Therapeutic Factor XII Antibodies

Abstract

A monoclonal antibody that binds (e.g., specifically binds) to the blood protein factor XII (FXII) is described. The monoclonal antibody (including an antigen-binding fragment thereof) is capable of forming an immune complex with human (FXII) and inhibiting (FXII) activity, thereby producing a safe anti-inflammatory and anti-thrombotic effect.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 772,235, filed on November 28, 2018, which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to potent therapeutic antibodies specific for the blood protein Factor XII (FXII) and their use in methods of medical treatment, including the treatment of thrombosis and hemostatic management. Background Art

[0004] Medical devices that contact blood, including catheters, stents, grafts, filters, and extracorporeal organ support systems (ECOS), often result in device-related thromboembolism even with medical thromboprophylaxis by administration of anticoagulants, because existing anticoagulants are not administered at their maximally effective doses due to adverse bleeding side effects (Lavery et al., Adv Drug Deliv Rev 2017; 112: 2–11). To maintain patency, perfused devices require prophylactic anticoagulation, which may increase the incidence and / or severity of bleeding. Inhibition of contact activation of blood, including FXII inhibition, has been proposed as a safe alternative to therapeutic anticoagulation (PCT Publication No. WO 2013 / 013423; Gruber and Hanson, Blood 2003; 102: 953–955; Yau et al., Blood 2014; 123: 2102–2107; Tillman and Gailani, Semin Thromb Hemost 2018; 44: 60–69). Extracorporeal membrane oxygenation (ECMO) is used for short-term management of respiratory failure; however, bleeding associated with anticoagulation reduces its benefits (Oliver, Semin Cardiothorac Vasc Anesth 2009; 13: 154–175; Murphy et al., Transfus Med Rev 2015; 29: 90–101; Barbaro et al., Am J Respir Crit Care Med 2015; 191: 894–901). ECOS systems, such as ECMO, cardiopulmonary bypass, left ventricular assist device (LVAD), total artificial heart (TAH), or hemodialysis, have several components that promote contact and platelet activation (Sniecinski and Chandler, Anesth Analg 2011; 113: 1319–1333).

[0005] In vivo activation of coagulation factors other than FXII, plasma kallikrein (PK), or high molecular weight kininogen (HK) leads to thrombin generation, and subsequently platelet activation and fibrin formation to support hemostasis (Smith et al., Crit Rev Biochem Mol Biol 2015; 50: 326-336). However, pathological coagulation within the vascular lumen may also involve FXII, PK, and HK, and may lead to vascular occlusive thrombosis / thromboembolism. Pharmacological treatment or thromboprophylaxis of thrombosis is achieved by antithrombotic agents (e.g., heparin), which may cause bleeding because they inhibit key hemostatic plasma proteins, such as thrombin or factor Xa (Abraham et al., BMJ 2015; 350: h1857; Ruff et al., Lancet 2014; 383: 955–962). Therefore, due to dose-limiting anticoagulant toxicity, these drugs cannot be administered at their full efficacy doses, and thrombotic vascular occlusion remains the leading cause of death in developed countries. In order to address the issue of antithrombotic safety, it is proposed that inhibition of contact activation will be a safer alternative to current antithrombotic treatments (PCT Publication No. WO 2013 / 013423; Gruber and Hanson, Blood 2003; 102: 953–955). Both industry and academia are constantly working to develop inhibitors to contact activated complex components to improve the safety of anticoagulation (Tillman et al., Blood Rev 2018; 32: 433-448).

[0006] The plasma contact activation complex promotes pathological thrombosis. It involves FXI, plasma kallikrein (PK), and the cofactor high molecular weight kininogen (HK), which are activated after blood is exposed to negatively charged surfaces, including various biomolecules and artificial materials (Schmaier, Thromb Res 2014;133:S41–S44; Schmaier, J Thromb Haemost 2016;14:28–39; Tillman and Gailani, Semin Thromb Hemost 2018;44:60–69). The plasma concentration of FXII is 30-40 μg / ml (375-500 nM) (Hanson and Tucker, Chapter II.2.6-Blood Coagulation and Blood-Materials Interactions. In: Ratner BD, Hoffman AS, Schoen FJ, et al., editor (s). Biomaterials Science (Third Edition). Academic Press; 2013. p. 551-557). Surface catalytic cleavage of FXII (80 kDa single-chain zymogen) after Arg353 produces protease α-FXIIa. Cleavage of α-FXIIa after Arg334 produces β-FXIIa, which contains the FXIIa catalytic domain. α-FXIIa activates FXI to FXIa, ultimately leading to the generation of thrombin (FIIa) and mutual activation of FXII. At the same time, α-FXIIa activates zymogen PK to α-kallikrein, which converts additional FXII into α-FXIIa. β-FXIIa activates components of the complement system and cleaves the cofactor HK as part of the kallikrein-kinin system to release bradykinin, which leads to the production of NO and activation of complement and other systems, such as the renin-angiotensin system (Ivanov et al., Curr Opin Hematol 2017; 24: 411–418). Thus, contact activation initiates prothrombotic, vasomodulatory, and proinflammatory processes (Renné, Semin Immunopathol 2012; 34: 31–41).

[0007] In the activated partial thromboplastin time (aPTT) coagulation test, contact activation drives thrombin formation, and the aPTT of plasma or blood from mammals lacking the contact protein is prolonged (Turi and Peerschke, Am J Clin Pathol 1986; 85: 43–49). Although hereditary FXI deficiency may cause a mild bleeding disorder in humans (hemophilia C), FXII, PK or HK deficiency is asymptomatic (Tagariello et al., Blood Transfus 2017; 15: 557–561; Kitchens, J Thromb Haemost 2005; 3: 2607–2611; Renne et al., Blood 2012; 120: 4296–4303). Importantly, although these proteins play little or no role in hemostasis, they appear to contribute to thrombosis in some experimental animal models (Renné et al., J Exp Med 2005;202:271–281; Kleinschnitz et al., J Exp Med 2006;203:513–518; Crosby et al., Arter Thromb Vasc Biol 2013;33:1670–1678). In a rabbit ECMO model, inhibition of FXIIa using the anti-FXIIa antibody 3F7 reduced fibrin deposition within the membrane oxygenator cartridge, whereby venous blood was extracted from the animal's vein and pumped through the device using a roller pump without significantly increasing bleeding caused by experimental injury (Kenne and Renne, Drug Discov Today 2014; 19: 1459–1464; Larsson et al., Sci Transl Med 2014; 6: 222ra17; Worm et al., Ann Transl Med 2015; 3: 247). Summary of the invention

[0008] Monoclonal antibodies that bind (e.g., specifically bind) to the blood protein factor XII (FXII) are described. The monoclonal antibodies (including antigen-binding fragments thereof) are capable of forming immune complexes with human FXII and inhibiting FXII activity, thereby producing anti-inflammatory and anti-thrombotic effects.

[0009] Provided herein are monoclonal antibodies or antigen-binding fragments thereof that bind to a catalytic domain and block the catalytic activity of FXII. The monoclonal antibody or antigen-binding fragment comprises a variable heavy (VH) domain and a variable light (VL) domain. In some embodiments, the VH domain of the monoclonal antibody or antigen-binding fragment comprises a complementary determining region (CDR) sequence of SEQ ID NO: 2, and / or the VL domain of the monoclonal antibody or antigen-binding fragment comprises a CDR sequence of SEQ ID NO: 4.

[0010] Also provided are fusion proteins, antibody conjugates, and compositions comprising FXII-specific monoclonal antibodies or antigen-binding fragments thereof as disclosed herein.

[0011] Further provided are nucleic acid molecules and vectors encoding the disclosed monoclonal antibodies or antigen-binding fragments. Also provided are isolated cells comprising the nucleic acid molecules or vectors disclosed herein.

[0012] Also provided are methods of detecting FXII in a sample by contacting the sample with a monoclonal antibody or antigen-binding fragment disclosed herein and detecting binding of the antibody to the sample.

[0013] Further provided are in vitro methods of inhibiting activation and / or activity of FXII (eg, activity of FXIIa) in a sample by contacting the sample with an FXII-specific antibody or antigen-binding fragment disclosed herein.

[0014] Also provided are methods of inhibiting the activation and / or activity of FXII in a subject, methods of treating pathological hypercoagulation involving the activation and / or activity of FXII in a subject, and methods of inhibiting thrombosis or inflammation involving the activation and / or activity of FXII in a subject, by administering to the subject an effective amount of an antibody or antigen-binding fragment disclosed herein. In some instances, the method involves inhibiting the activity of activated FXII (FXIIa).

[0015] The present disclosure further provides kits comprising the disclosed monoclonal antibodies, antigen-binding fragments, fusion proteins, antibody conjugates, compositions, nucleic acid molecules, vectors or isolated cells.

[0016] The foregoing and other objects, features and advantages of the present disclosure will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-1C : Effect of anti-factor XII antibody 5C12 on plasma coagulation. Standard activated partial thromboplastin time (aPTT) assay was performed using a synthetic lipid reagent. Figure 1A-1B) Before the aPTT assay, increasing concentrations of 5C12 (○) and a control non-inhibitory anti-FXII antibody (●) were incubated with human ( Figure 1A ) or baboon ( Figure 1B ) Platelet-poor plasma was incubated for 5 minutes. Data are the average of 2-6 coagulation times. Figure 1C ) Comparison of the dose-dependent aPTT prolongation of corn trypsin inhibitor (CTI), a known FXII inhibitor (◆) with that of FXII inhibition by 5C12 (○).

[0018] Figures 2A-2F : Effect of FXII concentration on plasma coagulation. Platelet-poor plasma containing 0.006–100% FXII was obtained by serial dilution of normal platelet-poor plasma from different species into FXII-deficient human plasma. Plasma was incubated for 5 min with buffer alone (●) or with 20 μg / ml 5C12 (○) before aPTT measurement. The following species were tested for cross-reactivity of 5C12 with FXII from these species: human ( Figure 2A ),baboon( Figure 2B )、cynomolgus monkey( Figure 2C )、rhesus macaque ( Figure 2D )、Mouse( Figure 2E ) and rats ( Figure 2F ). A rightward shift of the curve indicates cross-reactivity of 5C12 with the species tested.

[0019] Figures 3A-3C : Species specificity of 5C12. Figure 3A ) Western blot of platelet-poor plasma from different species (indicated above each lane) size fractionated by SDS-PAGE chromatography. The species listed in alphabetical order include: African green monkey, baboon, beagle, cat, cow, chicken, cynomolgus monkey, dog, elephant, giant anteater, horse, human, FXII-deficient human, llama, marmoset, pig, rabbit, raccoon, rat, red kangaroo, rhesus monkey, and tiger. Human plasma and FXII-deficient plasma were used as positive and negative controls, respectively, in all gels. Antibody 5C12 recognizes recombinant and plasma-derived FXII in its non-reduced state on SDS-PAGE gels, but not recombinant or plasma FXII in its reduced form ( Figure 3B 5C12 also recognizes the α (including both the light and heavy chains of FXII) and β (including only the nonapeptide fragment of the light and heavy chains) forms of activated FXII (FXIIa) ( Figure 3C ). The primary antibody for all Western blots was mouse 5C12 and the secondary antibody was goat anti-mouse polyclonal.

[0020] Figures 4A-4E:5C12 inhibits FXII activation and FXIIa activity. ( Figure 4A-4B )5C12 or control non-inhibitory FXII antibody was preincubated with FXII for 10 min at room temperature (RT) and then added to short polyphosphate ( Figure 4A ) or long polyphosphate ( Figure 4B ), high molecular weight kininogen (HK) and prekallikrein (PK) in a solution with a final concentration of 50nM FXII, 10μM polyphosphate, 12.5nM PK and 12.5nM HK, and different concentrations of antibodies (0-100nM). The samples were incubated at 37°C for 60 minutes. PK was then inhibited with soybean trypsin inhibitor (50μg / ml), polyphosphate was neutralized with polybrene (6μg / ml), and FXIIa was determined by adding the chromogenic substrate Spectrozyme FXIIa (final concentration of 0.5mM). ( Figure 4C )5C12 or control antibody (0-80 nM) was pre-incubated with FXII (40 nM) at RT for 10 minutes. Dextran sulfate was then added to each sample at a final concentration of 1 μg / ml, and the samples were incubated at 37°C for 20 minutes. FXIIa was determined by adding the chromogenic substrate SpectrozymeFXIIa (final concentration of 0.5 mM). Figure 4D )5C12 or control antibody (0-40 nM) was pre-incubated with FXIIa (20 nM) for 10 minutes. Then FXIIa activity was determined by adding the chromogenic substrate Spectrozyme FXIIa (final concentration was 0.5 mM). Symbols are (○) 5C12, (●) control antibody. Figure 4E ) Human FXII (100nM) was incubated with 5nM human kallikrein and 10μg / ml dextran sulfate at 37°C in the presence or absence of 100nM 5C12 for 0, 15, 30 and 60 minutes. 40μg / ml CTI was added to each sample to block the activity of FXIIa. The samples were separated by SDS-PAGE under reducing conditions, transferred to PVDF membranes, and immunoblotted with anti-FXII antibodies. Proteins were detected using ECL (GE Healthcare, Piscataway, NJ, USA).

[0021] Figures 5A-5H : Time course of 5C12 antibody activity in vivo. Individual baboons (Papio anubis) were injected intravenously with 6 doses of 1 mg / kg 5C12, 40 minutes apart, for a combined dose of 6 mg / kg. Blood samples were drawn at designated time points into 1 / 10 volume of 3.2% sodium citrate and processed to platelet-poor plasma. A second blood sample was drawn into an EDTA-K2 coated tube for complete blood count measurement. The aPTT ( Figure 5A )、Prothrombin time (PT) ( Figure 5B ) and activated clotting time (ACT) ( Figure 5C ) was used to measure plasma coagulation. Thrombin-antithrombin time (TAT) was measured using the Enzygnost TAT micro ELISA kit (Siemens Medical Diagnostics). Figure 5D ). Perform a complete blood count (CBC) each day that blood samples are collected. Display platelet count ( Figure 5E ), hematocrit ( Fig. 5F ), white blood cell count ( Figure 5G ) and red blood cell count ( Figure 5H ). All measurements were within the normal range for baboons. The normal ranges for these parameters are indicated by the dashed lines in each inset.

[0022] Figures 6A-6E : Baboon model of surface-induced thrombosis. Fig. 6A ) An infant extracorporeal membrane oxygenator (ECMO, Capriox Baby RX05, Terumo Corporation) was inserted into the chronic AV shunt of a young male baboon (Papio Anubis) and placed in the center of a GE-Brivo NM 615 nuclear medicine imaging system. Blood flow through the ECMO device was regulated by a distal clamp to a flow rate of 100 ml / min, and the experiment lasted for 60 minutes. 111 In labels platelets so that platelet deposition in the ECMO device can be monitored in real time. Except for the control study (●), treatment was given before each experiment. 20U / kg heparin was administered by bolus intravenous injection 15 minutes before the experiment (◆). 5mg / kg 5C12 was administered by intravenous bolus injection 30 minutes before the experiment (○). The combination study of 5C12 and heparin (◇) was conducted 48 hours after the first 5C12 administration, and a second "top off" dose of 2mg / kg 5C12 and a new dose of 20U / kg heparin were given. Figure 6B Platelet deposition rates (billions of platelets deposited per minute) during the second half of the study (30-60 minutes) are shown. Figure 6C-6D The terminal platelet count and fibrin content of thrombi formed in the ECMO device are shown, respectively. Fig. 6E The correlation between these two parameters is shown.

[0023] Figures 7A-7C : Plasma coagulation measurements during ECMO shunt studies. Blood samples were drawn at the beginning (white bars) and end (black bars) of the ECMO thrombosis study and placed in 1 / 10 volume of 3.2% sodium citrate. ACT was measured directly in whole blood ( Figure 7B ). aPTT was measured using platelet-poor plasma ( Fig. 7A ) and PT( Figure 7C ).

[0024] Figures 8A-8B : Measurement of thrombotic markers at the end of the ECMO bypass study. The thrombotic marker measured was thrombin-antithrombin (TAT) ( Fig. 8A ), and platelet factor 4 ( Figure 8B ). The data shown are measurements taken at the end of each study. Baseline measurements taken before the study did not differ from each other and are shown to the left of each inset.

[0025] Figures 9A-9C : Hemostasis Assessment During AV Shunt Studies. In each shunt thrombosis study, two hemostatic measurements were performed over a 15-minute period during the study. Bleeding time ( Fig.9A ) Skin bleeding time test using a standard template ( International Technidyne Corp, Piscataway, NJ) and recorded using a manual stopwatch. Blood drops from the wound were collected every 30 seconds using Whatman blotting paper ( Fig. 9B ). The amount of bleeding was assessed using this blotting paper and Drabkin's reagent (Sigma), which is a quantitative colorimetric chemical that can measure the hemoglobin concentration in whole blood. The dried blood sample on the blotting paper was soaked in 2.5 mL of Drabkin's reagent until it was completely dissolved. The absorbance was measured at 540 nm and compared to a standard curve using blood from the test animal. The bleeding rate was calculated as the ratio of bleeding volume to time ( Fig. 9C ). Each data point represents a single measurement.

