynucleotides as described herein are further provided. The polynucleotides encode the binding molecules of this disclosure, preferably monoclonal antibodies or their antigen-binding fragments. A “vector” is a nucleic acid molecule used, for example, as a vehicle for transferring (external) genetic material into a host cell that can replicate and / or express it.

[0123] The term "vector" includes, but is not limited to, plasmids, viral vectors (retroviral vectors, lentiviral vectors, adenovirus vectors, vaccinia virus vectors, polyomavirus vectors, and adenovirus-associated vectors (AAVs)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). Generally, a vector itself is a nucleotide sequence, and a vector is generally a DNA sequence containing an insert (transgene) and a larger sequence that acts as a "backpawn" of the vector. An engineered vector may include a starting point for autonomous replication in host cells (if stable expression of polynucleotides is desired), a selection marker, and restriction enzyme cleavage sites (e.g., multiple cloning sites, MCSs). A vector may further include a promoter, a genetic marker, a reporter gene, a targeting sequence, and / or a protein purification tag. A vector called an expression vector (expression construct) is specifically designed for the expression of a transgene in target cells and generally contains a regulatory sequence. Numerous suitable vectors are known to those skilled in the art, and many are commercially available. Examples of suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012).

[0124] [Targeted vector] Targeted vectors can be used to integrate polynucleotides into the chromosomes of host cells (Sambrook et al., 2012). In short, appropriate methods include homologous recombination or the use of hybrid recombinases that specifically target the sequence of the integration site. Targeted vectors can be circular and linearized before use for homologous recombination. Alternatively, the exogenous polynucleotide may be a DNA fragment ligated by fusion PCR, or a synthetically constructed DNA fragment subsequently recombined into the host cell. Non-homologous recombination, resulting in random or untargeted integration, is also possible.

[0125] [Manufacturing] [Expression vector] An “expression vector” or “expression construct” may be used for the transcription of heterologous polynucleotide sequences (e.g., those encoding the binding molecules of this disclosure) and for the translation of their mRNA in a suitable host cell (this process is also referred to herein as “expression” of the binding molecules of this disclosure). An expression vector may include, in addition to an origin of replication, a selection marker, and restriction enzyme cleavage sites, one or more regulatory sequences operably ligated to the heterologous polynucleotide to be expressed.

[0126] The term "regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a operably linked coding sequence of (heterogeneous) polynucleotides in a host organism, and therefore includes transcriptional and translational regulatory sequences. Regulatory sequences necessary for the expression of heterogeneous polynucleotide sequences in prokaryotes include, for example, promoters, optionally operator sequences, and ribosome-binding sites. In eukaryotes, promoters, polyadenylation signals, enhancers, and optionally splice signals may be required. Furthermore, specific initiation and secretion signals may also be introduced into the vector to enable the secretion of the polypeptide of interest into the culture medium.

[0127] Nucleic acids are "operably ligated" when they are functionally related to another nucleic acid sequence, for example, on the same polynucleotide molecule. For example, a promoter is operably ligated to a heterologous gene's coding sequence if it can influence the expression of that coding sequence. A promoter is placed upstream of the gene encoding the polypeptide of interest and can regulate the gene's expression.

[0128] Exemplary regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV) (CMV promoter / enhancer, etc.), Simianvirus 40 (SV40 promoter / enhancer, etc.), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyomas. For further descriptions of viral regulatory elements and their sequences, see, for example, Stinski's U.S. Patent No. 5,168,062; Cousens et al.'s U.S. Patent No. 4,510,245; and Koszinowski et al.'s U.S. Patent No. 4,968,615. Expression vectors may also include origins of replication and selection markers.

[0129] The vectors of this disclosure may further include one or more selection markers. Suitable selection markers for use with eukaryotic host cells include, but are not limited to, the herpes simplex virus thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and adenine phosphoribosyltransferase (APRT) genes. Other genes include DHFR (methotrexate resistance), GPT (mycophenolate resistance), NEO (G-418 resistance), and HYGR (hygromycin resistance). Vector amplification may be used to increase expression levels. Generally, the selection marker gene may be directly ligated to the polynucleotide sequence to be expressed or introduced into the same host cell by co-transformation.

[0130] Accordingly, in consideration of the foregoing, the Disclosure further provides one or more of the polynucleotide sequences described herein that can be inserted into a vector. Accordingly, the Disclosure provides a replicable vector comprising a nucleotide sequence encoding the binding molecule of the Disclosure, or its heavy or light chain, or a variable domain of the heavy or light chain, operably linked to a promoter. Such a vector may comprise a nucleotide sequence encoding a constant region of the binding molecule, and the variable domain of the binding molecule may be cloned into such a vector for expression of the entire heavy or light chain.

[0131] [Host cell] In general, various host cells can be used to express the binding molecules of this disclosure from an expression vector. As used herein, “host cell” means a cell that may be / is a recipient of a polynucleotide or vector encoding the binding molecules of this disclosure. Specifically, host cells may further express and optionally secrete the binding molecules. In describing the process of obtaining the binding molecules from host cells, the terms “cell” and “cell culture” are used interchangeably to indicate the source of the binding molecules unless otherwise specified. The term “host cell” also includes “host cell line.”

[0132] Generally, this term includes prokaryotic or eukaryotic cells. It also includes, but is not limited to, bacterial, yeast, fungal, and plant cells, as well as animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells. The polynucleotides and / or vectors of this disclosure may be introduced into host cells using conventional methods known in the art (e.g., transfection, transformation, etc.).

[0133] "Transfection" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term is often used for non-viral methods in eukaryotic cells. Transduction is often used to describe the transcription of nucleic acid molecules or polynucleotides via viruses. In animal cell transfection, it means creating a transient pore or "hole" in the cell membrane to allow for the uptake of a substance. Transfection can be carried out by using calcium phosphate, by electroporation, by compressing cells, or by mixing cationic lipids with a material to produce liposomes (which fuse with the cell membrane and deposit their carriers inside). Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0134] The term "transformation" is used to describe the nonviral transcription of nucleic acid molecules or polynucleotides (including vectors) into bacteria and into non-animal eukaryotic cells, including plant cells. Therefore, transformation is a genetic change resulting from the direct uptake of a bacterial or non-animal eukaryotic cell across its cell membrane from its surroundings, followed by the incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be influenced by artificial means. For transformation to occur, the cell or bacterium must be in a state of competence. Competence can arise as a time-limited response to environmental conditions such as starvation and cell density. Prokaryotic transformation techniques may include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. Plant transformation techniques include Agrobacterium-mediated transcription, such as by A. tumefaciens, rapidly propelled tungsten or gold microprojectiles, electroporation, microinjection, and polyethylene glycol-mediated uptake.

[0135] Accordingly, in consideration of the foregoing, this disclosure further provides a host cell comprising at least one polynucleotide sequence and / or vector described herein.

[0136] For the expression of the binding molecule of this disclosure, a host cell may be selected that, if desired, regulates the expression of the inserted polynucleotide sequence and / or modifies and processes the gene product (i.e., RNA and / or protein). Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the gene product may contribute to the function of the binding molecule. Different host cells have characteristic and specific mechanisms with respect to post-translational processing and modification of gene products. A suitable cell line or host system may be selected to ensure the precise modification and processing of the product. For this purpose, eukaryotic host cells having cellular mechanisms for appropriate processing of the primary transcript, glycosylation of the gene product, and phosphorylation may be used.

[0137] Exemplary mammalian host cells that may be used to express the binding molecules provided herein include Chinese hamster ovary (CHO cells), NSO, COS(SV40), DHFR-negative CHO cells such as DG44 and DUXB1 (used with DHFR selection markers such as those described in U.S. Patent No. 5,179,017, for example), DG44 and DUXB1 (used with DHFR selection markers such as those described in U.S. Patent No. 4,634,665, for example), NSO, and COS(SV40). This includes CVI derivatives with the T antigen, HEK293 (human kidney), and SP2 (mouse myeloma) cells. Other exemplary host cell lines include, but are not limited to, HELA (human cervical cancer), CVI (monkey kidney), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), and RAJI (human lymphocytes). Host cell lines are available from commercial services, the American Tissue Culture Collection, or published literature.

[0138] Non-mammalian cells such as bacteria, yeasts, insects, or plant cells are also readily available and, in principle, can be used for the expression of the binding molecules of this disclosure. Exemplary bacterial host cells include enterobacteria, such as Escherichia coli, Salmonella; Bacillaceae, including Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae.

[0139] Other host cells include yeast cells such as Saccharomyces cerevisiae and Ichiapastoris. Insect cells include, but are not limited to, Spodopterafrugiperda cells.

[0140] Based on the above, possible expression systems (i.e., host cells containing expression vectors) include the following microorganisms: Bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors (e.g., Escherichia coli, Bacillus subtilis); yeast transformed with recombinant yeast expression vectors (e.g., Saccharomyces, Pichia); insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus); plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid); or mammalian cell lines having recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) (e.g., COS, CHO, BLK, 293, 3T3 cells).

[0141] Eukaryotic cells are preferred for the expression of the binding molecule of this disclosure. Therefore, CHO cells containing a eukaryotic vector having a polynucleotide sequence encoding the binding molecule of this disclosure (for example, which can be operably ligated to the major early promoter (MIEP) of human cytomegalovirus (CMV)) are a useful expression system for producing the binding molecule of this disclosure.

[0142] 〔culture〕 Host cells containing an expression vector are grown under conditions suitable for the production of the binding molecules described herein (e.g., light and heavy chains as described herein), and assays are performed for heavy and / or light chain protein synthesis. Accordingly, the present disclosure includes host cells containing a polynucleotide encoding the binding molecule of the present disclosure, or its heavy or light chain, operably linked to a promoter. For the expression of a double-chain antibody, vectors encoding both the heavy and light chains may be co-expressed in the host cell for the expression of the entire molecule.