[0026] Figures 10A-10F : Coagulation evaluation of spiked human blood samples. Blood samples were collected from 6 volunteers and placed in 1 / 10 volume of 3.2% sodium citrate. ( Fig. 10A ) Platelet-poor plasma was generated and doped with vehicle (●) or 40 μg / ml 5C12 (○) and incubated for 5 min before measuring aPTT. Fig. 10B) Whole blood was spiked with vehicle (●) or 40 μg / ml 5C12 (○) and incubated for 15 min before centrifugation to obtain platelet-poor plasma and aPTT measurement. Figures 10C-10F )use Thromboelastography was measured using a dalta whole blood analyzer (Tem Systems, Inc. Research Triangle Park, NC). Whole blood was doped with vehicle (●) or 40 μg / ml 5C12 (○) and incubated for 5 minutes. In a cuvette, mix 20 μl of 200 mM calcium chloride and 300 μl of blood and pipette using an automated pipetting system and The delta analyzer starts measuring. The parameters shown are: Fig. 10C ) Clotting time (CT), the time until a clot begins to form; Fig. 10D ) Clot formation time (CFT), the time from the beginning of clotting to the time when the clot reaches the strength of 20 mm force; Fig. 10E ) Maximum clot strength (MCF), the maximum strength of the clot; and ( Fig.10F )α angle, a measure of the time to reach maximum velocity of clot formation.

[0027] Fig.11 : ELISA curve fitting of humanized antibodies and chimeric antibodies. The binding data of each humanized antibody, chimeric parent antibody and human Fc isotype control antibody to human FXII are plotted on a semi-logarithmic scale. Samples were performed in duplicate, and each data point represents the mean ± SEM of the replicates.

[0028] Fig.12 : Effect of humanized antibodies on concentration-response aPTT curves. All nine humanized antibodies prolonged aPTT in a concentration-dependent manner. The highest possible final concentration of the antibody was 75 or 100 μg / ml, depending on the stock concentration. Each sample was run in duplicate, and each data point represents the mean ± SEM of the replicates.

[0029] Fig.13 : Effect of humanized antibodies on FXIIa activity. All humanized antibodies (10 nM) inhibited FXIIa (20 nM) activity. Each sample was performed in duplicate. The Vmax (control) produced by FXIIa activity was set to 100%, and each antibody was compared with the control. The data shown are the mean ± SEM of the replicates.

[0030] Figures 14A-14B : Comparison of AB053 (5C12) and AB054 on human and baboon plasma aPTT. Fig.14A ) and humanized AB054 ( Fig. 14B) Comparison of human (filled circles) and baboon (open circles) plasma aPTT. Data represent mean ± SEM of 1 to 5 experiments.

[0031] Figures 15A-15C : aPTT, ACT, and PT measurements during membrane oxygenation perfusion experiments. At the start (0 min) and end (60 min) of perfusion, coagulation parameters were assessed for each treatment group, including aPTT ( Fig.15A )、ACT( Fig. 15B ) and PT( Fig. 15C ). If heparin was given, the measurements were performed before heparin administration (pre-heparin). Data are expressed as mean ± SEM.

[0032] Figures 16A-16C : Measurement of primary hemostasis in membrane oxygenator perfusion experiments. Approximately 15 minutes after the start of perfusion, the effect of AB054 on bleeding time ( Fig.16A )、Blood loss( Fig. 16B ) and bleeding rate ( Fig. 16C ). Two measurements were performed in each perfusion experiment and the mean value is shown by the black line.

[0033] Figures 17A-17B : Platelet aggregation in a membrane oxygenator. Real-time measurement of platelet aggregation in a membrane oxygenator during a 60-minute perfusion experiment. Fig.17A ) The oxygenator was placed on top of a gamma camera and the emitted radioactivity was recorded over a 5-minute frame. Fig. 17B ) Average deposition rate per minute during the second half of the experiment (30 to 60 minutes) when thrombus formation developed. Data are expressed as mean ± SEM.

[0034] Figures 18A-18B : Measurement of terminal platelets and fibrin in a membrane oxygenator. Fig.18A Final platelet deposition was measured after 60 min of membrane oxygenator perfusion in a flushed, saline-filled device. Fig.18B ) The fibrin content labeled with iodine-125 was measured after the radioactive decay of indium-111. Data are expressed as mean ± SEM.

[0035] Fig.19: Experimental timeline. 72 thrombosis experiments were performed in a single baboon for 7 weeks. On each study day, two perfusion experiments were performed using one collagen-coated and one tissue factor-coated graft in series (i.e., 4 graft experiments per day). Baboons received one dose of AB054 (9 mg / kg) (arrow) per week and experiments were performed on the same day and the following two days. Experimental days and treatment groups are indicated by vertical lines. After the last dose of AB054, experiments were performed 1 week and 2 weeks after dosing. Saline controls were performed at week 1 and week 7 after antibody clearance. N=6 for all groups.

[0036] Figures 20A-20D : aPTT and ACT measurements in graft perfusion experiments. Coagulation parameters, including aPTT ( Fig. 20A , 20B ) and ACT( Fig. 20C , 20D ). Data by time ( Fig. 20A , 20C ) or treatment group ( Fig. 20B , 20D ) Grouping. N=6 for all groups. Data are expressed as mean±SEM. ***p<0.001 compared with normal saline.

[0037] Figures 21A-21B : PT measurements during the graft perfusion experiment. Hemostasis assessment using PT was measured for each treatment group at the start of perfusion (0 min) and at the end of perfusion (60 min). Data were sorted by time ( Fig.21A ) or treatment group ( Fig. 21B ) Grouping. N=6 for all groups. Data are expressed as mean±SEM.

[0038] Fig. 22 : Bleeding measurements during graft perfusion experiments. The effect of AB054 on bleeding time was assessed approximately 15 minutes after the start of perfusion. One measurement was performed in each perfusion experiment. Data are shown as scatter plots of mean ± SEM.

[0039] Figures 23A-23D : Platelet aggregation in collagen-coated grafts. Real-time platelet aggregation in collagen-coated grafts during a 60-minute perfusion experiment. ( Fig.23A )Graft thrombosis within the graft itself. Fig. 23B ) Tail thrombosis distal to the graft. Fig.23C ) Total platelet aggregation of graft and tail combination. N = 6 ( Fig.23D ) Total platelet aggregation in all experimental combinations after AB054 administration (N=30 after AB054). Data are presented as mean ± SEM. Statistical analysis was performed only for complete thrombi ( Fig.23C and 23D ); ***p<0.001 compared with normal saline.

[0040] Figures 24A-24D : Platelet aggregation in tissue factor-coated grafts. Real-time platelet aggregation in tissue factor-coated grafts during a 60-minute perfusion experiment. ( Fig.24A )Graft thrombosis within the graft itself. Fig. 24B ) Tail thrombosis distal to the graft. Fig.24C ) Total platelet aggregation of graft and tail combination. N = 6 per group. ( Fig.24D ) Total platelet aggregation of all experimental combinations after AB054 administration (N=30 after AB054). Data are presented as mean±SEM.

[0041] Figures 25A-25F : Measurement of terminal platelet aggregation and fibrin content in collagen-coated grafts. After 60 minutes of perfusion, the final platelet deposition in the collagen-coated grafts and distal tail was measured in a flushed, saline-filled shunt loop (shunt loop). Figures 25A-25C ).exist 111 Measurement of radioactive decay of In 125 I-labeled fibrin content ( Figures 25D-25F ). Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 compared with saline.

[0042] Figures 26A-26F : Measurement of terminal platelet aggregation and fibrin content in tissue factor-coated grafts. After 60 minutes of perfusion, the final platelet deposition in the tissue factor-coated graft and distal end of the tail was measured in a flushed, saline-filled shunt loop ( Figures 26A-26C ).exist 111 Measurement of radioactive decay of In 125 I-labeled fibrin content ( Figures 26D-26F ). Data are expressed as mean ± SEM.

[0043] Figures 27A-27E : Complete blood count. Complete blood counts were measured for each treatment group at the beginning (0 min) and end (60 min) of perfusion. Parameters measured included platelet count ( Fig.27A ), hematocrit ( Fig.27B ), white blood cell count ( Fig.27C ), red blood cell count ( Fig.27D ) and mean platelet volume ( Fig.27E). During active thrombosis, white blood cell counts increased and platelet counts decreased, while other parameters remained unchanged. Repeated administration of AB054 had no effect on complete blood cell counts in baboons. Data are expressed as mean ± SEM.

[0044] Sequence Listing

[0045] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand is shown for each nucleic acid sequence, but any reference to the displayed strand is understood to include the complementary strand. The sequence listing is submitted as an ASCII text file, created on November 1, 2019, 43.2 KB, and is hereby incorporated by reference. In the accompanying sequence listing:

[0046] SEQ ID NO: 1 is the amino acid sequence of the 5C12 heavy chain.

[0047] SEQ ID NO: 2 is the amino acid sequence of the 5C12 VH domain.

[0048] SEQ ID NO: 3 is the amino acid sequence of the 5C12 light chain.

[0049] SEQ ID NO: 4 is the amino acid sequence of the 5C12 VL domain.

[0050] SEQ ID NO: 5 is the cDNA sequence of the 5C12 heavy chain.

[0051] SEQ ID NO: 6 is the cDNA sequence of the 5C12 light chain.

[0052] SEQ ID NO: 7 is the amino acid sequence of the humanized HC1 heavy chain.

[0053] SEQ ID NO: 8 is the amino acid sequence of the humanized HC1 VH domain.

[0054] SEQ ID NO: 9 is the amino acid sequence of the humanized LC1 light chain.

[0055] SEQ ID NO: 10 is the amino acid sequence of the humanized LC1 VL domain.

[0056] SEQ ID NO: 11 is the cDNA sequence of humanized HC1 heavy chain.

[0057] SEQ ID NO: 12 is the cDNA sequence of humanized LC1 light chain.

[0058] SEQ ID NO: 13 is the amino acid sequence of the humanized HC2 heavy chain.

[0059] SEQ ID NO: 14 is the amino acid sequence of the HC2 VH domain.

[0060] SEQ ID NO: 15 is the amino acid sequence of the humanized HC3 heavy chain.

[0061] SEQ ID NO: 16 is the amino acid sequence of the HC3 VH domain.

[0062] SEQ ID NO: 17 is the amino acid sequence of the humanized LC2 light chain.

[0063] SEQ ID NO: 18 is the amino acid sequence of the LC2 VL domain.

[0064] SEQ ID NO: 19 is the amino acid sequence of the humanized LC3 light chain.

[0065] SEQ ID NO: 20 is the amino acid sequence of the LC3 VL domain.

[0066] SEQ ID NO: 21 is the cDNA sequence of humanized HC2 heavy chain.

[0067] SEQ ID NO: 22 is the cDNA sequence of humanized LC2 light chain.

[0068] SEQ ID NO: 23 is the cDNA sequence of humanized HC3 heavy chain.

[0069] SEQ ID NO: 24 is the cDNA sequence of humanized LC3 light chain. DETAILED DESCRIPTION

[0070] I. Abbreviations

[0071] ACT activated clotting time

[0072] aPTT activated partial thromboplastin time

[0073] AV arteriovenous

[0074] bpm billion per minute

[0075] BT bleeding time

[0076] CBC complete blood count

[0077] CTI corn trypsin inhibitor

[0078] ECMO Extracorporeal membrane oxygenation

[0079] ECOS extracorporeal organ support systems

[0080] FXII blood protein factor XII

[0081] FXIIa Activated FXII

[0082] HK high-molecular-weight kininogen

[0083] iv intravenous

[0084] MAb monoclonal antibody

[0085] PF4 Platelet factor 4

[0086] PK prekallikrein

[0087] PT prothrombin time

[0088] TAT thrombin-antithrombin complex

[0089] II. Terminology and Methodology

[0090] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes X, published by Jones & Bartlett, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH, Volume 16, 2008; and other similar references.

[0091] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0092] Administration: The introduction of a composition into a subject via a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing it into a vein of the subject.

[0093] Antibody: A polypeptide ligand that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an antigenic epitope. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each of which has a variable region, called a variable heavy (V H ) area and variable lightness (V L ) area. H Area and V L The domains are responsible for binding to the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the main antibody produced by birds and reptiles and is similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0094] The variable region of an antibody consists of a "framework" region and a hypervariable region, the latter of which is called a "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the antigen epitope. The framework region of an antibody is used to position and arrange the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Rabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; the "Kabat" numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273, 927-948, 1997; the "Chothia" numbering scheme), and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; the "IMGT" numbering scheme). The Kabat and IMGT databases are maintained online.

[0095] "Single domain antibody" refers to an antibody with a single domain (the variable domain) that is capable of specifically binding to an antigen or antigenic epitope in the absence of additional antibody domains. H Domain antibodies, V NAR Antibodies, Camelidae V H H antibody and V L Domain antibodies. V NAR Antibodies are produced by cartilaginous fishes such as the nurse shark, wobbegong shark, spiny dogfish, and bamboo shark. H H antibodies are produced by several species, including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that naturally lack light chains.

[0096] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell that has been transfected with the coding sequence of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0097] "Chimeric antibodies" have framework residues from one species (eg, human) and the CDRs (which typically confer antigen binding) from another species.

[0098] "Humanized" antibodies are immunoglobulins that include a human framework region and one or more CDRs from non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulins. The non-human immunoglobulin that provides the CDRs is referred to as a "donor," and the human immunoglobulin that provides the framework is referred to as an "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region need not be present, but if present, they must be substantially consistent with the human immunoglobulin constant region, e.g., at least about 85-90%, e.g., about 95% or more consistent. Therefore, all parts of the humanized immunoglobulin may be substantially consistent with the corresponding parts of the natural human immunoglobulin sequence, except for the CDRs. The humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that have little effect on antigen binding or other immunoglobulin functions.

[0099] Anticoagulant: A compound (eg, a pharmaceutical agent or molecule) that prevents or inhibits blood clotting. Pharmaceutical anticoagulants are useful in treating thrombotic disorders such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

[0100] Antigen binding fragment: A portion of a monoclonal antibody that retains its ability to specifically bind to the antigen to which the monoclonal antibody is directed. Antigen binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins (scFv), and disulfide-stabilized Fv proteins.

[0101] Antithrombotic agent: Any compound (eg, a pharmaceutical agent or molecule) that prevents or inhibits the formation of a blood clot. Antithrombotic agents include anticoagulants (which limit the ability of platelets to clot), antiplatelet drugs (which limit the migration and aggregation of platelets), and thrombolytic drugs (which dissolve a clot after it has formed).

[0102] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated by the modified Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In another embodiment, antibody affinity is measured by flow cytometry. An antibody that "specifically binds" an antigen (e.g., FXII) is an antibody that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens.

[0103] Coagulation: The process of polymerization of fibrin monomers, resulting in the conversion of blood or plasma from a liquid to a gel state. Coagulation of liquid blood may occur in vitro, within blood vessels, or on exposed and damaged tissue surfaces. In vitro blood coagulation results in gelled blood that maintains cells and other blood components in essentially the same relative proportions as those found in unclotted blood, except for a decrease in the fibrinogen content and a corresponding increase in fibrin.

[0104] Complementarity Determining Region (CDR): Regions of hypervariable amino acid sequence that define the binding affinity and specificity of an antibody.

[0105] Complement system: A part of the immune system, particularly the innate immune system, that plays a major role in lysis of infectious organisms, activation of inflammation, opsonization, and immune clearance.

[0106] Conjugate: An antibody or antibody fragment (e.g., an antigen binding fragment) covalently linked to an effector molecule (e.g., a detectable label) or a secondary protein (e.g., a secondary antibody). The effector molecule can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, or a carbohydrate. Antibody conjugates are often referred to as "immunoconjugates."

[0107] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce the affinity of a protein (e.g., an antibody to FXII). For example, a monoclonal antibody that specifically binds to FXII may include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and specifically bind to FXII. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody specifically binds to FXII. Non-conservative substitutions are those that reduce activity or binding to FXII.

[0108] Conservative amino acid substitution tables that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions of each other:

[0109] 1) Alanine (A), serine (S), threonine (T);

[0110] 2) Aspartic acid (D), Glutamic acid (E);

[0111] 3) Asparagine (N), glutamine (Q);

[0112] 4) Arginine (R), Lysine (K);

[0113] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0114] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0115] Contact: Arranged in direct physical relation; includes both solid and liquid states.

[0116] Detectable label: A detectable compound or composition that is bound directly or indirectly to another molecule (e.g., an antibody or protein) to facilitate detection of that molecule. Specific non-limiting examples of detectable labels include fluorescent labels, enzyme linkages, and radioisotopes. In one example, a "labeled antibody" refers to the addition of another molecule to an antibody. For example, a label is a detectable marker, such as the addition of a radiolabeled amino acid or the attachment of a polypeptide with a biotinylated moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of polypeptide labels include, but are not limited to, the following: radioisotopes or radionucleotides (e.g., 35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H. 14 C. 15 N. 90 Y. 99 Tc, 111 In and 125I), fluorescent tags (e.g., fluorescein isothiocyanate (FUC), rhodamine, lanthanide phosphors), enzyme tags (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinylation groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags), or magnetic agents (e.g., gadolinium chelates). In some embodiments, the tags are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0117] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). Antibodies specifically bind to specific antigenic epitopes on polypeptides (e.g., FXII).

[0118] Factor XII (FXII or fXII): Any variant, isoform and / or species homolog of human coagulation factor XII, also known as Hageman factor, naturally expressed by cells and present in plasma. FXII is a plasma glycoprotein involved in coagulation, fibrinolysis, and the production of bradykinin and angiotensin. Prekallikrein is cleaved by FXII to form kallikrein, which then cleaves FXII to form α-FXIIa, which is then cleaved by trypsin to form β-FXIIa. α-FXIIa consists of an NH2-terminal heavy chain (coagulation factor XIIa heavy chain) and a COOH-terminal light chain (coagulation factor XIIa light chain) linked by a disulfide bond. α-FXIIa activates FXI to FXIa. It also drives the selective cleavage of the Arg-|-Ile bond in FVII to form FVIIa. β-FXIIa consists of two chains linked by a disulfide bond: a light chain (β-FXIIa part 2), corresponding to the COOH-terminal light chain (coagulation FXIIa light chain), and a nonapeptide (β-FXIIa part 1). FXII interacts with histidine-rich glycoprotein (HRG); this interaction is enhanced in the presence of zinc ions and inhibited by heparin binding, which inhibits fXII autoactivation and contact-initiated coagulation.

[0119] FXII deficiency is the cause of FXII deficiency (FA12D), also known as Hageman factor deficiency. This is characterized by asymptomatic in vitro coagulation abnormalities. Its diagnosis is based on the finding of low plasma activity of this factor in coagulation tests. It is usually discovered only accidentally during preoperative blood tests. FXII deficiency is divided into two categories: cross-reacting material (CRM) negative group (negative FXII antigen test) and CRM positive group (positive FXII antigen test).

[0120] Framework region: The amino acid sequence between the CDRs. The framework region includes the variable light framework region and the variable heavy framework region. The framework region is used to keep the CDRs in the proper orientation for antigen binding.

[0121] Fusion protein: A protein that contains at least portions of two different (heterologous) proteins.

[0122] Hemostasis: The physiological process of bleeding cessation. Hemostatic agents are those that prevent, treat or alleviate abnormal bleeding (e.g., abnormal bleeding caused by a bleeding disorder or a bleeding episode). Hemostatic disorders include, for example, platelet diseases, such as idiopathic thrombocytopenic purpura, and coagulation disorders, such as hemophilia. Hemostasis can also refer to the complex interaction between blood vessels, platelets, coagulation factors, coagulation inhibitors, and fibrinolytic proteins to maintain the blood in the vascular cavity in a fluid state. The purpose of the hemostatic system is to maintain intravascular integrity by achieving a balance between bleeding and thrombosis. As used herein, "promoting hemostasis" refers to the process of helping or improving the hemostasis of a subject. For example, the agent promoting hemostasis can be an agent that reduces abnormal bleeding, such as by stopping bleeding faster or reducing blood loss.

[0123] Heterologous: Originating from a separate genetic source or species.

[0124] Increased bleeding tendency: Bleeding that is spontaneous, excessive, and / or delayed in onset following tissue injury.

[0125] Inflammation: Part of the biological defense response of body tissues to harmful stimuli (e.g., pathogens, damaged cells, or irritants). This protective response involves immune cells, blood vessels, and molecular mediators. The function of inflammation is to help eliminate the initial cause of cellular damage, remove necrotic cells damaged by the original injury or the inflammatory process, and then initiate tissue repair pathways.

[0126] Inhibitory amount or inhibitory dose: The amount of a specific substance sufficient to achieve inhibition of the activity of a specific molecule or inhibition of the activation of a specific molecule. For example, this can be the amount necessary to inhibit the activation and / or activity of FXII in a subject or sample (e.g., a plasma or serum sample). In some embodiments, the inhibitory amount of a FXII-specific monoclonal antibody or antigen-binding fragment thereof is the amount required to inhibit FXII activation by at least 50%. In some instances, the inhibitory amount is the amount necessary to inhibit FXII activation by 90-100%.

[0127] Ischemic organ disease: A disease, condition, state or disorder in which the blood supply to an organ is deprived, restricted or impeded, including arteriosclerosis obliterans, Buerger's disease, Raynaud's disease, myocardial infarction, angina pectoris, diabetic neuropathy, spinal stenosis, cerebrovascular accident, cerebral infarction, pulmonary hypertension, bone fractures and Alzheimer's disease.

[0128] Isolated: An "isolated" biological component, such as a nucleic acid, protein (including an antibody), or organelle, has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0129] Linker: In some cases, a linker is a peptide within an antibody binding fragment (e.g., an Fv fragment) that is used to indirectly bind the variable heavy chain to the variable light chain. "Linker" may also refer to a peptide used to connect a targeting moiety (e.g., an antibody) to an effector molecule (e.g., a detectable label).

[0130] The terms "conjugating", "joining", "bonding" or "linking" refer to making two polypeptides into one continuous polypeptide molecule, or covalently attaching a radionuclide or other molecule to a polypeptide (e.g., scFv). In certain contexts, the terms include reference to linking a ligand (e.g., an antibody portion) to an effector molecule. Linking can be performed chemically or recombinantly. "Chemical" refers to the reaction between the antibody portion and the effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0131] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter (e.g., a CMV promoter) is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, in the case of two protein coding regions that need to be linked, are in the same reading frame.

[0132] Parenteral: refers to administration other than through the digestive tract (digestive tube), for example, subcutaneously, intramuscularly, intrasternal, or intravenously.

[0133] Pathological Hypercoagulability: A genetic or acquired disorder, disease or condition involving pathologically rapid or excessive blood clotting or an increased tendency to clot, such as may be seen in disseminated intravascular coagulation, heart attack, stroke, coronary artery disease, deep vein thrombosis, thromboembolic disease, pulmonary embolism, vascular disease, surgery, trauma, malignancy, the presence of vascular prosthetic devices, general anesthesia, pregnancy, use of oral contraceptives, systemic lupus erythematosus, infection, sepsis, diabetes, autoimmune disease, pancreatitis, cirrhosis, diverticulitis, antiphospholipid antibody syndrome, activated protein C resistance, protein C deficiency, protein S deficiency, elevated levels of coagulation factor VIII, sticky platelet syndrome, hyperhomocysteinemia, antithrombin deficiency, fibrinolytic disorders, prothrombin G20210A mutation, and thrombomodulin mutation.

[0134] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers used are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the antibodies disclosed herein.

[0135] Generally, the character of carrier will depend on the specific mode of administration adopted.For example, parenteral preparations generally include injectable fluids, and the injectable fluids include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solution, aqueous glucose solution, glycerol, etc. as carriers.For solid compositions (such as powder, pill, tablet or capsule form), conventional nontoxic solid carriers can include for example pharmaceutical grade mannitol, lactose, starch or magnesium stearate.Except biological neutral carriers, pharmaceutical compositions to be administered can also include a small amount of nontoxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0136] Prevent, treat or alleviate disease: "Preventing" a disease means inhibiting the full development of the disease. "Treatment" means therapeutic intervention to relieve the signs or symptoms of a disease or pathological condition after it has begun to develop. "Allowing" means reducing the number or severity of the signs or symptoms of a disease.

[0137] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than in its natural environment within a cell. In one embodiment, the preparation is purified so that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to purification from other proteins or cellular components. A substantially purified protein has a purity of at least 60%, 70%, 80%, 90%, 95%, or 98%. Thus, in a specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

[0138] Sample (or biological sample): A biological specimen obtained from a subject containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.

[0139] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed in terms of similarity between the sequences, also known as sequence identity. Sequence identity is often measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of polypeptides or nucleic acid molecules will have a higher degree of sequence identity when aligned using standard methods.

[0140] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0141] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from multiple sources including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are available from the NCBI website on the Internet.

[0142] V of an antibody that specifically binds to a FXII polypeptide H Homologues and variants of the invention are generally characterized by having at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, the sequence identity being counted using NCBI Blast 2.0 in the full-length comparison with the amino acid sequence of the antibody, with the gap blastp being set to default parameters. In order to be relatively greater than about 30 amino acid amino acid sequences, the default BLOSUM62 matrix being set to default parameters (gap existence penalty is 11, each residue gap penalty is 1) is used to adopt the Blast 2 sequence function. When comparing short peptides (less than about 30 amino acids), the PAM30 matrix being set to default parameters (gap 9, extension gap 1 penalty) should be used to compare using the Blast 2 sequence function. When evaluated by this method, the protein having even greater similarity to the reference sequence will show an increased percentage consistency, for example at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities of less than the entire sequence, homologues and variants will generally have at least 80% sequence identity over a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the NCBI website on the Internet. Those skilled in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain very important homologues beyond the ranges provided.

[0143] Solid tumor cancer: A type of cancer that forms abnormal masses of tissue, usually without cysts or areas of fluid, and includes sarcomas, carcinomas, and lymphomas. Solid tumor cancers can include cancers of the pancreas, breast, ovary, prostate, bone, bladder, cervix, colon and rectum, endometrium, kidney, lip and oral cavity, brain, kidney, liver, skin, mesothelium, lung, and thyroid, or their metastases.

[0144] Subject: A living multicellular vertebrate organism, including humans and the class of veterinary subjects, including human and non-human mammals. In some embodiments, the subject is any mammal, such as a human, a non-human primate, or a veterinary subject, such as a dog.

[0145] Synthetic: Produced by artificial means in a laboratory. For example, synthetic nucleic acids or proteins (such as antibodies) can be chemically synthesized in a laboratory.

[0146] Therapeutically effective amount (or pharmaceutically effective amount): The amount of a particular substance sufficient to obtain a desired effect in a treated subject. For example, this may be the amount required to inhibit the activation of Factor XII. The term may also indicate the amount of an anti-FXII monoclonal antibody or a combination of such an antibody and another anticoagulant or antithrombotic agent that is effective to inhibit thrombosis in vivo or otherwise produce a measurable benefit in a patient in need thereof. When administered to a subject, a dose that achieves a target tissue concentration that has been demonstrated to achieve the desired in vitro effect will generally be used. The exact amount will depend on many factors, including, but not limited to, the ingredients and physical properties of the therapeutic composition, the intended patient population, individual patient considerations, etc., and can be readily determined by one skilled in the art.

[0147] Thrombosis: The formation or presence of a clot (also called a "thrombus") within a blood vessel, obstructing the flow of blood through the circulatory system. Thrombosis is usually caused by abnormalities in the composition of the blood, the quality of the vessel wall, and / or the nature of blood flow. The formation of a clot is usually caused by damage to the vessel wall (for example, due to trauma or infection) and slow or stagnant blood flow through the point of injury. In some cases, abnormalities in coagulation lead to thrombosis. A clot that breaks through the confinement and begins to move through the body is called an embolus. When a thrombus occupies more than 75% of the cross-sectional area of ​​the arterial lumen, blood flow to the tissues is reduced enough to cause symptoms due to reduced oxygen (hypoxia) and the accumulation of metabolic products (such as lactate). Obstruction of more than 90% can lead to hypoxia, complete oxygen depletion, and infarction (a form of cell death). Thromboembolism is a combination of thrombosis and its major complication (embolism).

[0148] Vector: A nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

[0149] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an" and "the" include plural references. "Including A or B" means including A or B or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for description. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or test of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are cited in their full text form and added herein. In the event of a conflict, this specification (including term explanation) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be restrictive.

[0150] III. FXII-specific monoclonal antibodies

[0151] Disclosed herein are monoclonal antibodies that bind to human blood protein factor XII (FXII), such as mouse monoclonal antibody 5C12 (also referred to herein as "AB053") or a humanized form thereof (referred to as "humanized 5C12," "h5C12," or "AB054"). The antibody (or its antigen-binding fragment) is capable of forming an immune complex with FXII and inhibiting the activity of FXII. Administration of the disclosed antibodies inhibits inflammation and thrombosis and is therefore useful for treating diseases and disorders characterized by pathological activation of FXII.

[0152] Provided below are the amino acid sequences of the 5C12 heavy chain, 5C12 VH domain, 5C12 light chain, and 5C12 VL domain. In the VH and VL domain sequences, the position of each CDR is underlined according to IMGT. One skilled in the art can easily determine the CDR boundaries using any other numbering scheme (e.g., Kabat or Chothia numbering scheme). The underlined residues in the heavy and light chain sequences represent constant domains. The cDNA sequences of the heavy and light chains are also provided.

[0153] 5C12 heavy chain (SEQ ID NO: 1):

[0154]

[0155] 5C12 VH domain (SEQ ID NO:2):

[0156]

[0157] CDR1: GYSITSGYS (residues 26-34 of SEQ ID NO: 2)

[0158] CDR2: IQYSGNT (residues 52-58 of SEQ ID NO: 2)

[0159] CDR3: ARWGSFDY (residues 97-104 of SEQ ID NO: 2)

[0160] 5C12 light chain (SEQ ID NO:3):

[0161]

[0162] 5C12 VL domain (SEQ ID NO:4):

[0163]

[0164] CDR1: SSVNY (residues 27-31 of SEQ ID NO: 4)

[0165] CDR2: DTS (residues 49-51 of SEQ ID NO: 4)

[0166] CDR3: QQWSGNPPT (residues 88-96 of SEQ ID NO: 4)

[0167] 5C12 heavy chain cDNA sequence (SEQ ID NO: 5)

[0168]

[0169] 5C12 light chain cDNA sequence (SEQ ID NO:6)

[0170] CAAATTGTTCTCACCCAGTCTCCAGCTATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAATTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGGAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTGGTAACCCACCGACGTTCGGTGGAGGCACCATACTGGAAATCAAAC GGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTAACCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAA CAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0171] Humanized heavy and light chain sequences were generated for antibody 5C12. The h5C12 heavy chain, light chain, VH domain, and VL domain sequences are shown below.

[0172] Humanized 5C12 heavy chain ("HC1" SEQ ID NO:7)

[0173]

[0174] Humanized 5C12 (HC1) VH domain (SEQ ID NO: 8)

[0175]

[0176] CDR1: GYSITSGYS (residues 26-34 of SEQ ID NO: 8)

[0177] CDR2: IQYSGNT (residues 52-58 of SEQ ID NO: 8)

[0178] CDR3: ARWGSFDY (residues 97-104 of SEQ ID NO: 8)

[0179] Humanized 5C12 light chain ("LC1" SEQ ID NO:9)

[0180]

[0181] Humanized 5C12 (LC1) VL domain (SEQ ID NO: 10)

[0182] EIVLTQSPATLSLSPGERATLSCRASSSVNYLHWYQQKPGQAPRRLIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGNPPTFGGGTKVEIK

[0183] CDR1: SSVNY (residues 27-31 of SEQ ID NO: 10)

[0184] CDR2: DTS (residues 49-51 of SEQ ID NO: 10)

[0185] CDR3: QQWSGNPPT (residues 88-96 of SEQ ID NO: 10)

[0186] Humanized 5C12 heavy chain (HC1) cDNA sequence (SEQ ID NO: 11)

[0187]

[0188] Humanized 5C12 light chain (LC1) cDNA sequence (SEQ ID NO: 12)

[0189] GAGATCGTGCTGACCCAGAGCCCAGCAACCCTGAGCTTGAGCCCCGGTGAGAGGGCCACCCTGTCATGCAGGGCCAGCAGCAGCGTGAACTACCTGCACTGGTATCAGCAGAAGCCCGGTCAAGCCCCCAGGAGGCTGATCTACGACACCAGCAAGCTGG CCACCGGCATCCCCGCCAGGTTTTCCGGCAGCGGGTCAGGCACCGACTACACCCTCACCATAAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGTCAGCAGTGGAGCGGCAACCCACCTACCTTTGGCGGAGGCACTAAGGTGGAGATCAAGCGG ACCGTGGCCGCCCCACGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA

[0190] An alignment of the mouse ("murine") and humanized ("human") 5C12 heavy and light chain amino acid sequences is shown below. Amino acid residues shared between the mouse and humanized forms are indicated with asterisks.

[0191] Heavy chain alignment

[0192]

[0193] Light chain alignment

[0194]

[0195]

[0196] Alternative humanized heavy and light chain sequences

[0197] During the humanization process, a total of 3 humanized heavy chain (HC1, HC2, HC3) and 3 humanized light chain (LC1, LC2, LC3) sequences were developed. Humanized antibodies containing all 9 possible combinations of humanized heavy and light chains were generated and tested: (1) HC1 + LC1; (2) HC1 + LC2; (3) HC1 + LC3; (4) HC2 + LC1; (5) HC2 + LC2; (6) HC2 + LC3; (7) HC3 + LC1; (8) HC3 + LC2; and (9) HC3 + LC3. All antibodies tested showed similar binding affinity for FXII and had similar FXII inhibitory activity (determined by aPTT assay).

[0198] The humanized 5C12 antibody (also referred to as "AB054") consists of HC1 + LC1 (SEQ ID NOs: 8 and 10). The heavy chain, VH domain, light chain, VL domain, and cDNA sequences of HC2, HC3, LC2, and LC3 are shown below. The CDR sequences of murine 5C12 HC, humanized 5C12 HC, HC2, and HC3 are identical, and the CDR sequences of murine 5C12 LC, humanized 5C12 LC, LC2, and LC3 are identical; only the framework residues vary in the different heavy and light chain sequences.