[0143] 〔purification〕 If the binding molecules of this disclosure are recombinantly expressed, they can be purified by any purification method known in the art. For example, they can be purified by chromatography (e.g., ion exchange chromatography (e.g., hydroxyapatite chromatography), affinity chromatography, protein A, protein G, or lectin affinity chromatography, sizing column chromatography), centrifugation, differential solubility, hydrophobic interaction chromatography, or any other standard technique for protein purification. Those skilled in the art can easily select an appropriate purification method based on the individual characteristics of the recovered binding molecules.

[0144] Accordingly, in light of the foregoing, the present process also provides a method for producing a binding molecule, comprising the steps of culturing host cells as defined herein under conditions that enable the expression of the binding molecule, and optionally, recovering the binding molecule produced from the culture.

[0145] [Pharmaceutical composition] The Disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of the binding molecule, nucleic acid, vector and / or host cell of the Disclosure, and optionally one or more pharmaceutically acceptable excipients or carriers. A preferred pharmaceutical composition comprises the antibody of the Disclosure and optionally one or more pharmaceutically acceptable excipients.

[0146] Accordingly, in one embodiment, this disclosure relates to a pharmaceutical composition comprising a conjugating molecule described herein, preferably an anti-FXI antibody or its antigen-binding fragment, as an active substance. Accordingly, the use of conjugating molecules for the manufacture of pharmaceutical compositions is also assumed herein. The term “pharmaceutical composition” preferably means a composition suitable for administration to a subject, more specifically to a human. However, compositions suitable for administration to non-human animals are also included in this term.

[0147] The pharmaceutical composition and its components (i.e., the active substance and any excipients) are preferably pharmaceutically acceptable, i.e., capable of eliciting the desired therapeutic effect without causing undesirable local or systemic effects in the recipient. The pharmaceutically acceptable compositions of this disclosure may, for example, be sterile and / or pharmaceutically inactive. Specifically, the term “pharmaceutically acceptable” may mean approved by a regulatory authority or other generally recognized pharmacopoeia for use in animals, preferably humans.

[0148] The binding molecules described herein are preferably present in the pharmaceutical composition in a therapeutically effective amount. “Therapeutically effective amount” means the amount or dose of the binding molecule that elicits the desired therapeutic effect. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell culture, experimental animals, or clinical trials. For example, in clinical trials, ED 50 (A therapeutically effective dose for 50% of the population) and LD 50 (A dose that is lethal to 50% of the population) is used. The dose ratio between therapeutic effect and toxic effect is the therapeutic index, or ratio ED. 50 / LD 50 It can be expressed as such. Pharmaceutical compositions exhibiting a large therapeutic index are preferred.

[0149] As described herein, the pharmaceutical composition may optionally contain one or more excipients and / or additional active substances.

[0150] Antibodies and their fragments are generally administered parenterally, preferably intravenously (by 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 oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous solvents may be selected from the group consisting of water, alcohol / aqueous solutions, emulsions or suspensions containing physiological saline, and buffering media such as phosphate-buffered physiological saline, but are not limited. Parenteral vehicles further include aqueous sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or non-volatile oils. Other suitable pharmaceutically acceptable carriers, diluents, and / or excipients are well known in the art. The conjugated molecules according to this disclosure (e.g., antibodies or antibody fragments thereof) may be combined with the above-mentioned excipients for forming pharmaceutically acceptable carriers, diluents, and / or pharmaceutically acceptable compositions. The pharmaceutical composition may contain the binding molecule of this disclosure in an aqueous carrier containing a buffer selected from the group consisting of histidine buffer, acetate buffer, citrate buffer, and histidine / HCl buffer. Further buffer and formulation information is available to those skilled in the art, for example, Wang W This information is from et al., J. Pharmaceutical Sci. 2007 Jan(1):1-26. For example, preservatives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present.

[0151] The pharmaceutical composition may further contain a protein carrier, preferably of human origin, such as serum albumin or immunoglobulin. In one embodiment, the pharmaceutical composition comprises the binding molecule in lyophilized form, which is preferably reconstituted into a solution or suspension before administration. In other embodiments, the pharmaceutical composition comprises the binding molecule and is in a liquid state.

[0152] After preparing the pharmaceutical compositions of this disclosure, and optionally appropriate excipients, they can be placed in appropriate containers and labeled for treatment of the indicated condition. Such labels may include, for example, the dosage, frequency of administration, and method of administration.

[0153] [Additional active substances] This disclosure further provides pharmaceutical or pharmaceutically active compositions comprising the compounds of the present invention and one or more further active ingredients, including treatment and / or prevention of the disorders described herein. Preferred examples of active ingredients suitable for combination include, but are not limited to, the following: - Lipid-lowering substances, particularly HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors. These include, but are not limited to, lovastatin (Mevacol), simvastatin (Zocol), pravastatin (Pravacol), fluvastatin (Rescol), and atorvastatin (Lipitor); - Coronary artery treatment drugs / vasodilators, especially ACE (angiotensin-converting enzyme) inhibitors (including but not limited to captopril, lisinopril, enalapril, ramipril, cilazapril, benazepril, hosinopril, quinapril and perindopril), or AII (angiotensin II) receptor antagonists (including but not limited to embsartan, losartan, valsartan, irbesartan, candesartan, eprosartan and temisartan), or adrenergic receptor antagonists (including but not limited to carvedilol, alprenolol, bisoprolol, acebutolol, atenolol, betaxolol, carteolol, metoprolol, nadolol, penbutolol, pindolol, propanol and timolol), or α-1-adrenergic receptor antagonists (prazosin, bunazosin, This includes, but is not limited to, bunazosin, doxazosin, and terazosin; or diuretics (including, but not limited to, hydrochlorothiazide, furosemide, bumetanide, pyretanide, torasemide, amiloride, and dihydralazine); or calcium channel blockers (including, but not limited to, verapamil and diltiazem); or dihydropyridine derivatives (including, but not limited to, nifedipine (adalat) and nitrendipine (bayotensin)); or nitro preparations (including, but not limited to, isosorbide 5-mononitolate, isosorbide dinitrate, and glycerol trinitrate); or substances that cause an increase in cyclic guanosine monophosphate (cGMP) (including, but not limited to, riociguat, and soluble guanylate cyclase stimulants); - Plasminogen activators (thrombolytic agents / fibrinolytic agents) and compounds that promote thrombolysis / fibrinolysis (including, but not limited to, inhibitors of plasminogen activator inhibitors (PAI inhibitors)), or thrombin-activated fibrinolysis inhibitors (TAFI inhibitors) (including, but not limited to, tissue plasminogen activator (t-PA), streptokinase, leteplase, and urokinase); -Anticoagulants (anticoagulants) (including, but not limited to, low molecular weight heparins (LMW), including, but not limited to, heparin (UFH), tinzaparin, sertoparin, parnaparin, nadroparin, alderoparin, enoxaparin, reviparin, dalteparin, danaparoid, semloparin (AVE 5026), admiparin (M118), and EP-42675 / ORG42675); - Direct thrombin inhibitors (DTIs), including but not limited to Pradaxa (dabigatran), atesegatran (AZD-0837), DP-4088, SSR-182289A, argatroban, bivalirudine, and tanogitran (BIBT-986 and prodrug BIBT-1011), and hirudin; - Direct factor Xa inhibitors, including but not limited to rivaroxaban, apixaban, edoxaban (DU-176b), betrixaban (PRT-54021), R-1663, dalexaban (YM-150), otamixaban (FXV673 / RPR-130673), retaxaban (TAK-442), razakisaban (DPC-906), and inhibitors DX-9065a, LY-517717, tanogitran (BIBT-986, prodrug: BIBT-1011), hydraparinux and fondaparinux, and platelet aggregation inhibitors (platelet aggregation inhibitors); - Platelet aggregation inhibitors (platelet aggregation inhibitors) including, but not limited to, acetylsalicylic acid (e.g., aspirin), ticlopidine (Ticlid), clopidogrel (Plavix), prasugrel, ticagrelor, cangrelol, erinogrel, and borapaxal; - Fibrinogen receptor antagonists (glycoprotein-IIb / IIIa antagonists) (including, but not limited to, absiximab, eptifivatide, tyrofiban, ramifiban, refuradafiban, and fludafiban); - Antiarrhythmic drugs; - Various antibiotics or antifungal drugs as either a calculated treatment (prior to the presence of a microbiological diagnosis) or a specific treatment; - Vasopressors (including, but not limited to, norepinephrine, dopamine, and vasopressin); - Treatment of muscle contractions (including, but not limited to, dobutamine); - Recombinant human activated protein C, e.g., xygris; - Blood products (including, but not limited to, red blood cell concentrates, platelet concentrates, erythropietin, and fresh frozen plasma); - Inhibitors of platelet adhesion such as GPVI and / or GPIb antagonists (including, but not limited to, revacept or caplacizumab); - Inhibitors of VEGF and / or PDGF-dependent signaling pathways (including, but not limited to, ranibizumab, bevacizumab, KH-902, pegaptanib, ramucirumab, SqualaminoderBevasiranib, apatinib, axitinib, brivanib, sediranib, dovitinib, lenvatinib, linifanib, motesanib, pazopanib, regorafenib, sorafenib, sunitinib, tivozanib, vandetanib, batalanib, Vargatef, or E-10030); - Inhibitors of the angiopoietin-Tie signaling pathway (including, but not limited to, AMG386); - Inhibitors of Tie2 receptor tyrosine kinase activity; - Inhibitors of integrin-dependent signaling pathways, including boroxiximab, sirengitide, or ALG1001; - Inhibitors of PI3 kinase-AKT-mTor-dependent signaling (including, but not limited to, XL-147, perifosine, MK2206, sirolimus, temsirolimus, or everolimus); - Corticosteroids (including, but not limited to, hydrocortisone, fludrocortisone, anecoltaben, betamethasone, dexamethasone, triamcinolone, fluocinolone, or fluocinolone acetonide); - Inhibitors of the ALK1-Smad1 / 5-dependent signaling pathway (including, but not limited to, ACE041); - Cyclooxygenase inhibitors (including, but not limited to, bromfenac or nepafenac); - Kallikrein-kinin inhibitors (including, but not limited to, safotibant or ecalantide); - Inhibitors of the Sphingosin-1-phosphat-dependent signaling pathway (including, but not limited to, sonepcizumab); - C5a receptor inhibitors (including, but not limited to, eculizumab); - 5HT1a receptor inhibitors (including, but not limited to, tandospirone); - Inhibitors of the Raf-Mek-Erk-dependent signaling pathway, inhibitors of the MAPK signaling pathway; inhibitors of the FGF signaling pathway, inhibitors of endothelial cell proliferation; and compounds that can induce apoptosis; or, - A photodynamic therapy consisting of exposure to an active substance and light, the active substance being, for example, verteporfin.