[0199] HC2 heavy chain (SEQ ID NO: 13)

[0200] DVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYSWHWIRQHPGKGLEWMGYIQYSGNTNSNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARWGSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0201] HC2 VH domain (SEQ ID NO:14)

[0202]

[0203] CDR1: GYSITSGYS (residues 26 - 34 of SEQ ID NO:14)

[0204] CDR2: IQYSGNT (residues 52 - 58 of SEQ ID NO:14)

[0205] CDR3: ARWGSFDY (residues 97 - 104 of SEQ ID NO:14)

[0206] HC3 heavy chain (SEQ ID NO:15)

[0207] DVQLQESGPGLVKPSETLSLTCTVSGYSITSGYSWHWIRQPPGKGLEWIGYIQYSGNTNSNPSLKSRVTISRDTSKNQFSLRLSSVTAADTAVYYCARWGSFDYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0208] HC3 VH domain (SEQ ID NO:16)

[0209]

[0210] CDR1: GYSITSGYS (residues 26 - 34 of SEQ ID NO:16)

[0211] CDR2: IQYSGNT (residues 52 - 58 of SEQ ID NO:16)

[0212] CDR3: ARWGSFDY (residues 97 - 104 of SEQ ID NO:16)

[0213] LC2 light chain (SEQ ID NO:17)

[0214] EIVLTQSPATLSLSPGERATLSCRASSSVNYLHWYQQKPGQAPKRWIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] LC2 VL domain (SEQ ID NO: 18)

[0216]

[0217] CDR1: SSVNY (residues of SEQ ID NO: 18)

[0218] CDR2: DTS (residues of SEQ ID NO: 18)

[0219] CDR3: QQWSGNPPT (residues of SEQ ID NO: 18)

[0220] LC3 light chain (SEQ ID NO: 19)

[0221] DIVLTQTPATLSLSPGERATLSCRASSSVNYLHWYQQKPGQAPKRLIYDTSKLATGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGNPPTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0222] LC3 VL domain (SEQ ID NO:20)

[0223]

[0224] CDR1: SSVNY (residues 27-31 of SEQ ID NO: 20)

[0225] CDR2: DTS (residues 49-51 of SEQ ID NO: 20)

[0226] CDR3: QQWSGNPPT (residues 88-96 of SEQ ID NO: 20)

[0227] HC2 heavy chain cDNA sequence (SEQ ID NO: 21)

[0228]

[0229] LC2 light chain cDNA sequence (SEQ ID NO:22)

[0230] GAGATCGTGCTGACCCAGAGCCCAGCAACCCTGAGCTTGAGCCCCGGTGAGAGGGCCACCCTGTCATGCAGGGCCAGCAGCAGCGTGAACTACCTGCACTGGTATCAGCAGAAGCCCGGTCAAGCCCCCAAGAGGTGGATCTACGACACCAGCAAGCTGGCCACCGGCATCCCCGCCAGGTTTTCCGGCAGCGGGTCAGGCACCGACTACACCCTCACCATAAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGTCAGCAGTGGAGCGGCAACCCACCTACCTTTGGCGGAGGCACTAAGGTGGAGATCAAGCGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA

[0231] HC3 heavy chain cDNA sequence (SEQ ID NO:23)

[0232]

[0233] LC3 light chain cDNA sequence (SEQ ID NO: 24)

[0234] GACATCGTGCTGACCCAGACCCCAGCAACCCTGAGCTTGAGCCCCGGTGAGAGGGCCACCCTGTCATGCAGGGCCAGCAGCAGCGTGAACTACCTGCACTGGTATCAGCAGAAGCCCGGTCAAGCCCCCAAGAGGCTGATCTACGACACCAGCAAGCTGG CCACCGGCATCCCCGCCAGGTTTTCCGGCAGCGGGTCAGGCACCGACTACACCCTCACCATAAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTGTCAGCAGTGGAGCGGCAACCCACCTACCTTTGGCCAGGGCACTAGGTTGGAGATCAAGCGG ACCGTGGCCGCCCCACGTGTTCATCTTCCCTCCCAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGA GCGTGACCGAGCAGGACTCCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCTAA

[0235] Provided herein are monoclonal antibodies or antigen-binding fragments that bind (eg, specifically bind) FXII. In some embodiments, the monoclonal antibody or antigen-binding fragment comprises both a VH domain and a VL domain.

[0236] In some embodiments, the monoclonal antibody or antigen-binding fragment that binds FXII comprises at least one CDR sequence from antibody 5C12. In some embodiments, the CDR sequences are identified using the IMGT, Kabat, or Chothia numbering schemes.

[0237] In some embodiments, the FXII-specific monoclonal antibody or antigen-binding fragment comprises a VH domain and a VL domain, and the VH domain of the antibody comprises one, two, or all three CDR sequences of SEQ ID NO: 2, and / or the VL domain of the antibody comprises one, two, or all three CDR sequences of SEQ ID NO: 4. In some examples, the VH domain comprises residues 26-34, 52-58, and 97-104 of SEQ ID NO: 2, and the VL domain comprises residues 27-31, 49-51, and 88-96 of SEQ ID NO: 4.

[0238] In specific examples, the VH domain includes the CDR sequence of SEQ ID NO: 2, and the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14 or SEQ ID NO: 16; and the VL domain includes the CDR sequence of SEQ ID NO: 4, and the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 18 or SEQ ID NO: 20. In specific non-limiting examples, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, or SEQ ID NO: 16, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 18, or SEQ ID NO: 20. In one non-limiting example, the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 8, and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

[0239] In some embodiments, the monoclonal antibody or antigen-binding fragment comprises a complete heavy chain and / or a complete light chain. In some examples, the heavy chain comprises the CDR sequence of SEQ ID NO: 2, and the amino acid sequence of the heavy chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 15; and / or the light chain comprises the CDR sequence of SEQ ID NO: 4, and the amino acid sequence of the light chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 19. In specific non-limiting examples, the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 15; and / or the amino acid sequence of the light chain comprises or consists of SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 19. In one non-limiting example, the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO: 7, and the amino acid sequence of the light chain comprises or consists of SEQ ID NO: 9.

[0240] The FXII-specific antigen-binding fragment can be, for example, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain variable fragment (scFv), or a disulfide-stabilized variable fragment (dsFv). In some embodiments, the antigen-binding fragment is a scFv.

[0241] The FXII-specific monoclonal antibody can be of any isotype, such as IgG, IgM, IgA, IgD, or IgE. In some embodiments, the monoclonal antibody is IgG.

[0242] In some embodiments, the monoclonal antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment. In some examples, the amino acid sequence of the VH domain of the humanized antibody includes the CDR sequence of SEQ ID NO: 2, and is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 8; and / or the amino acid sequence of the VL domain of the humanized antibody includes the CDR sequence of SEQ ID NO: 4, and is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 10. In a specific non-limiting example, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 8; and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.

[0243] In some embodiments, the humanized monoclonal antibody or antigen-binding fragment comprises a complete heavy chain and / or a complete light chain. In some examples, the heavy chain comprises the CDR sequence of SEQ ID NO: 2, and the amino acid sequence of the heavy chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 7; and / or the light chain comprises the CDR sequence of SEQ ID NO: 4, and the amino acid sequence of the light chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9. In a specific non-limiting example, the amino acid sequence of the heavy chain comprises or consists of SEQ ID NO: 7; and / or the amino acid sequence of the light chain comprises or consists of SEQ ID NO: 9.

[0244] In some embodiments, the monoclonal antibody or antigen-binding fragment is a chimeric or synthetic antibody or antigen-binding fragment.

[0245] Also provided herein is a fusion protein comprising a monoclonal antibody or antigen binding fragment disclosed herein and a heterologous protein or peptide. In some embodiments, the heterologous protein is an Fc protein. In some instances, the Fc protein is a mouse Fc or human Fc protein.

[0246] Further provided are antibody conjugates comprising a monoclonal antibody or antigen-binding fragment disclosed herein and a detectable label, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope.

[0247] Also provided are compositions comprising a pharmaceutically acceptable carrier and the monoclonal antibodies or antigen-binding fragments disclosed herein.

[0248] Also provided are nucleic acid molecules encoding the monoclonal antibodies or antigen-binding fragments disclosed herein. In some embodiments, the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In some examples, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and / or SEQ ID NO: 24. In a specific non-limiting example, the nucleic acid molecule comprises SEQ ID NO: 11 and / or SEQ ID NO: 12. In some embodiments, the nucleic acid molecule is operably linked to a promoter. A vector comprising the described nucleic acid molecule is further provided. Also provided are cells comprising a nucleic acid molecule or vector disclosed herein.

[0249] Further provided are methods for detecting FXII in a sample. In some embodiments, the method comprises contacting the sample with a monoclonal antibody or antigen binding fragment disclosed herein; and detecting binding of the antibody to the sample. In some instances, the monoclonal antibody or antigen binding fragment is directly labeled. In other instances, the method further comprises contacting the monoclonal antibody or antigen binding fragment with a secondary antibody, and detecting binding of the secondary antibody to the monoclonal antibody or antigen binding fragment.

[0250] Also provided is an in vitro method for inhibiting the activation and / or activity of FXII in a sample comprising FXII. In some embodiments, the method comprises contacting the sample with an FXII-specific antibody or antigen-binding fragment disclosed herein. In a specific example, the sample comprises a blood or plasma sample. In some examples, the method is a method for inhibiting the activity of FXIIa.

[0251] Further provided is a method of inhibiting the activation and / or activity of FXII in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antigen binding fragment disclosed herein. Also provided is a method of treating a pathological process involving the activity and / or activation of FXII in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antigen binding fragment disclosed herein. Further provided is a method of inhibiting thrombosis involving the activation and / or activity of FXII in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antigen binding fragment disclosed herein. In some instances, the method comprises inhibiting the activity of FXIIa.

[0252] In some embodiments of these methods, the subject suffers from or is at risk of developing bacterial infection, fungal infection, viral infection, parasitic infection, ischemic organ disease, microvascular thrombosis, macrovascular thrombosis, thromboembolism, disseminated intravascular coagulation, severe systemic inflammatory response syndrome, allergic or inflammatory response involving FXIIa activity, acute respiratory distress syndrome, cancer, amniotic fluid embolism, trauma, transplant rejection, sickle cell disease, autoimmune disease, or has received a medical device implant. In some instances, the ischemic organ disease is myocardial infarction or ischemic stroke. In some instances, the cancer is non-metastatic solid tumor cancer, metastatic solid tumor cancer or leukemia. In some instances, the medical device implant is an implanted catheter, heart valve, stent or graft. In some instances, the method further comprises administering to the subject an effective amount of a second anticoagulant therapy, or antithrombotic or thrombolytic therapy.

[0253] In some embodiments of the disclosed method, the monoclonal antibody or antigen binding fragment is administered by parenteral administration. In some embodiments, the monoclonal antibody or antigen binding fragment is administered at a dosage of about 0.1 mg / kg to about 2 g / kg, or about 0.1 mg / kg to about 20 mg / kg, for example, about 0.5 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 5 mg / kg. In some instances, the monoclonal antibody or antigen binding fragment is administered at a dosage of about 0.1, about 0.25, about 0.5, about 0.75, about 1.0, about 2.5, about 5.0, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 200 mg / kg, about 500 mg / kg, about 1000 mg / kg or about 2 g / kg.

[0254] The present disclosure further provides a kit, which includes the disclosed monoclonal antibody, antigen-binding fragment, fusion protein, antibody conjugate, composition, nucleic acid molecule, vector or isolated cell. The kit may further include, for example, instruction materials, buffer, cell culture medium, pharmaceutically acceptable carrier or diluent, etc.

[0255] IV. Antibody Conjugates

[0256] Antibody conjugates are provided herein, comprising monoclonal antibodies or antigen binding fragments coupled to reagents (e.g., detectable labels) disclosed herein. For diagnostic and therapeutic purposes, reagents can be connected or covalently bound to antibodies or antigen binding fragments disclosed herein. Such molecules or moieties can be, but are not limited to, effector molecules or reporter molecules. Reporter molecules are defined as any moiety that can be detected by analysis. Non-limiting examples of reporter molecules that have been coupled to antibodies include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.

[0257] Some examples of antibody conjugates are those in which the antibody is linked to a detectable label. Detectable labels are compounds and / or elements that can be detected due to their specific functional properties and / or chemical properties, the use of which allows the antibody to which they are attached to be detected and / or further quantified if desired.

[0258] Antibody conjugates are useful as diagnostic reagents. Antibody diagnostics are generally divided into two categories, those used for in vitro diagnostics, such as in various immunoassays, and / or those used for in vivo diagnostic protocols, often referred to as "antibody-directed imaging."

[0259] Many suitable imaging agents are known in the art, as are methods of attaching them to antibodies (see, e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moiety can be, for example, a paramagnetic ion; a radioisotope; a fluorescent dye; or an NMR detectable substance.

[0260] Exemplary paramagnetic ions include, but are not limited to, chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and / or erbium (III). Useful ions in other contexts (e.g., X-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II), and bismuth (III).

[0261] Exemplary radioisotopes for therapeutic and / or diagnostic applications include, but are not limited to,35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H. 14 C. 15 N. 90 Y. 99 Tc, 111 In and 125 I. Radiolabeled monoclonal antibodies can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contacting with sodium iodide and / or potassium iodide and a chemical oxidant (e.g., sodium hypochlorite) or an enzymatic oxidant (e.g., lactoperoxidase). Monoclonal antibodies can be iodinated by a ligand exchange process. 99m Tc labeling, for example, by reducing pertechnetate with stannous solution and chelating the reduced technetium to SEPHADEX TM The antibody is applied to the column. Alternatively, direct labeling techniques can be used, for example, by incubating pertechnetate, a reducing agent (e.g., SNCl2), a buffer solution (e.g., sodium potassium phthalate solution), and the antibody. The intermediate functional group commonly used to bind radioisotopes in the form of metal ions to antibodies is diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0262] Among the fluorescent labels contemplated for use as conjugates include, but are not limited to, Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0263] Other types of antibody conjugates contemplated are those used primarily in vitro, in which the antibody is linked to a secondary binding ligand and / or an enzyme (enzyme tag) that will produce a colored product when contacted with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, horseradish peroxidase, or glucose oxidase. Secondary binding ligands include biotin and / or avidin and streptavidin compounds. The use of such tags is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.

[0264] Molecules containing an azide group can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atertone et al., 1985). 2- and 8-azido nucleotides have also been used to identify nucleotide-binding domains of purified proteins (Khatoon et al., 1989 and Dholakia et al., 1989) and can be used as antibody binders.

[0265] Several methods for attaching or coupling antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelates, which employ, for example, organic chelators as described in U.S. Patents 4,472,509 and 4,938,948 (each incorporated herein by reference). Monoclonal antibodies can also react with enzymes in the presence of coupling agents (e.g., glutaraldehyde or periodate). Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reacting with isothiocyanates. In U.S. Patent 4,938,948, imaging of breast tumors was achieved using monoclonal antibodies, and detectable imaging moieties were bound to antibodies using joints (e.g., methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate).

[0266] In other embodiments, it is contemplated to derivatize immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method are disclosed to show improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules is also disclosed in the literature, wherein the reporter or effector molecule is coupled to a carbohydrate residue in the Fc region. This method reportedly produces antibodies with diagnostic and therapeutic promise that are currently under clinical evaluation.

[0267] In another embodiment, modified immunoglobulin can be selected to improve its stability and half-life in vivo. PEGylation is a process that involves covalently bonding a polyethylene glycol (PEG) polymer chain to an antibody. PEGylation is conventionally achieved by incubating a reactive derivative of PEG with a target molecule. The covalent bonding of PEG can "mask" antibodies from the host immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the reagent, thereby extending its circulation time by reducing renal clearance. PEGylation can also provide water solubility. Other polymers used to modify antibodies include polyethyleneimine and polylysine, which are usually connected by a succinic acid group.

[0268] V. Immunoassay Methods

[0269] In further embodiments, immunoassays are provided for binding, purifying, removing, quantifying and / or otherwise generally detecting biological components, using antibodies that immunoreact with such components. Some immunoassays include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays, and Western blots, etc. The steps of various useful immunoassays are well known in the art and can be easily performed by those of ordinary skill in the art.

[0270] Generally, immunobinding methods involve obtaining a sample containing a target of interest and contacting the sample with a primary antibody that immunoreacts with the target under conditions effective to form an immune complex. Binding of the antibody to the target can then be assessed using a variety of different formats.

[0271] In one format, the antibody can be attached to a solid support, such as in the form of a column matrix, and a sample suspected of containing the target is applied to the immobilized antibody. Unwanted components are washed from the column, so that the target immunocomplexed to the immobilized antibody is eluted.

[0272] Immunobinding methods also include methods for detecting and quantifying the amount of target in a sample and detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing the target is obtained and contacted with an antibody against the target, and then the amount of immune complexes formed under specific conditions is detected and quantified.

[0273] In the case of antigen detection, the biological sample analyzed can be any sample suspected of containing the target, for example, a body fluid such as blood, serum, plasma, mucus, urine, saliva, tears or semen. Alternatively, tissue can be used. Contacting the selected biological sample with the antibody under effective conditions and within a time period sufficient to allow the formation of an immune complex (primary immune complex) is usually a simple addition of the antibody composition to the sample and incubating the mixture for a time sufficient to allow the antibody to form an immune complex with the existing antibodies (to bind to the target that immunoreacts with the antibody). After this period of time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or protein blot, is usually washed to remove any non-specifically bound species, so that only those molecules that are specifically bound in the primary immune complex are detected.

[0274] In general, detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods are generally based on the detection of labels or markers, such as any of those radioactive, fluorescent, biological and enzymatic labels. U.S. patents on the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference. However, as is known in the art, secondary binding ligands, such as secondary antibodies and / or biotin / avidin ligand binding arrangements, may also be used.

[0275] The antibody used for detection can itself be connected to a detectable label, wherein the label can then be simply detected, thereby allowing the amount of the primary immune complex in the composition to be determined. Alternatively, the primary antibody bound in the primary immune complex can be detected by a secondary binding ligand having a binding affinity for the antibody. In these cases, the secondary binding ligand can be connected to a detectable label. The secondary binding ligand itself is usually an antibody and can therefore be referred to as a "secondary" antibody. Under effective conditions and within a time period sufficient to allow the formation of a secondary immune complex, the primary immune complex is contacted with a labeled secondary binding ligand or antibody. The secondary immune complex is then typically washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining labels in the secondary immune complex are then detected.

[0276] A further method comprises detecting the primary immune complex by a two-step method. As described above, a secondary binding ligand (e.g., an antibody) having a binding affinity to the antibody is used to form a secondary immune complex. After washing, the secondary immune complex is contacted with a tertiary binding ligand or antibody having a binding affinity to the secondary antibody again under effective conditions and within a time period sufficient to allow the formation of an immune complex (tertiary immune complex). The tertiary ligand or antibody is connected to a detectable label, allowing detection of the tertiary immune complex thus formed. If desired, the system can provide signal amplification.

[0277] An immunoassay method designed by Charles Cantor uses two different antibodies. The first step uses a biotinylated monoclonal or polyclonal antibody to detect the target antigen, and then the second step uses an antibody to detect the biotin attached to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubating in a continuous solution of streptavidin (or avidin), biotinylated DNA and / or complementary biotinylated DNA, adding additional biotin sites to the antibody / antigen complex at each step. The amplification steps are repeated until a suitable amplification level is reached, at which time the sample is incubated in a solution containing an anti-biotin second step antibody. The second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. With appropriate amplification, a macroscopically visible conjugate can be produced.

[0278] Another known immunoassay method utilizes an immuno-PCR approach. The PCR approach is similar to the Cantor approach, but rather than using multiple rounds of streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer that releases the antibody prior to incubation with biotinylated DNA. The resulting wash solution is then used to perform a PCR reaction with appropriate primers under appropriate controls. The enormous amplification capacity and specificity of PCR can be used to detect single antigen molecules.

[0279] Another type of ELISA with an immobilized antigen involves the use of antibody competition in the assay. In this ELISA, a labeled antibody to the antigen is added to the wells, allowed to bind and / or detected by its label. The amount of antigen in the unknown sample is then determined by mixing the sample with the labeled antibody to the antigen during incubation with the coated wells. The presence of the antigen in the sample reduces the amount of antibody to the antigen available for binding to the wells, thereby reducing the final signal. This also applies to the detection of antibodies to the antigen in an unknown sample, where unlabeled antibodies bind to the antigen-coated wells and also reduce the amount of antigen available for binding to the labeled antibody.

[0280] ELISAs typically have certain common features, such as coating, incubation and binding, washing to remove non-specifically bound species and detecting bound immune complexes. When the plate is coated with an antigen or antibody, the wells on the plate are usually incubated with an antigen or antibody solution overnight or for a specific period of time. The wells on the plate are then washed to remove incompletely adsorbed material. Any remaining available surface of the wells is then "coated" with a non-specific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein or milk powder solutions. The coating allows the non-specific adsorption sites on the fixed surface to be blocked, thereby reducing the background caused by the non-specific binding of the antiserum to the surface.

[0281] In many ELISAs, secondary or tertiary detection is used rather than a direct process. Thus, after the protein or antibody is bound to the wells, coated with a non-reactive material to reduce background, and washed to remove unbound material, the fixed surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complex then requires a labeled secondary binding ligand or antibody, as well as a secondary binding ligand or antibody coupled to the labeled tertiary antibody or tertiary binding ligand.

[0282] "Under conditions effective to allow immune complex (antigen / antibody) to form" means that the conditions may include diluting the antigen and / or antibody with a solution (e.g., BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween). These added reagents also help reduce nonspecific background.

[0283] "Suitable" conditions also refer to incubation at a temperature or for a period of time sufficient to allow effective binding. In some examples, the incubation step is from about 1 to about 4 hours and at a temperature of about 25°C to about 27°C, or overnight at about 4°C.

[0284] VI. Antibody Purification

[0285] In some embodiments, the anti-FXII antibody can be purified. As used herein, the term "purified" is intended to refer to a composition that can be separated from other components, wherein the protein is purified to any extent relative to its naturally obtainable state. Therefore, a purified protein also refers to a protein that is not affected by the environment in which it is naturally found. When the term "substantially purified" is used, the name will refer to a composition in which the protein or peptide forms the major component of the composition, for example, constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0286] Protein purification techniques are well known to those skilled in the art. These techniques involve, at one level, the crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After the polypeptide is separated from other proteins, the target polypeptide can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity).

[0287] Analytical methods particularly suitable for preparing pure peptides are ion exchange chromatography, size exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc. or by heat denaturation followed by centrifugation, gel filtration, reverse phase, hydroxyapatite and affinity chromatography; and combinations of these and other techniques.

[0288] When purifying antibodies against FXII, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tag portion of the polypeptide. As is generally known in the art, it is believed that the order in which the various purification steps are performed may be changed, or certain steps may be omitted, and still result in a suitable method for preparing a substantially purified protein or peptide.

[0289] Typically, intact antibodies are fractionated using reagents that bind to the Fc portion of the antibody (e.g., protein A). Alternatively, an antigen can be used to simultaneously purify and select suitable antibodies. Such methods typically utilize a selection agent that is bound to a support (e.g., a column, a filter membrane, or a bead). The antibody is bound to the support, contaminants are removed, and the antibody is released by applying specific conditions (e.g., salt, heat, etc.).

[0290] According to the present disclosure, a variety of methods for quantifying the purity of proteins or peptides will be known to those skilled in the art. These include, for example, determining the specific activity of active fractions, or evaluating the number of polypeptides in fractions by SDS / PAGE analysis. Another method for evaluating the purity of fractions is to calculate the specific activity of the fractions, compare it with the specific activity of the initial extract, and thus calculate the purity. Of course, the actual unit used to represent the amount of activity will depend on the specific assay technique selected for purification and whether the expressed protein or peptide exhibits detectable activity. The migration of known polypeptides can vary with different conditions of SDS / PAGE, sometimes significantly. Therefore, it should be understood that under different electrophoretic conditions, the apparent molecular weight of a purified or partially purified expression product can vary.

[0291] VII. Pharmaceutical Compositions

[0292] Pharmaceutical compositions may include an effective amount of one or more antibodies, therapeutic agents or other agents dissolved or dispersed in a pharmaceutically acceptable carrier. Aqueous compositions include an effective amount of antibodies dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The terms "pharmaceutically acceptable", "therapeutically acceptable", etc. refer to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when administered to animals or humans (as the case may be).

[0293] The terms "pharmaceutically acceptable carrier" and "therapeutically acceptable carrier" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes and the like materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, they are considered for use in therapeutic compositions. Supplementary active ingredients may also be added to the composition. For human administration, the preparation should meet sterility, pyrogenicity, and general safety and purity standards.

[0294] Where appropriate, the biological material should be dialyzed extensively to remove unwanted small molecular weight molecules, and / or lyophilized to facilitate formulation into the desired carrier. The active compound is then typically formulated for parenteral administration, such as for injection or other means by intravenous, intramuscular, subcutaneous, intranasal, intrapulmonary, intrathecal or intraperitoneal routes. Typically, such compositions can be prepared as injections, or as liquid solutions or suspensions; solid forms suitable for preparing solutions or suspensions after addition of liquids prior to injection can also be prepared; and the preparations can also be emulsified.

[0295] The pharmaceutical forms suitable for injection purposes include sterile aqueous solutions or dispersions; formulas include sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injections or dispersions. In all cases, the form must be sterile and must have a certain degree of fluidity to facilitate injection, or aerosol delivery exists. It can be added to other drug delivery vehicles designed for extended release, or modified to have an extended biological half-life. It must remain stable under manufacturing and storage conditions, and must be preserved to prevent contamination by microorganisms (such as bacteria and fungi).

[0296] Solutions of free alkali or pharmacologically acceptable salts of the active compound can be prepared in water by mixing with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under conventional storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0297] Antibodies against FXII can be formulated as free base, neutral or salt forms. Pharmaceutically acceptable salts include acid salts (formed with free amino groups of proteins) and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0298] The carrier can also be a solvent or dispersion medium, which comprises, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size under dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugar or sodium chloride, can be included. The absorption of the injectable composition can be extended by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0299] Sterile injectable solutions are prepared by adding the required amount of active compound and various other ingredients listed above (as required) to an appropriate solvent and then filtering and sterilizing. Typically, dispersions are prepared by adding various sterilized active ingredients to a sterile carrier, which contains a basic dispersion medium and other ingredients required from those listed above. As for the sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying technology, which produces powders of active ingredients and any other required ingredients from its previously sterile filtered solution. It is also expected that more or highly concentrated solutions are prepared for direct injection, wherein it is envisioned that DMSO is used as a solvent to produce extremely rapid penetration, and high concentrations of active agents are delivered to smaller areas.

[0300] After formulation, the solution will be administered in a manner compatible with the dosage form and in such amount as to be therapeutically effective. The formulation is readily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be employed.

[0301] For example, for parenteral administration in aqueous solution, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intranasal and intraperitoneal administration. In this regard, those skilled in the art will know the sterile aqueous media that can be used in light of the present disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection or injected at the recommended infusion site (see, e.g., Remington's Pharmaceutical Sciences, 15 th Ed. Mack Printing Company, pages 1035-1038 and 1570-1580, 1975). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for an individual subject.

[0302] In addition to formulating the compound for parenteral administration (e.g., intravenous or intramuscular injection), other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomal formulations; time-release capsules; and any other forms currently used, including creams.

[0303] The therapeutic may include different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether sterility is required for routes of administration such as injection. Anti-FXII antibodies can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, local perfusion, bathing target cells directly, by catheter, by lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods known to one of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0304] The actual dosage of the composition administered to an animal or human patient can be determined by physical and physiological factors, such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic conditions of the patient, and route of administration. The practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0305] In some embodiments, the pharmaceutical composition may include, for example, at least about 0.1% of the active compound. In other embodiments, the active compound may include, for example, about 2% to about 75% of the unit weight, or about 25% to about 60%, and any range derivable therein.

[0306] In some embodiments, the composition includes various antioxidants to prevent oxidation of one or more components. In embodiments where the composition is in liquid form, the carrier can be a solvent or dispersion medium, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. It can be, for example, by using a coating such as lecithin; by being dispersed in a carrier (e.g., a liquid polyol or lipid) to maintain the desired particle size; by using a surfactant such as hydroxypropyl cellulose; or a combination thereof to maintain appropriate fluidity. In many cases, isotonic agents such as sugars, sodium chloride, or a combination thereof can be included.

[0307] In specific embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin, or a combination thereof.

[0308] Considering that factor XII lacks and is asymptomatic in humans and animals (such as mice), the antibodies of the present disclosure that suppress FXII activity are essentially safe. This is a major advance relative to the use of other anticoagulant antibodies, because the antibodies other than those discussed herein (other than those that suppress the contact activation of FXII), regardless of their route of administration or dosage, are expected to cause hemorrhagic diseases. Considering that factor XII does not participate in important hemostasis, antibodies similar to those disclosed herein will not have such adverse effects on hemostasis. In fact, the danger of overdose is theoretically zero, because it has no additional impact on its target saturation. Therefore, the antibodies of the present application are safe antithrombotic agents (anticoagulants, blood thinners).

[0309] The pharmaceutical composition can be administered parenterally to a subject suffering from a disease in which FXII activation brings problems. The composition can be administered to a patient in need as a bolus or by continuous infusion. For example, the amount of the bolus administration of the antibody or antibody fragment can be 0.0025 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg or 0.10 to 0.50 mg / kg. For continuous infusion, the antibody of the present invention present as a Fab fragment can be administered at 0.001 to 100 mg / kg body weight / minute, 0.0125 to 1.25 mg / kg / min, 0.010 to 0.75 mg / kg / min, 0.010 to 1.0 mg / kg / min or 0.10 to 0.50 mg / kg / min for 1-24 hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours or 1-2 hours. The antibody can be administered by bolus injection, repeated hourly, daily, weekly or monthly, depending on the dose. The frequency and duration of administration will depend on the severity of the condition and the need for continued therapeutic activity, without limitation. The frequency can also vary from three times a week to once every two weeks to six months.

[0310] In addition, the composition can be administered to the patient by subcutaneous, intravenous or intramuscular injection. For example, a dose of 10 to 100 mg of anti-FXII antibody can be administered to the patient by subcutaneous injection daily, weekly, biweekly or monthly.

[0311] Antibody disclosed herein can be used as monotherapy, but can be combined with other therapies.For example, co-administer one or more other antithrombotic agents or anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs (NSA1D), platelet inhibitors, anticoagulants or thrombolytic agents, are considered to be useful for treating certain diseases.These conjoint therapies are likely to reduce the necessary frequency or dosage of anticoagulant or anti-inflammatory drugs.Co-administer or conjoint therapies refer to the use of two therapeutic drugs prepared respectively or co-formulated in a kind of compositions, and when prepared respectively, at about the same time or at different times but in the same treatment cycle.

[0312] Combination antiplatelet agents include irreversible cyclooxygenase inhibitors (aspirin), adenosine diphosphate (ADP) receptor inhibitors (clopidogrel, prasugrel, ticagrelor, and ticlopidine), phosphodiesterase inhibitors (cilostazol), glycoprotein IIB / IIIA inhibitors (abciximab, eptifibatide, and tirofiban), adenosine reuptake inhibitors (dipyridamole), and thromboxane inhibitors, such as thromboxane synthetase inhibitors (ifetroban), thromboxane receptor antagonists (serostat, picotamide), and trutroban.

[0313] Anticoagulant drugs used in combination include vitamin K antagonists (warfarin), unfractionated and low molecular weight heparins (enoxaparin, dalteparin, heparin), thrombin inhibitors (bivalirudin, argatroban, dabigatran, antithrombin III), and factor Xa inhibitors (apixaban, fondaparinux, rivaroxaban, edoxaban).

[0314] Fibrinolytic agents used in combination include anistreplase, reteplase, streptokinase, kabikinase, alteplase, tenecteplase, urokinase, staphylokinase, plasmin, and rokinase.

[0315] Anti-inflammatory drugs used in combination include NSAIDs such as celecoxib, piroxicam, indomethacin, meloxicam, ketoprofen, sulindac, aspirin, diflunisal, nabumetone, and corticosteroids such as prednisone, betamethasone, cortisone, dexamethasone, hydrocortisone, methylprednisolone and prednisolone. These may also include inhibitors of the complement system or the kallikrein-kinin system.

[0316] VIII. Kit

[0317] Any composition described herein may be included in a kit. Thus, the kit will include the antibody (or antigen binding fragment) and / or additional reagents in a suitable container. Other components may be included in the kit. Diagnostic and therapeutic kits include a pharmaceutically acceptable formulation of the antibody in a pharmaceutically acceptable formulation in a suitable container. The kit may have a single container, and / or may have different containers for each compound.

[0318] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution may be an aqueous solution, with a sterile aqueous solution being an example of a particular embodiment. The antibody may also be formulated as an injectable composition, in which case the container itself may be a syringe, pipette, and / or other similar device from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or even applied to and / or mixed with other components of the kit.

[0319] However, the components of the kit may be provided as dry powders. When the reagents and / or components are provided as dry powders, the powders may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0320] The container will generally include at least one vial, test tube, flask, bottle, syringe and / or other container into which the antibody / antibody formulation is placed and appropriately dispensed. The kit may also include a second container for containing a sterile, pharmaceutically acceptable buffer and / or another diluent.

[0321] The kit may also include a device for hermetically containing the vials for commercial sale, for example, an injection and / or blow molded plastic container in which the desired vials are retained.

[0322] Regardless of the number and / or type of containers, the kit may also include and / or be packaged with an instrument for assisting in the injection / administration and / or placement of the final antibody in an animal. Such an instrument may be a syringe, a pipette, forceps and / or any such medically approved delivery vehicle.

[0323] IX. Therapeutic Uses

[0324] Considering that FXII deficiency is asymptomatic in humans and mice, the antibodies disclosed herein that prevent FXII activity are essentially safe. This is a major advance relative to the use of other anticoagulant antibodies, because antibodies other than those disclosed herein, regardless of their route of administration or dosage, are expected to cause hemorrhagic diseases. Considering that FXII is not involved in important hemostasis, antibodies similar to those disclosed herein will not have such adverse effects on hemostasis. In fact, the risk of excess is theoretically zero because it has no additional effects above its target saturation. Therefore, the antibodies disclosed herein are safe antithrombotic agents (anticoagulants, blood thinners) and safe anti-inflammatory molecules.

[0325] A. Thrombosis and thromboembolism

[0326] Thrombosis is the formation of a blood clot within a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is damaged, the body uses platelets (thrombocytes) and fibrin to form a blood clot to prevent blood loss. Even when a blood vessel is not damaged, a blood clot can form in the body under certain circumstances. A clot that breaks through the restriction and begins to move through the body is called an embolus. When a thrombus occupies more than 75% of the cross-sectional area of ​​the arterial lumen, blood flow to the tissues is reduced enough to cause symptoms due to reduced oxygen (hypoxia) and the accumulation of metabolic products such as lactic acid. Obstruction of more than 90% can lead to hypoxia, complete oxygen depletion, and infarction (a form of cell death). Thromboembolism is a combination of thrombosis and its major complication (embolism).

[0327] There are two different forms of thrombosis, venous thrombosis and arterial thrombosis, each of which can be manifested by several subtypes. Venous thrombosis is the formation of a thrombus (blood clot) within a vein. Several diseases can be classified into this category.

[0328] Deep vein thrombosis (DVT) is the formation of a blood clot in a deep vein. It most commonly affects veins in the legs, such as the femoral vein. Three factors are important in the formation of a blood clot in a deep vein: blood flow rate, blood thickness, and the quality of the vessel wall. Typical signs of DVT include swelling, pain, and redness in the affected area.

[0329] Portal vein thrombosis is a form of venous thrombosis affecting the hepatic portal vein, which can lead to portal hypertension and reduced blood flow to the liver. It usually has a pathological cause such as pancreatitis, cirrhosis, diverticulitis or bile duct cancer.

[0330] Renal vein thrombosis is a blockage of the renal vein by a blood clot. This often results in reduced drainage from the kidneys. Anticoagulation is the treatment of choice.

[0331] Jugular vein thrombosis is a condition that can occur as a result of infection, intravenous drug use, or malignancy. Jugular vein thrombosis can have a variety of complications, including: systemic sepsis, pulmonary embolism, and papilledema. Although characterized by severe pain at the site of the vein, it can prove difficult to diagnose because it can occur randomly.