[0154] For the purposes of this disclosure, “combination” does not mean only dosage forms containing all components (so-called fixed combinations) and combination packs containing components separated from each other. However, components are also components that are administered simultaneously or sequentially if they are used for the prevention and / or treatment of the same disease. Similarly, it is possible to combine two or more active ingredients with each other, meaning that they are each two-component or multi-component combinations.

[0155] [Administration] Various routes are applicable to the administration of the pharmaceutical compositions according to this disclosure. Administration can be achieved parenterally. Methods of parenteral delivery include, for example, topical, intra-arterial, intramuscular, subcutaneous, intramedullary, subarachnoid, intraventricular, intravenous, intraperitoneal, intrauterine, vaginal, sublingual, or intranasal administration.

[0156] Pharmaceutical formulations for parenteral administration contain an aqueous solution of the active compound. For injection, the pharmaceutical compositions of this disclosure may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiologically buffered saline. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, the suspension of the active compound may be prepared as a suitable lipophilic injection suspension. Exemplary lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. The suspension may also optionally contain a suitable stabilizer or agent that can increase the solubility of the compound to enable the preparation of a highly concentrated solution. For topical or intranasal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are known in the art. Further details regarding formulation and administration techniques can be found in the 22nd edition of Remington's Pharmaceutical Sciences (EdMaack Publishing Co, Easton, Pa, 2012).

[0157] [Treatment] The term “to treat” or “treatment” includes therapeutic or prophylactic measures for any disease described herein. “Therapeutic or prophylactic measures” include prophylactic measures aimed at delaying or completely preventing clinical and / or pathological signs, or therapeutic measures aimed at improving or relieving clinical and / or pathological signs. Thus, the term “treatment” also includes improvement or prevention of any disease described herein. Treatment may also mean extending survival compared to the survival expected without treatment. Those requiring treatment include those who already have a condition or disability, those prone to developing a condition or disability, or those for whom prevention of a condition or disability is desirable.

[0158] The terms “subject,” “individual,” “animal,” “patient,” or “mammal” are used interchangeably herein to refer to any subject, preferably a mammalian subject, for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, non-human primates, domesticated animals, companion animals, zoo animals, sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and so on.

[0159] As used herein, subject-related terms such as “beneficial” and “requiring treatment” include subjects who are beneficial to the administration of humanized monoclonal antibodies, their conjugated fragments, variants, or derivatives.

[0160] [Dosage] The precise dosage (therapeutic effective dose) of the binding molecule, polynucleotide, vector, or host cell can be determined by those skilled in the art using known techniques. The appropriate dosage provides a sufficient amount of the binding molecule and is preferably therapeutically effective, i.e., elicits the desired therapeutic or preventive effect.

[0161] As is well known in the field, the treatment (e.g., prevention, maintenance of remission, acute onset of disease), route, timing and frequency of treatment, timing and frequency of therapeutic agents, age, weight, general health, sex, diet, severity of the condition, drug combinations, sensitivity to response, and tolerance / response to treatment may be determined and adjusted. An appropriate therapeutic effective dose range can be determined using data obtained from cell culture assays and animal studies, and also from ED. 50It may include. Dosage may vary from 0.1 to 100,000 μg, total dose of approximately 2 g, depending on the route of administration. Exemplary doses of the conjugate molecule may range from approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, or approximately 0.1 mg / kg to approximately 1 mg / kg. Guidance on dosage and delivery methods is provided in the literature. It is recognized that treatment may require a single dose or multiple doses of the conjugate molecule, polynucleotide, vector, or host cells of this disclosure that are effective in treatment. For example, some pharmaceutical compositions may be administered as a single dose, regularly at specific time intervals, every 3-4 days, weekly, or every 2 weeks, once within 1 month, or once within 2 months, depending on the formulation, half-life, and clearance rate of the formulation. The determination of each applicable variable is well known to those skilled in the art.

[0162] 〔kit〕 This disclosure further relates to pharmaceutical packs and kits comprising one or more containers or vials filled with one or more active substances of the above-described pharmaceutical compositions of this disclosure, accompanied by instructions for use thereof. Accordingly, kits comprising the binding molecules, polynucleotides, vectors, host cells, and / or pharmaceutical compositions described herein are also provided herein. The kits described herein may be used for the treatment of the diseases described herein or for other purposes.

[0163] A notice in the form prescribed by the government agency regulating the manufacture, use, or sale of a pharmaceutical or biological product, reflecting approval by the agent for the manufacture, use, or sale of the product for human administration, may be associated with the above container.

[0164] The kit may contain one or more active substances (optionally formulated as a pharmaceutical composition comprising one or more excipients). Suitable active substances are listed above in the context of pharmaceutical compositions and may also be considered as part of the kit of the present invention. Additional active substances may be administered to the patient simultaneously or sequentially with respect to binding molecules, nucleic acid sequences, vectors, host cells, and / or pharmaceutical compositions. This disclosure further encompasses the administration of active substances via different routes, e.g., orally and intravenously.

[0165] Kits comprising polynucleotide sequences encoding the binding molecules of this disclosure are further conceivable herein. The polynucleotides may be provided in vectors such as plasmids suitable for transfection into and expression by host cells. Such vectors and host cells are described herein.

[0166] [Therapeutic use] This disclosure further provides the conjugation molecules of the present invention, preferably antibodies and their antigen-binding fragments, for use as agents for the treatment and / or prevention of diseases in humans and / or animals.

[0167] The Disclosure further provides conjugating molecules, preferably antibodies and their antigen-binding fragments, for use in the treatment and / or prevention of disorders (e.g., cardiovascular disorders, preferably thrombotic or thromboembolic disorders and / or thrombotic or thromboembolic complications, and inflammatory conditions, preferably autoimmune inflammation or infection-associated inflammatory responses).

[0168] FXIa is an enzyme involved in the coagulation process, as it can be activated by both thrombin and FXIIa in the context of coagulation. FXI is a central component of the transition from coagulation initiation to amplification and proliferation. Furthermore, FXIa is a component for the initiation and maintenance of pathological blood coagulation within blood vessels. The blood contact activation system can be activated on negatively charged vascular inner surfaces, including not only the exposure of flowing blood to subendothelial or extravascular material containing collagen and laminin, and the exposure of surface structures of foreign body cells (e.g., bacteria), but also artificial surfaces such as artificial blood vessels, catheters, stents, ventricular assist devices, valves, and extracorporeal life support systems such as artificial lungs, pumps, tubing, and dialyzers. On surfaces, factor XII (FXII) is activated to factor XIIa (FXIIa), followed by the activation of FXI attached to the surface of FXIIa. FXI can also be self-activated on negatively charged surfaces. This activation of FXI leads to downstream thrombin generation, as well as feedback amplification of all contact-activated complex enzymes, including FXI, FXII, and prekallikrein.

[0169] In contrast, hemostatic thrombin generation is driven by feedback activation of FXI by the TF / FVIIa complex and thrombin, which is not significantly affected by pharmacological interference of the function of the contact-activated complex; therefore, extravascular hemostatic thrombin generation in the blood escaping from blood vessels through wounds remains unaffected by inhibition of contact activation. In mammals with contact system dysfunction, the absence of an apparent bleeding tendency and a reduced tendency toward acute inflammation characterize the in vivo effect of complex formation between FXI and the binding molecule, which has a significant advantage over other types of FXI / FXIa inhibitors or other protease inhibitors for use in humans (e.g., patients with an increased risk of bleeding or inflammatory responses). Accordingly, the binding molecule of this disclosure, preferably an antibody and its antigen-binding fragment, is intended for use in the treatment and / or prevention of disorders or complications that may arise from the enzymatic and non-enzymatic activity of the contact-activated complex, including pathological contact-initiated thrombin and bradykinin production, which, in particular, lead to thrombosis and inflammation, respectively.

[0170] For the purposes of this disclosure, “thrombotic or thromboembolic disorder” includes, for example, disorders occurring in both arterial and venous vascular systems that can be treated with the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, and preferably includes not only disorders in the coronary arteries of the heart such as acute coronary syndrome (ACS), myocardial infarction with ST segment elevation (STEMI), myocardial infarction without ST segment elevation (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention such as angioplasty, stent implantation or aortic coronary artery bypass, but also thrombotic or thromboembolic disorders in further vascular systems, namely deep veins and renal veins of the lower extremities, transient ischemic attack, and thrombotic and thromboembolic strokes, as well as disorders in the coronary arteries of the heart such as acute coronary syndrome (ACS), myocardial infarction with ST segment elevation (STEMI), myocardial infarction without ST segment elevation (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention such as angioplasty, stent implantation or aortic coronary artery bypass, as well as peripheral artery occlusive disorders, pulmonary embolism, venous thromboembolism, and venous thrombosis.

[0171] For the purposes of this disclosure, “inflammatory disorder” includes pathological events supported or mediated by the activation of all associated contact system complexes (FXII / FXI / PK / HK); including excessive cleavage of HK (HMWK) and production of the most potent known pro-inflammatory peptide, bradykinin, resulting in or associated pathological vasodilation and increased vascular permeability, dysregulation of blood pressure, increased immune response to foreign bodies, excessive endogenous autoimmune responses; and other events resulting from antigen-antibody reactions, and therefore complement and plasminogen activation, and affecting either the local environment (cells, organs) or the systemic system.