[0332] Budd-Chiari syndrome is an obstruction of the hepatic veins or the inferior vena cava. This form of thrombosis presents with abdominal pain, ascites, and hepatomegaly. Management varies between therapy and surgical intervention with the use of a shunt.

[0333] Paget-Schroetter disease is caused by a blood clot that blocks an upper limb vein (such as the axillary or subclavian vein). The condition usually develops after strenuous exercise and usually occurs in young, healthy people. Men are more affected than women.

[0334] Cerebral venous sinus thrombosis (CVST) is a rare form of stroke caused by a blood clot blocking the dural venous sinus. Symptoms may include headache, vision abnormalities, any symptoms of a stroke (such as facial and limb weakness on one side of the body), and seizures. Diagnosis is usually made with a CT or MRI scan. Most people affected recover fully. The mortality rate is 4.3%.

[0335] Arterial thrombosis is the formation of a blood clot within an artery. In most cases, arterial thrombosis occurs after the rupture of an atheromatous plaque, and is therefore called atherothrombosis. Another common cause of arterial thrombosis is atrial fibrillation, which causes disturbed blood flow. In addition, it is well known that direct current cardioversion of atrial fibrillation carries a significant risk of thromboembolism, especially if it lasts for more than 48 hours. Thromboembolism occurs in about 5% of cases that are not treated with anticoagulation. The mechanism and pathogenesis of thromboembolism after cardiac cardioversion are not fully understood. Arterial thrombosis can embolize and is the main cause of arterial embolism, which can lead to infarction of almost any organ in the body.

[0336] Hepatic artery thrombosis often occurs as a serious complication after liver transplantation. Arterial embolism may also occur in the limbs.

[0337] Thus, actual bleeding in patients with thrombosis, such as those bleeding due to another drug or any other cause (genetic or acquired), can be safely treated. Moreover, thrombosis associated with foreign bodies, such as thrombosis associated with extracorporeal devices, oxygenators, dialysis membranes, catheters, intravascular objects (e.g., stents, grafts) can be safely treated, especially when other drugs are contraindicated.

[0338] B. Disseminated intravascular coagulation or consumptive coagulopathy

[0339] Consumption coagulopathy (CC), also known as disseminated intravascular coagulation (DIC) or disseminated intravascular coagulopathy, is a pathological activation of the coagulation (blood clotting) mechanism that occurs in response to a variety of diseases. DIC causes small blood clots to form in blood vessels throughout the body. Because the small clots consume coagulation proteins and platelets, normal clotting is disrupted and abnormal bleeding occurs in the skin (e.g., where blood samples are collected), the gastrointestinal tract, the respiratory tract, and surgical wounds. The small clots can also interfere with normal blood flow to organs (e.g., the kidneys), so the organs may malfunction.

[0340] DIC can occur acutely but can also occur on a slower chronic basis, depending on the underlying problem. It is common in critical illness and may be involved in the development of multi-organ failure, which can lead to death.

[0341] In the homeostasis state, the body maintains a delicate balance of coagulation and fibrinolysis. The activation of the coagulation cascade produces thrombin, which converts fibrinogen into fibrin; a stable fibrin clot is the final product of hemostasis. The fibrinolytic system then works to decompose fibrinogen and fibrin. The activation of the fibrinolytic system produces plasmin (in the presence of thrombin), which is responsible for the dissolution of the fibrin clot. The decomposition of fibrinogen and fibrin produces a polypeptide called fibrin degradation product (FDP) or fibrin cleavage product (FSP). In the homeostasis state, the presence of plasmin is crucial because it is the main proteolytic enzyme of coagulation and is also necessary for clot decomposition or fibrinolysis.

[0342] In DIC, the coagulation and fibrinolysis processes are dysregulated, resulting in extensive coagulation and bleeding. Regardless of the triggering event of DIC, once initiated, the pathophysiology of DIC is similar in all cases. A key mediator of DIC is the release of a transmembrane glycoprotein called tissue factor (TF). TF is present on the surface of many cell types (including endothelial cells, macrophages, and monocytes) and is usually not in contact with the general circulation, but is exposed to the circulation after vascular injury. For example, TF is released in response to exposure to cytokines (especially interleukin 1), tumor necrosis factor, and endotoxin. This plays a major role in the development of DIC under septic conditions. TF is also abundant in lung, brain, and placental tissues. This helps explain why DIC is prone to development in patients with extensive trauma. After activation, TF binds to coagulation factors and then triggers the extrinsic pathway (via FVII), followed by the intrinsic pathway of coagulation (XII to XI to IX).

[0343] The release of endotoxin is a mechanism by which Gram-negative sepsis induces DIC. In acute promyelocytic leukemia, treatment induces destruction of leukemic granulocyte precursors, resulting in the release of large amounts of proteolytic enzymes from their storage granules, causing microvascular damage. Other malignancies may enhance the expression of various oncogenes, leading to the release of TF and plasminogen activator inhibitor-1 (PAI-1), which prevents fibrinolysis.

[0344] Excessive thrombin in the circulation is caused by overactivation of the coagulation cascade. Excessive thrombin cleaves fibrinogen, ultimately leaving multiple fibrin clots in the circulation. These excess clots capture platelets and aggregate into larger clots, leading to microvascular and macrovascular thrombosis. This deposition of clots in the microcirculation, macrovessels, and organs is the cause of the ischemia, inflammation, impaired organ perfusion, and end-organ damage that accompanies DIC.

[0345] Coagulation inhibitors are also consumed in the process. Reduced inhibitor levels will allow more clotting, creating a feedback system where increased clotting leads to more clotting. At the same time, thrombocytopenia occurs, which is attributed to the entrapment and consumption of platelets. Consumption of clotting factors in the formation of multiple clots can lead to bleeding in DIC.

[0346] At the same time, excess circulating thrombin facilitates the conversion of plasminogen to plasmin, which results in fibrinolysis. The breakdown of the clot results in excess FDP, which has potent anticoagulant properties, leading to bleeding. Excess plasmin also activates the complement and kinin systems. The activation of these systems leads to many clinical symptoms exhibited by patients with DIC, such as shock, hypotension, and increased vascular permeability. The acute form of DIC is considered an extreme manifestation of the intravascular coagulation process, with a complete disruption of its normal homeostatic boundaries. DIC is associated with a poor prognosis and a high mortality rate.

[0347] However, recent challenges have been raised to the basic assumptions and explanations of the pathophysiology of DIC. Studies of sepsis and DIC in animal models have demonstrated that a highly expressed receptor on the surface of hepatocytes, termed the Ashwell-Morell receptor, contributes to the thrombocytopenia seen in bacteremia and sepsis caused by Streptococcus pneumoniae (SPN) and possibly other pathogens. The thrombocytopenia observed in SPN sepsis is not due to increased consumption of coagulation factors, such as platelets, but rather to the activity of this receptor, which enables hepatocytes to take up and rapidly clear circulating platelets. By removing prothrombotic components before they contribute to the coagulopathy of DIC, the Ashwell-Morell receptor reduces the severity of DIC, thereby reducing thrombosis and tissue necrosis and improving survival. The bleeding observed in DIC and in some tissues lacking this receptor may therefore be secondary to increased thrombus formation and loss of the mechanical vascular barrier. This finding has potentially important clinical implications for the development of new approaches to reduce the pathophysiology of DIC.

[0348] The only effective treatment is reversal of the underlying cause. Anticoagulants are used very rarely and only in situations where thrombosis is likely to result in imminent death (e.g., in coronary artery thrombosis or cerebrovascular thrombosis). If the platelet count is less than 5,000-10,000 / mm 3 If major bleeding occurs, platelet transfusions may be given, and administration of fresh frozen plasma to supplement coagulation and antithrombotic factors may be attempted, although these are only temporary measures and may result in increased development of thrombosis.

[0349] DIC results in low fibrinogen levels (because it has all been converted to fibrin), which can be measured in a hospital laboratory. The test is for "fibrin cleavage products" (FSP) or "fibrin degradation products" (FDP), which are produced when fibrin is broken down as fibrinolysis dissolves the clot. In some cases, an infusion of antithrombin may be necessary.

[0350] C. Trauma

[0351] Physical trauma is a severe and body-altering bodily injury, such as the removal of a limb. Blunt trauma, a physical trauma caused by an impact or other force applied to or by a blunt object, and penetrating trauma, a physical trauma in which the skin or tissue is pierced by an object. Trauma can also be described as unplanned (such as an accident) or planned (in the case of surgery). Both can manifest as mild to severe tissue damage, blood loss, and / or shock, and both can lead to subsequent infection, including sepsis. The present disclosure provides for the safe management of bleeding in trauma patients, including pre-treatment (in the case of medical procedures) and treatment after the trauma has occurred.

[0352] Surgeons use manipulative manual and instrumental techniques on patients to investigate and / or treat pathological conditions, such as disease or injury, to help improve body function or appearance, or sometimes for some other reason. The present disclosure can safely address the trauma caused by surgery, including perioperative and peri-interventional thromboprophylaxis, especially in situations where the risk of bleeding is high. Two areas of particular concern are surgeries related to the nervous system and the eye.

[0353] Generally speaking, a procedure is considered surgery when it involves cutting a patient's tissue or closing a previously sustained wound. Other procedures that do not necessarily fall into this category, such as angioplasty or endoscopy, may be considered surgery if they involve common surgical procedures or settings, such as the use of a sterile environment, anesthesia, sterile conditions, typical surgical instruments, and suturing or stapling. All forms of surgery are considered invasive procedures; so-called non-invasive surgery generally refers to ablations that do not penetrate the structures being treated (e.g., laser ablation of the cornea) or radiosurgical procedures (e.g., irradiation of a tumor). Surgery can last from a few minutes to several hours.

[0354] Surgical procedures are often classified by urgency, type of procedure, body systems involved, degree of invasiveness, and specialized instrumentation. Elective surgery is performed to correct a non-life-threatening condition and is done at the request of the patient, subject to the availability of the surgeon and surgical equipment. Emergency surgery is surgery that must be done quickly to save life, limb, or functional ability. Exploratory surgery is performed to aid or confirm a diagnosis. Therapeutic surgery treats a previously diagnosed condition.

[0355] Amputation involves cutting off a body part, usually a limb or finger / toe. Replantation involves reattaching a severed body part. Reconstructive surgery involves rebuilding an injured, mutilated or deformed part of the body. Cosmetic surgery is performed to improve the appearance of a previously normal structure. Excision is the removal of an organ, tissue or other body part from a patient. Transplant surgery is the replacement of an organ or body part by inserting another organ or body part from a different person (or animal) into the patient. Removing an organ or body part from a living person or animal for transplantation is also a surgery.

[0356] When surgery is performed on an organ system or structure, it can be classified by the organ, organ system, or tissue involved. Examples include cardiac surgery (performed on the heart), gastrointestinal surgery (performed within the digestive tract and its appendages), and orthopedic surgery (performed on bones and / or muscles).

[0357] Minimally invasive surgery involves small external incisions to insert tiny instruments into body cavities or structures, as in laparoscopic surgery or angioplasty. In contrast, open surgical procedures require larger incisions to access the target area. Laser surgery involves the use of lasers to cut tissue instead of scalpels or similar surgical instruments. Microsurgery involves the use of surgical microscopes to allow the surgeon to see small structures. Robotic surgery utilizes surgical robots (such as the da Vinci or Zeus surgical systems) to control instruments under the surgeon's guidance.

[0358] Traumatic hemorrhage accounts for a large proportion of the widespread international impact of injury, is responsible for a large proportion of deaths, and causes significant morbidity among those injured. Despite differences in prehospital care, the acute management of traumatic hemorrhage is similar worldwide and follows recognized published guidelines. Care of the critically injured patient is divided into four often overlapping phases: resuscitation, surgery, and critical care. Diagnosis and control of hemorrhage should be a high priority in all phases of trauma care and is particularly important for patients with hemorrhagic shock. Early attempts at hemorrhage control include direct control of visible sources of severe bleeding with directed pressure, compression bandages, or tourniquets; stabilization of long bone and pelvic fractures; and keeping the patient warm. During the resuscitation phase, warmed intravenous fluids are provided, hypotensive resuscitation is performed prior to surgical control of the hemorrhage, and appropriate transfusion of blood and blood products is performed. During the surgical phase, surgical control of the hemorrhage and any other injuries is performed, and additional transfusions are provided. Finally, the critical care phase provides postoperative support and tissue perfusion.

[0359] D. Device implantation

[0360] An implant is a medical device manufactured to replace a missing biological structure, to support a damaged biological structure, or to augment an existing biological structure. In contrast to a graft, a medical implant is a man-made device, whereas a graft is a transplanted biomedical tissue. The surface of the implant that contacts the body may be made of biomedical materials, such as titanium, silicon, or apatite, depending on what function is most desired. In some cases, implants contain electronic devices, such as artificial pacemakers and cochlear implants. Some implants are biologically active, such as subcutaneous drug delivery devices in the form of implantable pills or drug-eluting stents.

[0361] The most common types of medical implants are pins, rods, screws and plates used to anchor broken bones while they heal. More complex implants include artificial joints such as knees and hips, breast implants, artificial heart valves, stents and catheters.

[0362] E.Transplantation

[0363] The purpose of transplanting an organ from one body to another or from a patient's own donor site is to replace a damaged or missing organ in the recipient. The emerging field of regenerative medicine allows scientists and engineers to regenerate organs from a patient's own cells (stem cells or cells extracted from a failing organ). Organs and / or tissues transplanted within the same person are called autologous transplants. More recently transplants between two subjects of the same species are called allogeneic transplants. Allogeneic transplants can come from living or cadaveric sources.

[0364] Organs that are commonly transplanted are the heart, kidney, liver, lung, pancreas, intestine, and thymus. Tissues include bones, tendons (both called skeletal muscle transplants), corneas, skin, heart valves, and veins. Worldwide, the kidney is the most commonly transplanted organ, followed by the liver and then the heart. Cornea and skeletal muscle transplants are the most common tissues transplanted; these organs are transplanted more than 10 times as often.

[0365] Organ donors may be alive, or brain dead. Tissue can be recovered from donors who are cardiac dead (up to 24 hours after their heart has stopped beating). Unlike organs, most tissues (except corneas) can be preserved and stored for up to five years, meaning they can be "banked." In the United States, tissue transplants are regulated by the Food and Drug Administration (FDA), which has strict regulations regarding the safety of transplants, primarily aimed at preventing the spread of infectious diseases. Regulations include standards for donor screening and testing, as well as strict regulations for the handling and distribution of tissue transplants. Organ transplants are not regulated by the FDA.

[0366] Transplantation medicine is one of the most challenging and complex areas of modern medicine. In addition to the critical issue of transplant rejection, thrombosis caused by clotting at the surgical site and subsequent transport of clots into the vascular system is a major concern.

[0367] F. Cancer

[0368] Variable coagulopathy in cancer patients is caused by tumor-specific growth features, neovascularization of the damaged endothelial lining, poor myelopoiesis, hypoproteinemia, or metastatic growth with organ dysfunction. Recent studies have found clinical correlations between coagulopathy and tumor growth. These have prompted new therapeutic strategies focused on growth factors with the goal of controlling tumor metastasis, especially if used to treat micrometastatic disease. However, such treatments may result in life-threatening coagulation imbalances.

[0369] In fact, some cancers express more thrombogenic proteins than normal cells. These proteins include tissue factor, collagen, laminin, factors VII, XI and XII, plasminogen activator inhibitor, antithrombin, vitronectin, fibronectin and fibrinogen. These proteins may appear on the surface of cancer cells or may be secreted and may induce cancer-related thrombosis, which is very common in cancer patients. Safe anticoagulation with the antibodies disclosed herein can help some cancer patients with poor hemostasis.

[0370] After nonsurgical cancer treatment, especially preoperative radiotherapy, coagulation homeostasis may be further impaired, which can produce damage that exacerbates bleeding and thrombosis. Anticancer chemotherapy may affect liver function and reduce the synthesis of procoagulant and anticoagulant factors. Most chemotherapy regimens affect platelet synthesis, which is the main dose-limiting side effect. This is observed with combined systemic chemotherapy and local anticancer therapy. Although the side effects of chemotherapy are reversible, endothelial damage may persist for many years after anticancer treatment. In addition, some patients have low platelet counts during chemotherapy, and these patients are at risk for bleeding but may still need treatment for thrombosis.

[0371] G. Stroke and myocardial infarction

[0372] A stroke is a rapid decline in brain function due to a disturbance in the blood supply to the brain. This may be due to ischemia, thrombosis, embolus (trapped particles), or massive hemorrhage (bleeding). In a thrombotic stroke, a thrombus (blood clot) usually forms around an atherosclerotic plaque. Because the blockage of the artery is gradual, symptomatic thrombotic stroke develops more slowly. Thrombotic strokes can be divided into two categories: large vessel disease and small vessel disease. The former affects blood vessels such as the internal carotid artery, vertebral column, and the circle of Willis. The latter can affect smaller blood vessels such as branches of the circle of Willis.

[0373] Myocardial infarction (MI) is caused by infarction (tissue death due to lack of blood), usually due to a blood clot blocking a coronary artery. MI can be quickly fatal if not treated immediately with emergency medical treatment. If diagnosed within 12 hours of the initial attack (seizure), thrombolytic therapy is started.

[0374] H. Infection

[0375] If there is infection at the site of thrombosis, the clot may break apart, dispersing particles of infected material throughout the circulatory system (sepsis, septic emboli) and forming metastatic abscesses wherever they travel. In the absence of infection, the clot may break away and enter the circulation as an embolus, eventually becoming lodged and completely blocking the vessel, causing tissue necrosis (infarction) in the area behind the blockage unless treated quickly. If the blockage is in a coronary artery, myocardial ischemia is likely to occur, whereby the myocardial cells cannot function properly due to lack of oxygen. This lack of oxygen is then likely to cause a myocardial infarction.

[0376] I. Inflammation

[0377] Inhibition of FXIIa is expected to attenuate all pathological responses supported by contact activation. For example, contact activation can promote allergic reactions, angioedema, pulmonary reactions (e.g., asthma), skin reactions, intestinal reactions, ocular reactions, cardiovascular reactions (shock, edema, etc.), and systemic inflammatory response syndrome. FXIIa produces kallikrein by cleaving plasma prekallikrein, which in turn cleaves kininogen 1 and releases bradykinin. Bradykinin is one of the most potent inflammatory mediators known. Therefore, inhibition of FXIIa attenuates prekallikrein, thereby reducing bradykinin production, and is expected to interfere with inflammatory and vascular regulatory reactions involving the bradykinin pathway. Since contact activation can also trigger activation of the complement system, it is reasonable to expect that FXII inhibition will reduce inflammatory responses involving the complement system.

[0378] J. Combination therapy

[0379] The antibodies disclosed herein can be used as a monotherapy, but can also be combined with other therapies for treating the disease conditions that the FXII inhibitor is intended to treat. For example, co-administration of one or more additional anti-inflammatory or anti-thrombotic agents, such as NSAIDs, platelet inhibitors, anticoagulants or thrombolytic agents, is believed to be useful for treating certain diseases. These combination therapies have the potential to reduce the necessary frequency or dosage of treatment of other drugs for the same pathological condition. Co-administration or combination therapy refers to the administration of therapeutic drugs that are separately formulated or co-formulated in a composition and, when formulated separately, are administered at about the same time or at different times but within the same treatment cycle.

[0380] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0381] Example

[0382] Example 1: Materials and Methods

[0383] This example describes the experimental procedures and materials used for the studies described in Example 2.

[0384] Generation of anti-FXII monoclonal antibodies (MAbs)

[0385] FXII-deficient mice were immunized with recombinant human FXII (Ivanov et al., Blood 2017; 129: 1527–1537), and hybridomas were generated by standard methods. Clone 5C12 produces antibodies that bind to FXII and FXIIa. Hybridoma cells were subcloned and expanded, and 5C12 was purified using standard procedures, characterized in vitro, and then used for in vivo experiments.

[0386] Characterization of 5C12

[0387] FXII (40 nM) (Haematologic Technologies, Inc., Essex Junction, VT) was incubated with 0-80 nM 5C12 (10 min) and then with 1 μg / ml dextran sulfate (20 min). Spectrozyme FXIIa (0.5 mM) (Sekisui Diagnostics GmbH, Pfungstadt, Germany) was then added to measure hydrolysis by activated FXII (FXIIa). Next, FXII (100 nM) and 5C12 (0-200 nM) were incubated together (10 min) and then diluted 50 / 50 (60 min, final concentration) with kininogen-1 (HK, 12.5 nM), prekallikrein (PK, 12.5 nM) (Enzyme Research Laboratories, South Bend, IN) and short-chain or long-chain polyphosphate (10 μM) (Ivanov et al., Blood 2017; 129: 1527–1537). The amidolytic activity was quantified after the addition of polybrene (6 μg / ml) and soybean trypsin inhibitor (50 μg / ml). To test FXIIa inhibition, Spectrozyme FXIIa was added to a mixture of FXIIa (20 nM) and 5C12 (0-40 nM). To test the effect of 5C12 (0-100 nM) on FXII (100 nM) activation, corn trypsin inhibitor (CTI, 40 μg / ml), kallikrein (5 nM) and dextran sulfate (10 μg / ml) were co-incubated (0-60 min). Samples were separated by reducing SDS-PAGE and immunoblotted with anti-FXII polyclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX).

[0388] A Baboon Model of Thrombosis in an Extracorporeal Membrane Oxygenator

[0389] Baboons (n ​​= 3) with chronic external femoral arteriovenous (AV) shunts were used as previously described (Gruber and Hanson, Blood 2003; 102: 953–955; Crosby et al., Arter Thromb Vasc Biol 2013; 33: 1670–1678; Gruber et al., Thromb Res 2007; 119: 121–127). 111 In-labeled autologous platelets and 125I-labeled homologous fibrinogen, and then their AV shunt was extended to incorporate a saline-primed membrane oxygenator cartridge (Terumo- RX05, Terumo Cardiovascular Group, Ann Arbor, MI), the membrane oxygenator cartridge was perfused with blood driven by the AV pressure gradient, with a flow rate limited to 0.1 L / min. Platelet activity within the oxygenator was recorded using gamma camera imaging (GE-Brivo NM 615, GE Healthcare, Chicago, IL, interfaced with Xeleris 3.1 software) and calculated as described for other devices in the long-term 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). At 60 minutes, the cartridge was removed, rinsed, dried, and stored refrigerated for subsequent evaluation 125 I-fibrin content, as previously described (Gruber and Hanson, Blood 2003; 102: 953-955; Hanson et al., J Clin Invest 1993; 92: 2003-2012). Briefly, captured clots were lysed in an oxygenator filled with digestion buffer (10 mM Tris-H3PO4 pH 7.0, 35 mM SDS) for 3 days, and radioactivity was measured using a gamma counter (Wizard-3, PerkinElmer, Shelton, CT).

[0390] Anticoagulation in Baboons

[0391] In a dose-finding experiment, six consecutive 1 mg / kg doses of 5C12 were injected intravenously at 40-minute intervals, and aPTT prolongation was measured in plasma samples. It was found that 5 mg / kg of 5C12 produced a near-saturating effect on coagulation. It was also determined that low-dose heparin (20 U / kg) 15 minutes before perfusion significantly maintained some measurable real-time platelet deposition in the oxygenator, so that any changes after supplementation with 5C12 anticoagulation could be determined. In two oxygenator perfusion experiments, oxygenators were perfused without heparinization. In a definitive experiment, the effects of pretreatment with heparin (20 U / kg), 5C12 (5 mg / kg), or a combination thereof on platelet retention in the oxygenator barrel were evaluated.

[0392] Hemostasis assessment

[0393] Adult use Primary skin hemostasis was assessed using the CT Scanner (Instrumentation Laboratories, Bedford, MA). Bleeding time (BT) was recorded twice during each experimental period. Blood loss during the BT test was measured. Skin wounds were observed for re-bleeding. Plasma prothrombin time (PT) was measured using a KC4 coagulometer, as prolongation of the prothrombin time (PT) is the most widely used and clinically important marker of impaired hemostasis.

[0394] Blood analysis

[0395] Blood samples were collected into citrate and analyzed using SynthASil (Instrumentation Laboratory, Bedford, MA) and KC4 TM Plasma aPTT was measured using LupoTek KCT (r 2 The activated clotting time (ACT) of blood was determined using ELISA kits from Siemens (Marburg, Germany) and RnD Systems (Minneapolis, MN), respectively.

[0396] Data analysis

[0397] Values ​​are shown as mean and range in the text and mean and SEM in the figures. Means were compared for treatment by one-way ANOVA using GraphPad Prism 5 (GraphPad Software, San Diego, CA). Heparin and heparin + 5C12 results were compared over time for platelet deposition rate by repeated measures ANOVA using SigmaPlot 11 (Systat, Inc., Chicago, IL) for time and treatment. For differences between means, a probability of <0.05 was considered statistically significant.

[0398] Example 2: Antibody inhibition of factor XII in baboons reduces platelet deposition from blood in an extracorporeal membrane oxygenator

[0399] This example describes the characterization of FXII-specific monoclonal antibody 5C12 and its ability to act as an anticoagulant and reduce platelet aggregation. Furthermore, using a primate model, these studies demonstrate that FXII inhibition reduces platelet activation and deposition within membrane oxygenators in both heparinized and non-heparinized baboons.

[0400] 5C12 inhibits FXIIa

[0401] On Western blots, the 5C12 MAb recognized the α and β forms of human, baboon, and African green monkey FXII by binding to the protease domain, but not FXII from the other species tested ( Figures 3A-3C ), and prolonged the aPTT of human and baboon plasma mixed with FXII-depleted plasma ( Figure 2A-2B ), but not for other tested species ( Figure 2C-2F Increasing concentrations of 5C12 prolonged clotting times in human and baboon plasma ( Figure 1A-1B ), reaching an aPTT of FXII-depleted plasma at approximately 50 to 60 μg / ml (330-400 nM), and suggesting inhibition of FXII by a 1:1 complex with the antibody. Compared to the well-characterized FXIIa inhibitor CTI (Hansson et al., J Thromb Haemost 2014;12:1678–1686), 5C12 was more potent, with a measurable aPTT prolongation at 200 nM ( Figure 1C 5C12 prevents the generation of amidolytic activity when FXII is exposed to contact activators ( Figures 4A-4C ) and blocked the amidolytic activity of an equimolar amount of FXIIa, consistent with active site inhibition ( Figure 4D ). In the presence of dextran sulfate, 5C12 did not inhibit the activation of FXII by kallikrein ( Figure 4E). These data suggest that 5C12 inhibits FXIIa by binding to the catalytic domain of FXII.

[0402] 5C12 is an effective anticoagulant in vivo

[0403] Within the first few hours of dose escalation, 5C12 administration increased aPTT 4-fold and ACT 3-fold ( Figure 5A-5B ) without affecting prothrombin time or circulating TAT levels ( Figure 5C-5D Complete blood count parameters, including platelet count, hematocrit, and white and red blood cell counts remained within normal reference ranges ( Figure 5E-5H ) (Hainsey et al., Lab Anim Sci 1993;43:236–243).

[0404] 5C12 inhibits platelet deposition in extracorporeal membrane oxygenator

[0405] In baboons, heparin increased aPTT 1.6-fold (1.5 to 1.7) and ACT 1.3-fold (1.2 to 1.5) above baseline (Table 1). Administration of 5C12 at 5 mg / kg increased aPTT 2.6-fold (2.3 to 3.3) and ACT 1.8-fold (1.7 to 1.9) without increasing PT, a marker of hemostatic defects. The combination of heparin and 5C12 increased aPTT 3.9-fold (3.4 to 4.5) and ACT 2.3-fold (2.1 to 2.3) without increasing PT, a marker of hemostatic defects. The anticoagulant effect of 5C12 in the aPTT assay was measurable for 3 days ( Figure 5A-5B ).

[0406] Table 1: Changes in coagulation parameters during anticoagulation and extracorporeal membrane oxygenator perfusion. APTT, ACT and PT values ​​were determined in three baboons before exposure to anticoagulants and at the beginning (0 min) and end (60 min) of each experiment.

[0407]

[0408] In non-anticoagulated baboons, platelet deposition in the oxygenator was detected at 5 minutes ( Fig. 6A ), and the platelet retention rate averaged 1.64 ± 0.13 billion per minute (bpm) between 30 and 60 minutes from the start of perfusion ( Figure 6B In heparinized baboons, platelet deposition was measurable at 25 minutes and increased at a rate of 1.21 ± 0.08 bpm between 30 and 60 minutes ( Figure 6B). Pretreatment with 5C12 reduced platelet retention to 0.61±0.05 bpm (without heparin) and 0.56±0.04 bpm (with heparin) ( Figure 6B Supplementation of heparin with 5C12 reduced the endpoint platelet deposition from 52±11 to 24±4 billion platelets and the endpoint fibrin content of the oxygenator from 45±15 mg to 15±4 mg ( Figure 6C-6D ). Fibrinogen and platelet content showed a significant correlation (R 2 =0.99), indicating that platelet activation plays a key role in oxygenator thrombosis ( Fig. 6E For all treatment regimens, hemostatic parameters (bleeding time, bleeding volume, and bleeding rate) remained within normal ranges in baboons ( Figures 9A-9C ).

[0409] In non-anticoagulated baboons, plasma TAT levels increased 60-fold from a baseline of 12 ng / mL (range 8-20 ng / mL) to 726 ng / mL (range 521-938 ng / mL) after oxygenator perfusion ( Fig. 8A In heparinized baboons, TAT increased 37-fold, and when 5C12 was combined with heparin, TAT increased 14-fold. After 60 minutes of oxygenator perfusion, plasma PF4 levels increased 11-fold in untreated baboons, 7-fold in heparinized baboons, and 3-fold in baboons treated with the 5C12 + heparin combination ( Figure 8B ).

[0410] Therapeutic applications

[0411] The studies disclosed herein demonstrate for the first time that intravenous administration of the anticoagulant anti-FXII MAb 5C12 significantly reduces platelet accumulation in extracorporeal membrane oxygenators in non-human primates, even though 5C12 is an anticoagulant targeting a contact system. 5C12 binds to the FXII catalytic domain, where it blocks the protease active site when kallikrein converts FXII proenzyme to FXIIa. The 5C12 MAb inhibits coagulation in contact activation-induced coagulation tests (e.g., aPTT and ACT) primarily, if not exclusively, by limiting thrombin generation. Although inhibition of FXII activity is not expected to have a profound effect on initial platelet adhesion to the membrane and subsequent accumulation on the membrane, the data suggest that platelet activation in the oxygenator is strongly supported by FXIIa activity and, in addition to fibrin accumulation, may also lead to occlusive device failure, despite the low initial average wall shear rate calculated in the membrane oxygenator cartridge.

[0412] For the baboon experiments, a low dose of heparin was used in the membrane oxygenator, which produced a measurable anticoagulant effect in the aPTT assay but did not completely prevent quantifiable platelet accumulation. This dose prevented clogging of the cartridge while still demonstrating the antithrombotic effect of 5C12. High doses of heparin are used during pediatric ECMO, which can lead to bleeding complications (ELSO Anticoagulation Guideline. 2015). The finding that 5C12 has significant antithrombotic effects in the presence of heparin suggests that heparin can be administered at lower doses when used in combination with 5C12, which may provide a potentially safer anticoagulation method during ECOS that involves blood perfusion through a device such as ECMO, as FXII has no known role in hemostasis.

[0413] The physiological function of FXII has not been established. The FXII zymogen is present in most terrestrial vertebrates, but is absent in birds and aquatic cetaceans (Ponczek et al., J Thromb Haemost 2008;6:1876–1883). Among terrestrial vertebrates, mammals synthesize FXI alone, which serves as a bridge between FXII and downstream thrombin generation. However, FXIIa can shorten the contact initiation clotting time of FXI-deficient plasma in vitro (Puy et al., J Thromb Haemost 2013;11:1341–1352), probably because FXIIa can cleave several macromolecular substrates, including certain coagulation inhibitors such as antithrombin. Nonphysiological or pathogenic roles of FXII have been demonstrated by many researchers, mainly under experimental conditions and a few in the clinic. For example, FXII promotes experimental embryo implantation (Matsubayashi et al., Am J Reprod Immunol 2008;59:316–322; Kawato et al., Reprod Fertil Dev 2009;21:840–847), promotes tissue repair, angiogenesis, and mitosis (LaRusch et al., Blood 2010;115:5111–5120), and enhances vasodilation and inflammation through kallikrein activation and bradykinin production ( et al., Thromb Haemost 2013;110:399–407), may contribute to the pathogenesis of autoimmune diseases through dendritic cell interactions (Gobel et al., Nat Commun 2016;7:11626), may contribute to experimental thrombotic processes (U.S. Patent No. 9,574,013; Larsson et al., Sci Transl Med 6:222ra17, 2014; Worm et al., Ann Transl Med 3:247, 2015), and neutrophil-produced FXII appears to play a role in normal neutrophil function (Stavrou et al., J Clin Invest 2018;128:944–959). Despite all of these data, pathology has not been associated with FXII deficiency, although the absence of natural FXIIa inhibitors (e.g., C1Inh) can produce local and systemic responses consistent with reduced inhibition of contact activation and bradykinin overproduction (e.g., as seen in angioedema). The data disclosed herein support the premise that FXII inhibition may also be used in medical procedures where both thrombosis and bleeding are immediate risks. This may include ECOS systems, including hemodialysis, ECMO, left ventricular assist devices, or cardiopulmonary bypass (Cheng et al., Ann Thorac Surg 2014; 97: 610–616).

[0414] Based on epidemiological observations suggesting an association between low-normal FXII levels and cardiovascular disease, some researchers have inferred from these data that severe reductions in FXII activity (e.g., FXII deficiency) may be associated with an increased risk of thrombosis (Goodnough et al., Med 1983;62:248–255; Kuhli et al., Am J Ophthalmol 2004;137:459–464). However, the incidence, prognosis, or prevalence of cardiovascular disease in FXII deficiency has not been determined, and other observational studies of the general population have not found an association between FXII levels and susceptibility to thrombosis (Girolami et al., J Thromb Thrombolysis 2004;17:139–143; Zeerleder et al., Thromb Haemost 1999;82:1240–1246). Deficiency of FXI (a substrate for FXIIa) may result in mild to moderate hemorrhagic cerebrovascular disease but also reduces the risk of thrombosis (Salomon et al., Thromb Haemost 2011;105:269–273; Preis et al., Blood 2017;129:1210–1215; Salomon et al., Blood 2008;111:4113–4117). FXI inhibitors are in clinical development (Tillman et al., Blood Rev 2018;32:433-448; Gailani et al., J Thromb Haemost 2015;13:1383–1395), and FXI antisense oligonucleotides demonstrated improved results in a phase 2 trial for the prevention of venous thromboembolism after knee replacement surgery (Buller et al., N Engl J Med 2015;372:232–240). Inhibition of FXI or FXII with antisense oligonucleotides showed similar antithrombotic efficacy and safety in a rabbit model of catheter-induced thrombosis (Yau et al., Blood 2014; 123: 2102–2107). The development of FXII inhibitors is in the early stages. Some monoclonal anti-FXII / FXIIa antibodies have been described, but human trials have not yet been conducted. Decades ago, the monoclonal anti-FXII antibody C6B7 was found to attenuate hypotension in a baboon model of sepsis, but did not eliminate signs of disseminated intravascular coagulation (DIC); however, the antibody only inhibited FXII activity by about 60% in baboon plasma (Pixley et al., J Clin Invest 1993; 91: 61–68).Recently, the anti-FXIIa monoclonal antibody 3F7 reduced thrombosis in a rabbit ECMO model and was evaluated as a potential treatment for hereditary angioedema (Larsson et al., Sci Transl Med 2014; 6:222ra17; Worm et al., Ann Transl Med 2015; 3:247; Farkas et al., Expert Opin Investig Drugs 2018; 27:87–103). Other approaches to targeting FXII include CTI-based synthetic peptides (Baeriswyl et al., ACS Chem Biol 2015; 10:1861-1870), anti-FXII nanobodies (de Maat et al., Thromb Haemost 2013; 110:458–468), and nuclease-resistant FXII inhibitory RNA aptamers (Woodruff et al., J Thromb Haemost 2013; 11:1364–1373). FXII antisense oligonucleotides have also been tested in animal studies (Yau et al., Blood 2014; 123: 2102–2107). Since antisense oligonucleotides take several weeks to achieve efficacy and produce sustained FXII deficiency (Buller et al., N Engl J Med 2015; 372: 232–240), they may be useful for long-term thromboprophylaxis.