[0172] Activation of the contact system can occur due to a variety of causes or associated disorders. In particular, in association with surgical interventions and other tissue trauma, including but not limited to immobility, bedriddenness, infection, inflammation, cancer, tissue injury and necrosis, autoimmunity, temporary or chronic foreign body implantation, and ischemia, the contact system can be activated to cause thrombotic and inflammatory complications. Accordingly, the conjugating molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are useful in the prevention of thrombosis and inflammation in association with surgical interventions (e.g., in patients undergoing major surgeries known to be associated with or potentially associated with thrombosis and / or inflammation in the gastrointestinal tract, lungs, nervous system, urinary tract, orthopedics, and other major surgeries). Accordingly, the conjugating molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are also intended for use in the prevention of thrombosis in patients with an activated contact system.

[0173] Accordingly, the conjugating molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are also intended for use in the treatment and / or prevention of venous thromboembolism and cardiogenic thromboembolism (e.g., cerebral ischemia, stroke, and systemic thromboembolism and ischemia, in patients with acute, intermittent or persistent cardiac arrhythmias such as atrial fibrillation, patients undergoing electrical cardioversion, patients with heart valve disorders or those with artificial heart valves). Furthermore, the conjugating molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are also intended for use in the treatment and / or prevention of disseminated intravascular coagulation (DIC), which may be associated with sepsis (but not limited to sepsis), but can also be caused by surgical intervention, neoplastic disease, burns or other injuries, and which can lead to serious organ damage due to microthrombosis.

[0174] Thromboembolic and inflammatory complications also occur in microangiogenic hemolytic anemia, in association with extracorporeal circulation (e.g., cardiopulmonary bypass), hemodialysis, extracorporeal membrane oxygenation (ECMO), left ventricular assist devices (LVADs), and similar methods, as well as other life support systems such as AV fistulas, vascular and heart valve prostheses, resulting from blood contact with foreign body surfaces.

[0175] Furthermore, the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are intended for use in the treatment and / or prevention of disorders in which microclot formation or fibrin deposition in cerebral blood vessels may be involved and cause dementia-related disorders such as vascular dementia or Alzheimer's disease. Here, clots may contribute to the disorder both by binding to occlusion and further disorder-related factors.

[0176] Furthermore, the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, may be used for the prevention and / or treatment of thrombotic and / or thromboembolic complications (e.g., venous thromboembolism in cancer patients, including patients undergoing major surgical intervention or chemotherapy or radiotherapy).

[0177] In the context of this disclosure, the term “pulmonary hypertension” includes pulmonary artery hypertension, pulmonary hypertension associated with left heart dysfunction, pulmonary hypertension associated with lung dysfunction, and / or hypoxia and pulmonary hypertension due to chronic thromboembolism (CTEPH).

[0178] Furthermore, the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are also intended for use in the treatment and / or prevention of systemic inflammation and infection, and / or systemic inflammatory response syndrome (SIRS), septic organ dysfunction, septic organ failure and multiple organ failure, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), septic shock and / or disseminated intravascular coagulation (DIC) in association with septic organ failure. During the course of infection, systemic activation of the contact and coagulation systems may occur, leading to symptomatic DIC (with or without wasting coagulation disorders) and (micro)thrombosis and secondary hemorrhagic complications in various organs. In addition, endothelial damage may be present, with increased vascular permeability and diffusion of fluids and proteins into the extravascular space. As the infection progresses, organ failure (renal failure, hepatic failure, respiratory failure, central nervous system dysfunction, cardiovascular failure, etc.) and multiple organ failure may occur. In DIC, there is large-scale activation of the coagulation system on the surface of damaged endothelial cells, on the surface of foreign bodies, or in cross-linked extravascular tissues. As a result, there is coagulation in the small vessels of various organs, accompanied by hypoxia and subsequent organ dysfunction. Secondary effects include depletion of coagulation factors (wasting coagulation disorders), such as protein C, protein S, protein Z, FII, FV, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, as well as fibrinogen (FI) and platelets. These impair the control of the blood's homeostatic balance and can lead to severe, and even fatal, bleeding.

[0179] The binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, are also intended for use in the primary prevention of thrombotic or thromboembolic disorders and / or inflammatory disorders and / or diseases involving increased vascular permeability in patients (where gene mutations result in enhanced enzyme activity or increased zymogen levels, which are established by testing / measuring appropriate enzyme activity or zymogen concentration).

[0180] Furthermore, the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, may also be used to prevent coagulation ex vivo. For example, to protect transplanted organs from organ damage due to the formation of blood clots, to prevent thromboembolism of recipients from transplanted organs, to preserve blood and plasma products, to clean / pre-treat catheters and other medical aids and instruments, to coat synthetic surfaces of medical aids and instruments used in vivo or ex vivo, or for biological samples that may contain FXI / FXIa.

[0181] The disclosure further provides the use of the conjugating molecules of the disclosure, preferably antibodies and their antigen-binding fragments, for the treatment and / or prevention of disorders, in particular the disorders described above.

[0182] This disclosure further provides the use of the binding molecules of this disclosure, preferably antibodies and their antigen-binding fragments, for the manufacture of pharmaceuticals for the treatment and / or prevention of the disorders described herein, preferably for the manufacture of pharmaceuticals for the treatment and / or prevention of thrombotic disorders or thromboembolic disorders.

[0183] This disclosure further provides methods for treating and / or preventing disorders, particularly those described herein, using a therapeutically effective amount of the binding molecule of this disclosure (preferably an antibody and its antigen-binding fragment).

[0184] The Disclosure further provides the use of the conjugated molecule, preferably an antibody and its antigen-binding fragment, for the treatment and / or prevention of disorders, in particular the disorders described herein, using a therapeutically effective amount of the conjugated molecule, preferably an antibody and its antigen-binding fragment.

[0185] The Disclosure further provides a method for treating thrombotic or thromboembolic disorders in humans and / or animals by administering a therapeutically effective amount of at least one conjugating molecule of the Disclosure, preferably an antibody and its antigen-binding fragment, or a pharmaceutical composition of the Disclosure. The Disclosure further provides a method for inhibiting blood coagulation, platelet aggregation, and / or thrombosis in a subject by administering a therapeutically effective amount of at least one conjugating molecule of the Disclosure, preferably an antibody and its antigen-binding fragment, or a pharmaceutical composition of the Disclosure.

[0186] [Gene therapy] Further provided herein are transfer vectors for use in mammalian gene therapy, comprising polynucleotides as disclosed herein, and methods for treating or preventing a disease, comprising incorporating an exogenous nucleic acid described herein into the cells of a mammalian patient in need, such that the exogenous nucleic acid is expressed and the disease is prevented or treated. In one embodiment, a nucleic acid molecule encoding both heavy and light chains is administered to the patient. In a preferred embodiment, the nucleic acid molecule is administered so as to be stably incorporated into the chromosomes of B cells, because these cells are specialized to produce antibodies. In one embodiment, precursor B cells are infected transfect or ex vivo and re-implanted into a patient in need. In other embodiments, precursor B cells or other cells are infected in vivo with a recombinant virus known to infect the cell type of interest.

[0187] In a preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain or its antigen-binding moiety of an anti-FXI antibody disclosed herein, and expressing the nucleic acid molecule. In another preferred embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the light chain or its antigen-binding moiety of an anti-FXI antibody disclosed herein, and expressing the nucleic acid molecule. In yet another embodiment, the gene therapy method comprises the steps of administering an isolated nucleic acid molecule encoding the heavy chain or its antigen-binding moiety, and an isolated nucleic acid molecule encoding the light chain or its antigen-binding moiety of an anti-FXI antibody disclosed herein, and expressing the nucleic acid molecule.

[0188] Specific conditions for the uptake of exogenous nucleic acids are well known in the art. These include, but are not limited to, retroviral infection, adenovirus infection, plasmid-mediated transformation, transformation using liposomes containing exogenous nucleic acids, biolistic nucleic acid delivery (i.e., loading nucleic acids onto gold or other metal particles and shooting or injecting them into cells), adeno-associated virus infection, and Epstein-Barr virus infection. All of these can be considered “expression vectors” for the purposes of this disclosure. Expression vectors can be either extrachromosomal vectors or vectors integrated into the host genome. Generally, these expression vectors contain transcriptional and translational regulatory nucleic acids operably linked to the exogenous nucleic acid. Generally, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription start and stop sequences, translation start and stop sequences, and enhancer or activator sequences. In preferred embodiments, the regulatory sequences include promoters and transcription start and stop sequences. Furthermore, expression vectors may include additional elements. For example, to incorporate an expression vector, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences adjacent to the expression construct. The incorporation vector can be directed to a specific locus in the host cell by selecting appropriate homologous sequences for incorporation into the vector. Constructs for vector incorporation are well known in the art.

[0189] [experiment] Novel humanized antibodies were generated starting from mouse 14E11 anti-FXI antibodies. Candidate antibodies were tested by their FXI binding activity, as determined by competitive ELISA, and their ability to inhibit the conversion of FXI to its active form. The characteristics of a typical antibody, AB023, were determined. After humanization of 14E11 by CDR transplantation, tests were performed using AB023 to confirm that the binding and anticoagulant properties were maintained and equivalent. In vitro binding assays showed that AB023 bound to both mouse and human FXI with high affinity (0.16 nM and 3.2 nM, respectively), but with lower affinity than the mouse monoclonal antibody 14E11. In vitro aPTT assays confirmed that the anticoagulant activity was maintained after humanization as AB023, and that aPTT in human, baboon, cynomolgus monkey, and rat plasma was prolonged in a concentration-dependent manner.

[0190] Interestingly, several differences between 14E11 and AB023 were observed in several in vitro assays. Firstly, AB023 was able to inhibit FXI autoactivation in a concentration-dependent manner in the presence of both dextran sulfate and DNA, whereas 14E11 was unable to inhibit it at all concentrations tested. Secondly, AB023 inhibited FXIa-mediated FXII activation in a concentration-dependent manner, in contrast to 14E11. Finally, AB023 was more effective in inhibiting human FXI activation by human FXIIa in the presence of HK and dextran sulfate.

[0191] The antithrombotic effect observed in 14E11 (Cheng Q, Tucker EI, Pine MS, Sisler I, Matafonov A, Sun MF, White-Adams TC, Smith SA, Hanson SR, McCarty OJ, Renne T, Gruber A, Gailani D.Blood. 2010 Nov 11;116(19):3981-3989; Tucker EI, Verbout NG, Leung PY, Hurst S, McCarty OJ, Gailani D, Gruber A.Blood. 2012 May 17;119(20):4762-8;) was maintained after humanization (AB023) in both the mouse model of arterial thrombosis and the baboon model of arterial and venous thrombosis. In a mouse model of arterial thrombosis, AB023 prevented FeCl3-induced carotid occlusion equivalent to that caused by total FXI deficiency, although this effect was not as significant as that produced by the mouse monoclonal antibody (14E11) at the same dose. In a well-established baboon thrombosis model using thrombogenic grafts plus dilation chambers to mimic arterial and venous flow, respectively, administration of low-dose AB023 (0.2 mg / kg, iv) slightly reduced platelet accumulation in the vascular grafts compared to controls, but nearly complete inhibition of platelet accumulation was achieved in the dilation chambers. Fibrin deposition was also reduced in both arterial and venous thrombosis. Interestingly, using thrombogenic grafts without dilation chambers, 1.0 mg / kg of AB023 (intravenously) reduced both platelet and fibrin deposition within the vascular graft segment itself and inhibited the formation of downstream thrombus "tails," thereby demonstrating that AB023, like its mouse precursor 14E11, prevented venous thrombosis at all doses tested. Furthermore, this study suggests that AB023 also appeared to reduce arterial thrombosis at higher doses, as evidenced by the reduction in platelets and fibrin in collagen-coated thrombogenic grafts.

[0192] The anti-inflammatory effect of AB023 was tested in a septic baboon model. Furthermore, 14E11 had previously been tested in a mouse infection model (Silasi R et al., Inhibition of contact-mediated activation of factor XI protects baboons against aureus-induced organ damage and death. Blood Advances 2019 3:658-669, data not shown; Tucker EI et al., Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis. Blood. 2012 May 17;119(20):4762-8. All tests used to compare the activity of AB023 and 14E11 showed equivalence of effects. Furthermore, the anticoagulant effect of AB023 was evaluated in healthy subjects (Lorentz CU et al., Contact Activation Inhibitor and Factor XI Antibody, AB023, Produces Safe, Dose-Dependent). Anticoagulation in a Phase 1 First-In-Human Trial. Arterioscler Thromb Vasc Biol. 2019 Apr;39(4):799-809), data (not shown) indicated that AB023 is safe and exhibits dose-dependent anticoagulant activity.

[0193] Potential cross-reactivity of AB023 was evaluated using frozen sections of healthy human tissue, but none was found.

[0194] In summary, the studies conducted on AB023 demonstrated its anticoagulant and antithrombotic properties. Additional studies demonstrated a low risk of hemostatic impairment, low probability of cross-reactivity and off-target effects, low probability of immune activation and immunogenicity, and low probability of neurotoxicity and cardiotoxicity (data not shown).

[0195] <Characterization of mouse antibody 14E11> As described in the 14E11 patent and other publications cited herein, the binding and anticoagulant properties of monoclonal, mouse, anti-FXI antibody, and 14E11 were determined in the tests conducted. Briefly, binding and anticoagulant properties were tested in vitro. The results showed that 14E11 prolonged aPTT in mouse (white circles) or human (black circles) plasma in an aPTT assay (Figure 1), showing maximum inhibition at 25–50 nM. This is within the range of FXI concentrations in human plasma (20–45 nM). The prolongation of coagulation time was approximately half that of FXI-deficient human and mouse plasma. 14E11 did not affect prothrombin time in human plasma (data not shown).

[0196] 14E11 was found to bind to and recognize a single band of FXI homodimer (~160kDa) of expected size in normal mouse and human plasma, as well as recombinant mouse FXI (Figure 2A-C) plasma, but no binding was observed in FXI-deficient plasma. In solid-phase binding assays, the binding affinity of 14E11 to mouse and human FXI, as well as human FXIa, was determined (apparent K). d(~2-3 pM, Figure 2D). In most mammals, FXI is a homodimer of two 80 kDa subunits, each containing four apple domains (A1-A4) and a protease domain. Prekallikrein (PK), the monomeric homolog of FXI, has a nearly identical structure to the FXI monomer. Using a human FXI / PK chimera, we demonstrated that the A2 domain is required for 14E11 binding to FXI (Figure 2E). Western blotting using individual FXI apple domains linked to t-PA shows that the 14E11 binding site is likely located entirely within the A2 domain (Figure 2F).

[0197] As shown, the mouse monoclonal antibody 14E11 binds to the apple 2(A2) domain of human and mouse FXI, inhibiting FXI activation by FXIIIa and downstream thrombinogenesis, as well as FXI autoactivation. 14E11 binds to FXI in many different species and can prolong aPTT in the plasma of mouse, human, baboon, rabbit, rat, pig, and rhesus monkey (data not shown).

[0198] <Humanization of mouse antibody 14E11> Humanization of mouse monoclonal antibody 14E11 using complementarity-determining region (CDR) grafting technology (O'Brien S. and Jones T. 2001 Humanising Antibodies by CDR Grafting. In: Kontermann) This was performed at Abzena (also known as Antitope Limited, Cambridge, GB) using R, Dubel S. (Eds) Antibody Engineering. Pp. 567-590. Springer Lab Manuals. Springer, Berlin, Heidelberg). In detail, for the selection of germline acceptor family subsets, CDR residues of mouse antibodies were determined and annotated according to the Kabat numbering system (see http: / / www.bioinf.org.uk / abs / #kabatnum for details). In a CDR homology-based approach to antibody humanization, the standard structures of heavy and light chain CDRs were determined using human germline genes, and human germline framework acceptors with the same standard structures were selected (O'Brien and Jones, 2001; Hwang, 2005). Human germline framework acceptors with the same standard structures were selected.

[0199] The 14E11 variable (V) region gene was sequenced from RNA isolated from the 14E11 hybridoma cell line, and these sequences were used to generate chimeric antibodies and design a series of germline humanized antibody variants. By constructing in silico structural models of chimeric antibodies consisting of the 14E11 variable domain and human IgG4 and κ light chains using Swiss PDB, amino acids in the 14E11 variable region framework that could support antibody binding properties were identified, with consideration given to their incorporation into one or more CDR transplantation variants. The humanized V region gene was designed based on human germline sequences with the closest homology to mouse sequences and constructed by gene synthesis. Next, the humanized heavy chain variable region (VH) variant was cloned into a first vector containing the human IgG4 heavy chain common region (CH) 1-3 using a modified S241P hinge region with restriction enzymes Hind III and Mlu I. Humanized light chain variable region mutants (Vκ) were cloned into a second vector containing the human kappa common region (Cκ) using restriction enzymes BssH1 and BamH1. Chimeric antibodies were also produced by cloning the mouse variable region into the same vector. The humanized and chimeric antibodies were stably expressed in NS0 cells and tested for binding to the target antigen (recombinant human FXI) in a competitive ELISA compared to 14E11 (data not shown). Furthermore, the anticoagulant properties of the humanized antibodies were tested using the aPTT assay (Figure 3). The results of these assays identified the mutant VH3 / Vκ3 (clone 3G3) and AB023 as preferred antibody candidates, and the combined CHO TM Using advanced technology, we created a stable production cell line in Abzena.

[0200] Cells derived from stable cell lines can be used for seeding into production bioreactors. For example, the cells can be cultured using a batch feeding process and can be harvested, for example, on day 14. Next, the cells can be purified by one or more chromatographic column steps, a virus clearance step, and can be concentrated and diafiltered. After the final formulation in buffer, the antibody, i.e., AB023, can be filtered and stored frozen as a lyophilized product (e.g., 15 mg / mL after reconstitution), although this is not essential.

[0201] <Properties of AB023> AB023 was characterized using various analytical methods. Using these methods, the primary structure, partial conformational structure, binding, and post-translational modifications of the protein were understood. The analyses used were peptide mapping, mass spectrometry (MS), chip-based capillary electrophoresis, capillary isoelectric focusing (cIEF), size exclusion (SEC) chromatography, oligosaccharide mapping, fluorescence, circular dichroism (CD), and differential scanning calorimetry (DSC), each of which is well known in the art.

[0202] To verify the structure and post-translational modifications, the molecular weight of AB023, 146,560 Da, was determined by MS, demonstrating the integrity of the molecule. After reducing the disulfide bonds in the molecule, the molecular weights of the heavy and light chains (49,890 Da and 23,401 Da, respectively) were determined. The difference compared to the theoretical molecular weight (144,020 Da) is due to post-translational modifications, particularly glycosylation.

[0203] 〔Binding Affinity〕 The binding affinity of the humanized monoclonal antibody AB023 to human and mouse coagulation factor XI (FXI) was determined using a solid-phase binding assay. Further, the binding affinity of AB023 to activated human FXI (FXIa) to both AB023 and 14E11 was evaluated. Briefly, for this solid-phase binding assay, both 14E11 and AB023 were conjugated to EZ-Link according to the instructions. TMBiotinylation was performed using the Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific, Waltham, MA). Microtiter plates were coated with FXI or FXIa (2 μg / ml, 100 μL / well) in 50 mmol / L Na2CO3 (pH 9.6) and incubated overnight at 4°C in Immulon 2HB microtiter plates (Thermo Scientific). Wells were blocked at room temperature for 1 hour with 150 μL of phosphate-buffered saline (PBS) containing 2% BSA. 100 microliters of biotinylated 14E11 or AB023 (0.7 pmol / L to 6.7 μmol / L) were added to 90 mmol / L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.2, 100 mmol / L NaCl, 0.1% BSA, and 0.1% Tween-20 (HBS [HEPES buffered saline]), and incubated at room temperature for 90 minutes. After washing with PBS-0.1% Tween-20 (PBS-T), 100 μL of streptavidin-horseradish-peroxidase (ThermoFisher Scientific, 1:8000 dilution in HBS) was added, and incubated at room temperature for 90 minutes. After washing with PBS-T, 100 μL of substrate solution (12 mL of 30 mmol / L citrate; 100 mmol / L Na2HPO4, pH 5.0; and 1 o-phenylenediamine dihydrochloride tablets, 12 μL of 30% H2O2) was added. The reaction was stopped after 10 minutes with 50 μL of 2.5 M H2SO4. Absorbance at 495 nm was measured using a SpectroMax 340 microplate reader (Molecular Measurements were taken at Devices, San Jose, CA. The data were analyzed using nonlinear regression analysis, and the apparent K d The equilibrium dissociation constant was calculated using GraphPad Prism (v.5.0) as the concentration of AB023 required to achieve maximum half-max binding at equilibrium.

[0204] This solid-phase binding assay was used to determine the binding affinities of 14E11 and AB023 to mouse and human FXI. 14E11 bound to mouse FXI with lower affinity (as previously determined) (apparent K compared to ~2-3 pM). d (~0.07nM). 14E11 also showed lower binding affinity to human FXI and human FXIa than to mouse FXI (apparent K, respectively). d (~0.39 nM and 0.20 nM) (Table 1). AB023 also showed nanomolar affinity binding to both human and mouse FXI, although its binding affinity to both was lower than that of 14E11 (apparent K to mFXI). d -0.16nM, apparent K relative to hFXI d ~3.2nM, and apparent K for hFXIa d (~1.3 nM) (Figure 4C, Table 1). In summary, these results support the idea that high affinity binding to the same domain was maintained after humanization of the 14E11 antibody.

[0205] [Table 1]

[0206] [A2 Domain Binding Confirmation] To confirm that AB023 binds to the Apple 2 (A2) domain of FXI, immunoblotting was performed using standard techniques (Cheng et al., 2010). Figure 4A shows immunoblots of recombinant human FXI (lane 1) and human FXI. The A1, A2, A3, or A4 domains were substituted with corresponding domains derived from prekallikrein (PK), separated by electrophoresis, and immunoblotted with AB023, demonstrating that AB023 binds to the Apple 2 (A2) domain of FXI, similar to 14E11. Fusion proteins were also created by fusing individual Apple domains derived from FXI to tissue plasminogen activator (t-PA). These fusion proteins were then separated by electrophoresis and immunoblotted with AB023. Figure 4B shows that AB023 binds to the A2 domain of FXI.

[0207] [Suppression of FXI activation by FXIIa] In vitro assays were performed to demonstrate that AB023 maintains the ability to inhibit FXIIa activation of FXI after humanization of 14E11. Briefly, FXI (30 nmol / L) was incubated with 0.5 nmol / L α-FXIIa and dextran sulfate (0.1 μg / mL) at 37°C in 25 mmol / L HEPES, pH 7.4, 150 mmol / L NaCl, and 0.1% BSA, in or without AB023 (0 nmol / L to 300 nmol / L). After 30 minutes of incubation, the samples were removed and quenched with polyblen (6 μg / mL), and FXIIa was inactivated by neutralizing dextran sulfate and CTI (50 μg / mL). Subsequently, the production of FXIa was quantified by measuring the rate of S-2366 hydrolysis at 405 nm. The hydrolysis rate of S-2366 was converted to FXIa concentration using a standard curve. Figure 4D shows that AB023 inhibits FXI's FXIIa activation in a concentration-dependent manner. Similar to 14E11, AB023 does not inhibit FXI's thrombin-contact activation (Figure 4E). To determine this, an in vitro assay was performed in which FXI (30 nM) was incubated with 2.5 nM α-thrombin and dextran sulfate (0.1 μg / mL) at 37°C in 25 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% BSA, in or without AB023 (300 nM). After incubation for 0, 5, 15, 30, or 60 minutes, the samples were removed, quenched with polyblen (6 μg / mL), and thrombin was inactivated by neutralizing dextran sulfate and hirudin (10 U / mL). Subsequently, the generation of FXIa was quantified by measuring the rate of S-2366 hydrolysis at 405 nm. The rate of S-2366 hydrolysis was converted to FXIa concentration using a standard curve.

[0208] [Extends activated partial thromboplastin time (aPTT)] The ability of AB023 to prolong aPTT in several mammalian plasmas was tested. Pooled plasma from humans, baboons, rats, and cynomolgus monkeys (90 μL, from three individual subjects) anticoagulated with 0.38% sodium citrate was mixed with 10 μL of AB023 (0.183–1500 μg / mL) or control (PBS). The mixtures were incubated at room temperature for 5 minutes. Next, 40 microliters of plasma / antibody mixture were incubated with 40 μL of aPTT reagent (SynthASil, #0020006800, Instrumentation Laboratory, Bedford, MA) at 37°C for 3 minutes. After incubation, 40 μL of CaCl2 was added, and the time to coagulation was measured using a KC4® analyzer (TCoag, Bray, Ireland). Each sample was assayed in duplicate. For all the species tested, AB023 prolonged aPTT in a concentration-dependent manner (Figure 4F). APTT data are expressed as a multiple change from baseline, and again, the log of AB023 10 The concentrations were plotted.

[0209] In summary, these data demonstrate that the characteristics of 14E11 were maintained after humanization.

[0210] <Anticoagulant complex formation> These experiments investigated whether complexes are formed between a monoclonal antibody or any binding fragment, variant, or derivative with the anticoagulant FXI, using activated partial thromboplastin time (aPTT). In other examples (Figure 4), it has been well established that FXI immediately forms a stable immune complex with AB023, a high-affinity anti-FXI A2 domain antibody. Here, FXI-deficient human subjects (George King Bio-medical Inc, Overland Two separate experiments were conducted using FXI-deficient plasma from Park (KS), and the results are shown in Figure 5.

[0211] In the initial experiment, baseline aPTT was measured in FXI-deficient plasma. 40 microliters of plasma were incubated with 40 μL of aPTT reagent (SynthASil, #0020006800, Instrumentation Laboratory, Bedford, MA) at 37°C for 3 minutes. After incubation, 40 μL of CaCl2 was added, and the time to coagulation was measured using a KC4® analyzer (TCoag, Bray, Ireland). Each sample was assayed 8 times. The mean baseline aPTT of FXI-deficient plasma was 118.5 seconds. After baseline measurement, FXI (Enzyme Research Laboratories, #HCFXI-1111) was added to 400 μL of FXI-deficient plasma at a final concentration of 10 μg / mL. The FXI / FXI-deficient plasma was incubated at room temperature for 5 minutes, and the aPTT of 200 μL of the mixture was measured as described above. Figure 5 shows that, as expected, the addition of purified FXI (MW 160kD) to FXI-deficient plasma reduced the coagulation time to 32.3 seconds, since FXI is the zymogen for the coagulation enzyme FXIa. Finally, AB023 was added to the remaining 200 μL of FXI-deficient plasma + 10 μg / mL of FXI at a final concentration of 100 μg / mL (10 × excess antibody). This mixture was incubated at room temperature for 5 minutes, and aPTT was measured as described above. The addition of AB023 (MW 146kD) in a 10-fold molar excess of the added FXI to the FXI-deficient plasma prolonged the aPTT to an average of 66.6 seconds by approximately twofold, consistent with in vitro (Figure 4) and in vivo (Table 2) experiments where the AB023 concentration was excessive compared to FXI. The addition of AB023 to normal plasma containing FXI did not result in the same degree of aPTT prolongation observed in FXI-deficient plasma. This is thought to be because the procoagulant activity of FXIa generated in the aPTT assay is not inhibited by AB023 (Figure 4E).

[0212] In the second experiment, AB023 was added to 400 μL of FXI-deficient plasma at a final concentration of 100 μg / mL, incubated at room temperature for 5 minutes, and aPTT was measured for 200 μL of the mixture as described above. As a result of the addition of AB023, there was no change in the clotting time (the mean aPTT was 117.7 seconds compared to 118.5 seconds for the FXI-deficient plasma), indicating that AB023 alone does not form a complex with FXI and does not exhibit an anticoagulant effect. Finally, FXI was added to the FXI-deficient plasma / AB023 (100 μg / mL) mixture at a final FXI concentration of 10 μg / mL (a 10-fold excess of the antibody to FXI to ensure that all FXI forms immune complexes). The mixture was incubated at room temperature for 5 minutes, and aPTT was measured as described above. The addition of FXI decreased the aPTT from an average of 117.7 seconds to 59.1 seconds (Figure 5).

[0213] From these experiments, it was shown that: 1) addition of FXI to FXI-deficient plasma exhibits a procoagulant effect; 2) AB02 alone does not exhibit an anticoagulant effect; and 3) the immune complex formed between FXI and AB023 exhibits an anticoagulant effect, but does not produce an equivalent anticoagulant effect due to FXI deficiency.

[0214] <Characteristics of the Anticoagulant Effect of AB023> [Mouse Arterial Thrombosis Model] Using a well-established mouse model of experimental arterial thrombosis, the antithrombotic properties of AB023 were determined as previously done for 14E11 (Cheng et al., 2010). Briefly, mice were anesthetized with 50 mg / kg of IP pentobarbital. The right common carotid artery was exposed and a Doppler flow probe was attached. After injecting AB023 (1.0 mg / kg, i.v.) into the internal jugular vein over 15 minutes, injury was induced by applying two 1×1.5 mm filter papers saturated with FeCl3 (2.5%-10% solution) on both sides of the artery for 3 minutes. After removing the pads, the area was washed with phosphate-buffered saline and the flow was monitored for 30 minutes.

[0215] Intravenous infusion of 1.0 mg / kg of AB023 into wild-type mice protected them from carotid artery occlusion induced by 3.5%, 5.0%, and 7.5% FeCl3 (Figure 6). These results are comparable to those obtained from FXI- / - mice. This is consistent with the premise that FXI is activated by FXIIa in this thrombosis model and demonstrates that antithrombotic activity was maintained in mice after humanization of the 14E11 antibody.

[0216] [PK / PD in the baboon model] This experiment evaluated the plasma concentration of AB023 over time and demonstrated the correlation between AB023 exposure and aPTT. Six male baboons were administered 1.0 mg / kg of AB023 on day 1 by a single intravenous bolus or single subcutaneous injection. Blood samples were collected at several time points after administration and treated with anticoagulation using 0.32% sodium citrate (1 / 10 volume). Plasma AB023 concentration was determined using one aliquot, and aPTT measurement was performed using another aliquot. To measure aPTT, plasma (40 μL) was incubated with 40 μL of aPTT reagent at 37°C for 3 minutes. After incubation at 37°C for 3 minutes, 40 μL of CaCl2 was added, and the time to coagulation was measured using a KC4 Delta® analyzer (Tcoag Ireland, Ltd, Wicklow, Ireland). To detect free AB023 in plasma, plasma AB023 concentrations were measured using a partially validated enzyme immunosorbent assay (ELISA).

[0217] The results showed a rapid and immediate prolongation of aPTT, with all animals showing a prolongation of approximately twofold above baseline for at least one week (168 hours). Figure 7 shows the relationship between AB023 plasma concentration and aPTT. Similarly, SC administration with 1.0 mg / kg of AB023 also resulted in a rapid and immediate prolongation of aPTT. However, after SC administration, aPTT remained approximately twofold above baseline for at least two weeks (336 hours) (not shown). These studies demonstrate that aPTT prolongation is closely tracked by AB023 exposure.

[0218] [Prevention and Treatment in the Arterial and Venous Thrombosis Models of Baboons] These studies revealed the antithrombotic effect of inhibiting FXI activation by FXIIa using the humanized monoclonal antibody AB023 in well-established primate models of experimental arterial and venous thrombosis.

[0219] Non-terminal tests were conducted using young male baboons (Papio anubis) weighing 9 - 13 kg. All tests were approved by the Institutional Animal Care and Use Committee. Each baboon had a chronic arteriovenous (AV) shunt that connected the femoral artery and vein, which was surgically placed and had healed, as described elsewhere (Hanson SR, Griffin JH, Harker LA et al., Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates. J Clin Invest 1993 Oct;92(4):2003 - 12). The experiments were performed on unrestrained, awake, non-anticoagulated animals restrained in a sitting position. Restlessness was managed by intramuscular administration of a low dose of ketamine not exceeding 2 mg / kg up to once per hour. Experimental treatments were administered, and blood samples were collected from a silicone rubber extension tube incorporated into the AV shunt during the experiment. The red blood cell count and hematocrit value of each animal were measured daily, including before and after the experiment, and the calculated blood loss did not exceed 4% of the total blood volume on any experimental day. Multiple experiments were performed on the same animals on separate days, with or without treatment. In repeated experiments, only animals with shunts having a good unrestricted baseline flow (>250 mL / min) were used.

[0220] In this experimental thrombosis model, acute focal thrombosis was initiated by a prosthetically modified thrombogenic graft segment briefly intervened within the AV shunt, as previously described (Hanson SR, Griffin JH, Harker LA et al., Antithrombotic effects of thrombin-induced activation of Endogenous protein C in primates (J Clin Invest 1993 Oct;92(4):2003-12; Kelly AB, Marzec UM, Krupski W et al., Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood 1991 Mar 1;77(5):1006-12). Since vascular injury exposes flowing blood to the extracellular matrix (containing platelets and structural proteins such as collagen that induce FXII activation), the inventors made graft segments thrombogenic using fixed collagen coating. The lumen of a 20 mm long clinical vascular graft (expanded polytetrafluoroethylene, ePTFE, Gore-Tex; WLGore and Associates, Flagstaff, AZ) with an inner diameter (id) of 4 mm was coated with equine type I collagen (CHRONO-LOG Corporation, Haverton, PA) for 15 minutes and then dried overnight under sterile airflow. This method generates a uniform collagen coating within the graft lumen, as determined by scanning electron microscopy (data not shown). The collagen-coated (thrombus-forming) graft segment was incorporated into a silicone rubber tube and placed in a baboon's AV shunt for the entire duration of a 60-minute acute thrombosis experiment.

[0221] Blood flow through the shunt in non-anticoagulated baboons always induces acute thrombosis in the collagen-coated ePTFE graft segment. During each experiment, the maximum blood flow rate through the graft (about 250 mL / min) was limited to 100 mL / min by the distal clamp, resulting in an initial wall shear rate of 265 s-1 in the 4-mm graft. Flow was continuously monitored using an ultrasonic flowmeter (Transonics Systems, Ithaca, NY). These 4-mm-diameter grafts did not occlude, and the pulsatile flow remained at 100 mL / min during thrombosis. The graft segments (and thrombi) were removed from the shunt at 60 or 90 minutes, and the permanent shunt was restored after each experiment. Thrombosis was found to spread downstream from the collagen surface over time, so platelet accumulation was also measured in a 10-cm region of the arteriovenous shunt immediately distal to the graft. This model of thrombus growth on the proximal collagen surface (the thrombus "head") is accompanied by a thrombus that propagates distally to the collagen segment (forming the thrombus "tail").

[0222] Thrombosis was evaluated by quantitative gamma camera imaging of radiolabeled platelets in the graft segment during experiments of 60 - 90 minutes duration. Thrombosis was further evaluated by measurement of endpoint radiolabeled fibrin deposition after the end of each experiment, as described in (1). For quantification of platelet deposition, autologous baboon platelets were labeled with 1 mCi of 111 In and then reinjected into the animals and allowed to circulate for at least 1 hour and 4 days before the test. The accumulation of platelet-related radioactivity on the graft was measured at 5-minute intervals using a GE-400A-61 gamma scintillation camera connected to a NuQuest InteCam computer system, as described for other devices in the chronic AV shunt model (Gruber and Hanson, Blood 2003;102:953-955;Gruber et al., Thromb Res 2007;119:121-127;Hanson et al., J Clin Invest 1993;92:2003-2012;Tucker et al., Blood (2009;113:936-944). After 60-90 minutes, remove the graft, rinse, dry, and then, as previously described (Gruber and Hanson, Blood 2003;102:953-955; Hanson et al., J Clin Invest 1993;92:2003-2012), 125 The samples were refrigerated for evaluation of I-fibrin content. In short, homologous 125 I-labeled fibrinogen (5-25 μg, 4 μCi, >90% coagulation) was administered intravenously 10 minutes before each test. The fibrinogen attached to platelets... 111 To reduce In, the uptake of labeled fibrinogen / fibrin into the thrombus was evaluated at least 30 days after graft removal from the AV shunt using a gamma counter (Wizard-3, PerkinElmer, Shelton, CT). 125 I-radioactivity was measured and compared to the radioactivity of coagulating fibrin (fibrinogen) in plasma samples collected between the initial tests.

[0223] In the first experiment (Figure 8), the antithrombotic effect of AB023 was tested using a 4 mm diameter collagen-coated vascular graft segment with a 9 mm diameter silicone chamber positioned 20 mm downstream to mimic venous flow (0.2 mg / kg administered intravenously 1 hour before the experiment). A total of four non-terminal trials were performed using two young male baboons, and the data were compared with a control group (n=7 out of 4 baboons / group) used in the same 14E11 experiment.

[0224] The results from this study are shown in Figures 8A-D. The left panel shows platelet deposition, and the right panel shows terminal fibrin deposition. This study showed that the platelet accumulation rate within vascular grafts appeared to be slightly lower in AB023-treated animals than in untreated controls (Figure 8A), while almost complete inhibition of platelet accumulation was achieved in the dilated chamber in AB023-treated baboons (Figure 8C). These data are similar to those observed after 14E11 administration, demonstrating that the antithrombotic effect of AB023 is equivalent to that of the mouse precursor 14E11. Fibrin deposition was also lower in both arterial and venous thrombosis (Figures 8B and D).

[0225] In the second set of the experiment, acute focal thrombosis was induced for 60 minutes using a 20 mm long, 4 mm inner diameter collagen-coated artificial blood vessel graft segment placed in a chronic AV shunt in eight non-terminal trials involving four young male baboons. The baboons were treated with either AB023 (1.0 mg / kg, intravenously) or physiological saline (control, intravenously). The antithrombotic effect of AB023 was evaluated 24 hours after intravenous administration.

[0226] Platelet deposition within collagen-coated vascular grafts was lower in AB023-treated animals (Figure 9A), and near-complete inhibition of platelet deposition was achieved by AB023 (Figure 9B) in a 10 cm region immediately downstream of the graft ("tail"). Overall, platelet deposition (thrombogenic graft plus platelet deposition within the tail) was reduced after pretreatment with AB023 (1.0 mg / kg, iv, Figure 9C). These studies demonstrated that AB023, as with its mouse precursor, is antithrombotic in primate models of thrombosis. Furthermore, higher doses of AB023 at 1.0 mg / kg were effective in attenuating the rate of arterial thrombus growth in both the thrombogenic graft and the "tail." Fibrin deposition in both the graft and the "tail" was reduced after AB023 treatment.

[0227] APTT was monitored throughout the study period and was prolonged after AB023 treatment following infusion. Furthermore, in the AB023 treatment group, it remained elevated beyond 24 hours post-treatment (at 30 and 60 minutes of the experiment, Table 2), while no change in aPTT was observed in the control group. PT was also measured throughout the study. AB023 did not alter PT compared to the control (Table 2).

[0228] [Table 2]

[0229] These data demonstrate that the humanized AB023 antibody maintained its anticoagulant properties and reduced experimental thrombosis in both mouse and non-human primate models of thrombosis.

[0230] [Comparison between 14E11 and AB023] Following humanization, the anticoagulant effect of AB023 was compared with that of 14E11 using the aPTT assay described above. AB023 similarly prolonged aPTT in both human and baboon plasma compared to 14E11 (Figure 3).

[0231] The effects of 14E11 or AB023 on FXIa-mediated contact pathway activation, FXI self-activation, and mutual FXII activation were also investigated. Briefly, to compare the effects of 14E11 and AB023 on contact pathway activation, FXI (80 nM) in HEPES (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% PEG-8000) were incubated at 37°C for 15 minutes with or without 1280 nM 14E11 or 800 nM AB023. At zero, FXIIa, HK, and dextran sulfate in all HEPES were added to achieve final concentrations of FXI (30 nM), FXIIa (5 nM), HK (30 nM), dextran sulfate (0.1 μg / ml), and 480 nM 14E11 or 300 nM AB023. At various time points, maize trypsin inhibitor (CTI) (final concentration 500 nM) and polyblen 20 μg / ml (final concentration) were added to 5 μL aliquots. Then ΔOD 405nm This was tracked on a microplate reader in the presence of 250 μM S-2366.

[0232] To compare the effects of 14E11 and AB023 on FXIa self-activation, purified human FXI was mixed with DXS (0.1 μM) in the presence of 25 and 100 nM 14E11 or AB023. Alternatively, purified human FXI was mixed with purified leukocyte-derived DNA at 37°C for 60 minutes 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, 0.1% PEG-8000, and ZnCl2 (10 μM). FXI activation was terminated by mixing aliquots with polyblen (0.2 mg / mL), and FXIa amid degradation activity was measured using the chromogenic substrate S-2366 (1 mM). max The measurement was performed with an OD of 405 nm / min.

[0233] Finally, to compare the effects of 14E11 and AB023 on mutual FXII activation by FXIa, purified human FXII (200 nM) and FXIa (10 nM) were incubated with 14E11 or AB023 (0-200 nM) for 60 minutes at 37°C in 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% PEG-8000, and ZnCl2 (10 μM). FXII activation was stopped with aprotinin (10 μM), and FXIIa amidolite activity was measured using the chromogenic substrate S-2302 (1 mM). max The measurement was performed with an OD of 405 nm / min.

[0234] Despite its clearly lower affinity for FXI, AB023 was more effective in inhibiting FXI's FXIIa activation (Figure 10C). Furthermore, the attenuation of FXI self-activation by AB023 in the presence of a negative surface (sulfate dextran or DNA) was greater compared to 14E11 (Figure 10A-B).

[0235] Humanization of 14E11 to AB023 resulted in unexpected functional acquisition, namely a novel binding molecule with bidirectional inhibitory activity against the interaction of FXII and FXI (Figures 10C, D). Figure 10C shows FXI activation by FXIIa. Figure 10D shows the inhibitory effect of each anti-FXI IgG on FXIa-mediated FXII activation when human FXII (200 nM) is activated by 10 nM human FXIa in the presence of 14E11 or AB023. Not only is AB023 more effective than its mouse precursor in inhibiting FXIIa activation (Figure 10C), but AB023 also inhibits the mutual activation process between FXII and FXI, whereas 14E11 does not. The unexpected acquisition of inhibitory effect may be related to intermolecular sequence homology. Blast alignment showed that the sequence homology between 14E11 and AB023 is approximately 60-70% (data not shown).

[0236] [The sequence described] The nucleic acid and amino acid sequences listed in the attached Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the presented strand.

[0237] 〔LC CDR 1 (SEQ ID NO: 1)〕 KASQDVSTAVA 〔LC CDR 2 (SEQ ID NO: 2)〕 LTSYRNT 〔LC CDR 3 (SEQ ID NO: 3)〕 QQHYKTPYS 〔HC CDR 1 (SEQ ID NO: 4)〕 GYGIY 〔HC CDR 2 (SEQ ID NO: 5)〕 MIWGDGRTDYNSALKS 〔HC CDR 3 (SEQ ID NO: 6)〕 DYYGSKDY

[0238]

Chemical Formula

[0240] The above discussions in this disclosure are presented for illustrative and explanatory purposes only. The foregoing is not intended to limit this disclosure to the forms or configurations disclosed herein. For example, various features of this disclosure are grouped into one or more aspects, embodiments, and configurations to streamline the disclosure and facilitate understanding. Features of the aspects, embodiments, and configurations of this disclosure may be combined in alternative aspects, embodiments, and configurations other than those discussed. Therefore, this disclosure should not be interpreted as reflecting an intention that the claimed disclosure requires more features than are explicitly enumerated in each claim. Rather, as reflected in the following claims, aspects of the invention are within a smaller scope than all the features of a single disclosed aspect, embodiment, and configuration. Thus, the following claims are incorporated into the detailed description of the invention, and each claim exists in itself as a distinct preferred embodiment of this disclosure. Furthermore, the description of this disclosure includes, for example, descriptions of one or more aspects, embodiments, or configurations, and specific variations and modifications, other variations, combinations, and modifications, which may be within the scope of the art and knowledge of those skilled in the art after understanding this disclosure. Regardless of whether such alternative, compatible, and / or equivalent structures, functions, scopes, or processes are disclosed herein, the intention is to obtain rights to alternative forms, embodiments, and configurations, including alternative, interchangeable, and / or equivalent structures, functions, scopes, or processes to the extent permitted, to the extent permitted, and not to publicly contribute any patentable subject matter. [Brief explanation of the drawing]

[0241] [Figure 1] This graph shows the concentration-dependent effect of 14E11 (10⁻⁵-100 μM) on activated partial thromboplastin time (aPTT) in mouse plasma (white circles) and human plasma (black circles). [Figure 2]Figure 2, AF, is a blot and graph illustrating the binding properties of 14E11. [Figure 3] Figure 3, section AB, is a graph showing the effects of 14E11 (black circle) and its humanized version, AB023 (white circle), on in vitro aPTT. [Figure 4] Figures 4A-F are blots and graphs illustrating the binding properties of AB023. [Figure 5] Figure 5 shows the effect of AB023-FXI complex formation on in vitro aPTT. [Figure 6] Figure 6 shows the effects of 14E11 and AB023 in a mouse model of experimental arterial thrombosis. [Figure 7] Figure 7 shows the relationship between AB023 plasma concentration and aPTT in four baboons. [Figure 8] Figures 8A-D show the effects of AB023 in an in vivo baboon thrombosis model (transplant + dilation chamber). [Figure 9] Figures 9A-F show the effect of AB023 on platelet-rich thrombus growth in an in vivo baboon thrombosis model (collagen-coated transplant). [Figure 10] Figures 10A-D show a comparison of the activity of AB023 and the activity of mouse antibody 14E11.

Claims

1. A monoclonal antibody, or an antigen-binding fragment thereof, wherein the monoclonal antibody, or the antigen-binding fragment thereof, CDR1 of the light chain containing sequence KASQDVSTAVA (SEQ ID NO: 1), CDR2 of the light chain containing sequence LTSYRNT (SEQ ID NO: 2), CDR3 of the light chain containing the sequence QQHYKTPYS (SEQ ID NO: 3), Heavy chain CDR1 containing sequence GYGIY (SEQ ID NO: 4), Heavy chain CDR2 containing the sequence MIWGDGRTDYNSALKS (SEQ ID NO: 5), Heavy chain CDR3 containing sequence DYYGSKDY (SEQ ID NO: 6), It includes and specifically binds to human factor XI and / or human factor XIa, The above monoclonal antibody is an IgG4 antibody, The above monoclonal antibody is V as described in Sequence ID No.

8. Ｈ Region, and V as described in Sequence ID No. 9 Ｌ A monoclonal antibody containing a region, or its antigen-binding fragment.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the monoclonal antibody or its antigen-binding fragment comprises the light chain described in SEQ ID NO: 10 or the light chain encoded by SEQ ID NO:

12.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the monoclonal antibody or its antigen-binding fragment comprises the heavy chain described in SEQ ID NO: 11 or the heavy chain encoded by SEQ ID NO:

13.

4. A monoclonal antibody according to claim 1, or an antigen-binding fragment thereof, wherein the antibody is IgG.

5. The monoclonal antibody described above comprises an IgG4 isotype heavy chain, as described in claim 1, or an antigen-binding fragment thereof.

6. The monoclonal antibody described above comprises an IgG4 isotype heavy chain and a kappa (κ) light chain, as described in claim 1, or an antigen-binding fragment thereof.

7. A monoclonal antibody according to claim 1, or a polynucleotide encoding an antigen-binding fragment thereof.

8. A vector comprising the polynucleotide described in claim 7.

9. The vector according to claim 8, wherein the above-mentioned vector is an expression vector.

10. A culture comprising host cells having the polynucleotide described in claim 7.

11. A method for producing a monoclonal antibody or an antigen-binding fragment thereof, the method comprising the step of culturing the host cell described in claim 10 under conditions that enable the expression of the monoclonal antibody or the antigen-binding fragment.

12. A monoclonal antibody produced according to the manufacturing method described in claim 11, or an antigen-binding fragment thereof; and, A pharmaceutical composition containing pharmaceutically acceptable excipients.

13. A therapeutically effective amount of the monoclonal antibody according to claim 1, or its antigen-binding fragment; and A pharmaceutical composition containing pharmaceutically acceptable excipients.

14. The pharmaceutical composition according to claim 13, further comprising one or more additional active substances.

15. The above additional active substances are Plasminogen activator (thrombolytic agent / fibrinolytic agent); Inhibitors of plasminogen activator; Inhibitors of thrombin-activated fibrinolysis (TAFI) inhibitors, including tissue plasminogen activator (t-PA), streptokinase, leteplase, and urokinase; Unfractionated heparin; Low molecular weight heparin; Heparinoid; Hirudin; bivalirudin; and, Argatroban A pharmaceutical composition according to claim 14, selected from the group including the following.

16. The pharmaceutical composition according to claim 12, for use in the treatment and / or prevention of thrombotic or thromboembolic disorders, thrombotic or thromboembolic complications, or inflammatory diseases.

17. The pharmaceutical composition according to claim 12, for use in the treatment and / or prevention of blood coagulation, platelet aggregation, and / or thrombosis.

18. A method for using a monoclonal antibody or an antigen-binding fragment thereof as an anticoagulant in a blood sample, blood preservative, plasma preparation, biological sample, or medical additive or device, the method comprising the step of adding the monoclonal antibody or an antigen-binding fragment thereof to a sample requiring the anticoagulant.

19. A monoclonal antibody according to claim 1, or an antigen-binding fragment thereof; and, The kit includes the instruction manual.

20. The above monoclonal antibody, or its antigen-binding fragment, is in a lyophilized form. The kit according to claim 19, further comprising a pharmaceutically acceptable carrier for resuspending the monoclonal antibody and its antigen-binding fragment.

21. The manufacturing method according to claim 11, further comprising the step of recovering the manufactured antibody and its antigen-binding fragment from the culture.

Images