[0415] The results of this study demonstrate that 5C12 MAb forms an inhibitory complex with circulating FXII in vivo and effectively reduces platelet-dependent thrombus formation in an extracorporeal membrane oxygenator device without significant changes in template bleeding time. Importantly, the combination of 5C12 and heparin has been shown to improve the antithrombotic efficacy of the anticoagulant effect of heparin alone.

[0416] Example 3: Evaluation of FXIIa inhibition using 5C12 (AB053) in a baboon model of lethal S. aureus exposure

[0417] This example describes a study evaluating the effect of prophylactic 5C12 (hereinafter "AB053") in a baboon model of fatal systemic inflammatory response syndrome (SIRS). SIRS with or without intravascular coagulation and / or coagulopathy is sometimes observed in patients receiving bactericidal antibiotics to prevent or treat sepsis. Baboons were given a "saturation" dose of AB053 (10 mg / kg) or no antibody intravenously immediately before a 2-hour continuous intravenous infusion of 30 billion heat-killed Staphylococcus aureus. Two additional doses of AB053 were administered 8 hours (10 mg / kg) and 24 hours (5 mg / kg). Vital signs were monitored and blood samples were collected to measure organ function and markers of coagulation, inflammation, and organ damage. Untreated animals (n=3) developed severe SIRS and shock, with significant changes in almost all monitored markers within 6 to 24 hours, and the animals had to be euthanized within 48 hours. In contrast, baboons that received AB053 (n=3) survived without the development of SIRS, severe disease, and major changes in blood markers. Survivors were euthanized 7 days later for final analysis. The results of this study demonstrate that nearly complete inhibition of FXIIa with AB053 prevents the development of lethal SIRS induced by S. aureus exposure in baboons.

[0418] Biochemical tests

[0419] Blood glucose was measured using a Contour Next blood glucose meter (Bayer HealthCare LLC, Mishawaka, IN). Blood lactate was measured by Lactate Scout (EKF Diagnostics GmbH, Barleben, Germany). Serum alanine aminotransferase, aspartate aminotransferase, amylase, creatinine, blood urea nitrogen, potassium, phosphate, and lactate dehydrogenase levels were measured using standard clinical tests. Myeloperoxidase (MPO) activity in plasma was quantified using the FluoroMPO myeloperoxidase assay kit (Cell Technology, Fremont, CA).

[0420] CBC

[0421] Complete blood counts (CBC) were measured using blood collected into K2-EDTA coated vials and analyzed using an Abaxis Vetscan HM5 (Abaxis, Union City, CA).

[0422] aPTT

[0423] The activated partial thromboplastin time (aPTT) was measured in plasma samples anticoagulated with 0.32% sodium citrate (final concentration). 50 μL of plasma was incubated with 50 μL of aPTT reagent (Pacific Hemostasis Kontact, Cat#100312TS, Thermo Fisher Scientific, Waltham, MA) at 37°C for 3 minutes. After incubation, 50 μL of 25 mM CaCl2 (Pacific Hemostasis CaCl2, Cat#100314TS, Thermo Fisher Scientific, Waltham, MA) was added and the clotting time was measured on a KC4A analyzer (Amelung GmbH, Lemgo, Germany).

[0424] PT

[0425] Prothrombin time (PT) was measured in plasma samples anticoagulated with 0.32% sodium citrate. 50 μL of plasma was incubated at 37° C. for 1 minute. After incubation, 100 μL of Pacific Hemostasis Thromboplastin-DS (Cat#100354TS, Thermo Fisher Scientific, Waltham, MA) was added and the clotting time was measured on a KC4A analyzer.

[0426] Antithrombin ELISA assay

[0427] The complex of activated factor and antithrombin (FXIIa-AT, Kallikrein-AT, FXIa-AT, FVIIa-AT, TAT) was measured by custom enzyme-linked immunosorbent assay. Plates were coated overnight at 4°C with one of the following affinity purified antibodies (100 μL / well): (i) goat anti-human FXII (2 μg / mL); (ii) sheep anti-human FXI (2 μg / mL); (iii) sheep anti-human thrombin (2 μg / mL), all from Affinity Biologicals; or (iv) goat anti-human FVII (0.3 μg / mL, R&D Systems, Minneapolis, MN, USA) or (v) (sheep anti-human prekallikrein antibody, Affinity Biologicals). After each step, the plate was washed with PBS containing 0.1% Tween-20 and then blocked with the same buffer containing 1% BSA. EDTA plasma samples were incubated in PBS containing 1% BSA, 5mM EDTA, 5mM benzamidine hydrochloride, 0.1% Tween-20 for 60 minutes. Biotinylated affinity purified sheep anti-human antithrombin (0.5μg / mL, Affinity Biologicals) was used for detection, followed by streptavidin-HRP (400ng / mL, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and o-phenylenediamine (OPD) as substrates. The absorbance was recorded at 492nm.

[0428] Other ELISA assays

[0429] Plasma kininogen (all forms), plasminogen activator inhibitor 1 (PAI-1), and tissue plasminogen activator (tPA) were quantified using DuoSet ELISA kits (R&D Systems, Minneapolis, MN). For plasmin-antiplasmin complexes, affinity-purified goat anti-human plasminogen (2 μg / mL, Affinity Biologicals) was used as the capture and horseradish peroxidase-conjugated goat anti-human α2-antiplasmin (1 μg / mL, Affinity Biologicals) was used as the detection antibody. D-dimer ELISA was performed using mouse monoclonal anti-human D-dimer (clone DD1, 1 μg / mL, Novus Biologicals, Littleton, CO) as the capture and horseradish peroxidase-conjugated sheep anti-human fibrinogen (2 μg / mL, Affinity Biologicals) as the detection antibody. Plasma cytokines were quantified using the MILLIPLEX MAP non-human primate cytokine magnetic bead set (EMD Millipore, Billerica, MA). Mouse monoclonal anti-human C3b / c IgG, clone C3-28 (Cell Sciences) was used for capture, and goat anti-human C3 (Complement Technologies) was used as the detection antibody to measure C3b. Monoclonal antibody aE11 (Enzo Life Sciences) was used as the capture antibody to measure C5b-9, and anti-human C6 biotinylated antibody (Quidel Corporation, San Diego, CA) was used as the detection antibody. Nucleosomes were quantified using the Cell Death Detection ELISA PLUS Kit (Roche Diagnostics GmbH, Mannheim, Germany).

[0430] bacteria

[0431] Staphylococcus aureus subspecies aureus Rosenbach (ATCC 12598) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). To control the standard dose of bacteria and avoid possible confounding effects of live organisms, exponential phase cultures of Staphylococcus aureus were washed thoroughly with saline solution and counted, then heated at 70°C for 1 hour. Aliquots were stored at -80°C until use. For consistency, animals were challenged with bacteria from a single preparation.

[0432] animal

[0433] A total of 6 animals were used for these terminal studies. Six healthy juvenile baboons (Papio anubis and ursinus) weighing between 6.7 and 20.2 kg were used. As part of the study, data from control animals were published using the FXI antibody AB023, which inhibits contact activation of FXI (Silasi et al., Blood Advances 3(4):658–669 2019).

[0434] experiment

[0435] The experimental design included 2 arms: a control group (n=3) and a treatment group (n=3). Both groups of animals were challenged with 30 billion heat-killed Staphylococcus aureus (a lethal dose established in baboons), intravenously infused over 2 hours. The untreated control group received only bacterial infusion, while the treated group received a bolus of AB053 (10 mg / kg) 30 minutes before the start of bacterial infusion (pretreatment) and received two additional intravenous bolus injections 8 hours (10 mg / kg) and 24 hours (5 mg / kg) after the start of S. aureus infusion. The time point at which bacterial infusion began was designated as T0. Eight hours after the start of bacterial infusion (T+8), the animals were returned to the recovery cage and observed until they showed signs of irrecoverable organ failure and septic shock, and then they were humanely euthanized. Surviving animals were euthanized on day 7.

[0436] Intensive care

[0437] Respiratory and heart rate, temperature, and systemic mean arterial blood pressure (MSAP) were monitored using a Caidec Max-12 HD Duo monitor. These physiological data were recorded before each blood sample collection, every 15 min for the first 8 h until the animal was returned to the recovery cage, then once a day for 3 days and then on day 7.

[0438] Blood sample collection

[0439] Systemic blood samples were collected at T-0.5, 0, +2, +4, +6, +8, +24, +48, ​​+72, and +168 hours using the following anticoagulants and blood volumes: 3 ml of blood in a K2-EDTA coated vial, 2 mL of blood in a vial containing 0.32% sodium citrate, and 1 mL of blood for serum. The total amount of blood drawn during the initial 24 hour period was less than 10% of the animal's calculated blood volume (70 mL / kg).

[0440] Data and statistical analysis

[0441] Prism (GraphPad Software 7.0d) was used for statistical analysis. Data are presented as mean ± standard error of the mean. Two groups were compared using a 2-tailed Student's t-test at each time point. Results were considered significant when *p<0.05, **p<0.01, ***p<0.001. Log-rank Mantel-Cox test was used for comparison of survival curves.

[0442] The primary endpoint of this study was survival. In the untreated (negative control) group, 2 of 3 animals needed to be euthanized within 24 hours and the third animal was euthanized on the second day, while three animals treated with AB053 survived to 7 days. AB053 pretreatment increased aPTT to greater than 100 seconds, but did not enhance the increase in aPTT caused by S. aureus exposure observed in control animals. In addition, AB053 treatment attenuated the increase in PT induced by S. aureus. Coagulation activation was assessed as changes in the levels of antithrombin complexes with multiple thrombins. Antithrombin-enzyme complexes increased within hours of S. aureus exposure, and AB053 treatment attenuated these reactions. AB053 treatment also attenuated activation of fibrinogen consumption and fibrin(ogen) cleavage. Analysis of markers of complement activation and cell death (both elevated after S. aureus exposure) were reduced in the AB053 arm. AB053 significantly reduced the activation of several inflammatory cytokines (cytokine storm) observed within hours of S. aureus exposure. Vital signs of all animals were closely monitored during the first eight hours after exposure, indicating that AB053 treatment offset the hypotension caused by S. aureus exposure. AB053 also reduced liver, kidney and pancreatic damage or helped maintain their function. AB053 prevented the decrease in white blood cell and platelet counts and reduced systemic markers of platelet activation compared to untreated animals. AB053 also prevented hemoconcentration.

[0443] in conclusion

[0444] The results of this study demonstrate that AB053 provides effective protection against lethal SIRS induced by S. aureus exposure in baboons. All AB053-treated animals survived a lethal dose of S. aureus compared to untreated controls. The results suggest that FXII plays an important pathogenic role in the development of lethal SIRS induced by S. aureus exposure.

[0445] Example 4: Humanization of 5C12 (AB053)

[0446] This example describes the generation of a humanized antibody library based on the sequence of the murine antibody 5C12 (also referred to herein as "AB053").

[0447] Antibody

[0448] AB053 was generated as described in Example 1. Humanization of AB053 was performed by CDR-grafting technology of LakePharma (Belmont, CA).

[0449] Construction of expression vector

[0450] Full-length antibody genes were constructed by first synthesizing the variable region sequences designed in the humanization experiments. The sequences were optimized for expression in mammalian cells. These variable region sequences were then cloned into an expression vector that already contained the Fc domain of human IgG4 (S241P hinge modification).

[0451] For comparison, the variable regions of murine AB053 heavy and light chains were constructed as full-length chimeric chains using the same framework human Fc sequences as above.

[0452] Small-scale production

[0453] Nine humanized antibodies produced by the combination of three humanized heavy chains and three humanized light chains were produced in 0.01L. The chimeric parent antibodies were also scaled up for direct comparison. The plasmids of the heavy and light chains shown were transfected into suspension HEK293 cells and serum-free chemically defined medium was used for antibody production. The full-length antibodies were then purified from the conditioned medium using MabSelectSuRe protein A resin (GE Healthcare).

[0454] result

[0455] Three humanized heavy chains were designed based on two different human heavy chain acceptor frameworks, and three humanized light chains were designed based on two different human light chain acceptor frameworks (Table 2).

[0456] Table 2: Humanized chain information

[0457]

[0458] The first humanized heavy chain / light chain utilizes the respective first framework and comprises the most human sequences with the least parent antibody framework sequences (humanized HC1 and LC1; SEQ ID NOs: 7 and 9, respectively). The second humanized heavy chain / light chain uses the same framework as humanized HC1 / LC1, but comprises additional parent sequences (humanized HC2 and LC2; SEQ ID NOs: 13 and 17, respectively). The third humanized heavy chain / light chain utilizes the second corresponding framework and, similar to humanized HC2 / LC2, also comprises additional parent sequences fused to the humanized framework (humanized HC3 and LC3; SEQ ID NOs: 15 and 19, respectively).

[0459] Then humanized heavy and light chains were combined to produce nine different fully humanized antibodies, as well as chimeric antibodies comprising the variable regions of mouse monoclonal antibody AB053 and the human Fc region of S241P hinge-modified IgG4 (Table 3). These antibodies were transiently expressed in HEK293 and produced in a small-scale production format for testing and selection of lead humanized antibody candidates (see Example 5).

[0460] Table 3: Antibodies transiently expressed in HEK293 cells

[0461]

[0462] Example 5: Selection of lead humanization candidates

[0463] This example describes the identification of a lead humanized antibody from the nine antibodies described in Example 4. Antibodies were evaluated by humanness score, binding characteristics (ELISA, Octet), aPTT and inhibition of FXIIa activity.

[0464] Humanity Score

[0465] LakePharma has developed a tool for calculating the human score (T20 score) of monoclonal antibodies (Gao et al., BMC Biotechnology 13: 55, 2013). In short, the T20 score indicates the degree of humanization of the antibody by analyzing the primary sequence of the variable region. For the full-length heavy chain, a score of 79 or higher indicates that it looks like a human; for the full-length kappa light chain, a score of 86 or higher indicates that it looks like a human. Since the T20 score of the full-length variable region is significantly affected by the low humanity of the CDR region that remains unchanged during the humanization process, the T20 score of the humanized antibody skeleton is also calculated. For the heavy chain skeleton, a score of 84 or higher indicates that it looks like a human; for the kappa light chain skeleton, a score of 90 or higher indicates that it looks like a human.

[0466] ELISA

[0467] ELISA plates were coated with 1 μg / mL of human FXII (Enzyme Research Labs, #HFXII 1212) at 4°C overnight. The coated plates were blocked with 2% BSA in phosphate buffered saline (PBS) for 1 hour at room temperature and then washed 3 times with PBS. All antibodies to be tested (PP13892-PP13901 or human Fc isotype control) were diluted with sample diluent and diluted 11 points in 5-fold serial dilutions starting from 10 μg / mL. Each sample was performed in duplicate. Each concentration was incubated on the coated plate at room temperature for 1 hour and then washed 3 times with PBS. Bound antibodies were detected with goat anti-human IgG-HRP and washed six times with PBS. TMB substrate was added to each well and the reaction was terminated with 1M HCl. The absorbance was read at 450nm.

[0468] The EC of the concentration response curve was calculated by plotting the OD measured at 450 nm against the logarithm of the antibody concentration. 50 and calculated EC by nonlinear regression analysis 50 .

[0469] Octet

[0470] Multi-concentration kinetic experiments were performed on an Octet RED96 system (ForteBio). Anti-human Fc biosensors (ForteBio) were hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) and pretreated in glycine, pH 1.9. Antigen was diluted with sample diluent in a 3-fold serial dilution of 7 points starting from 200 nM. Antibodies were diluted to 1 μg / ml with sample diluent and then immobilized on the anti-human Fc biosensor. After establishing a baseline in sample diluent for 30 seconds, the biosensor was moved to the wells containing the serially diluted antigen to measure binding and dissociation. Binding was observed for 180 seconds and dissociation was observed for 300 seconds. Binding affinity was characterized by fitting the kinetic sensorgrams to a monovalent binding model (1:1 binding).

[0471] aPTT

[0472] The pooled human plasma (90 μL) (from three individual subjects) anticoagulated with 0.38% sodium citrate was mixed with 10 μL of antibodies or controls (PBS) at different concentrations (0-75 or 100 μg / mL) and incubated at room temperature for 5 minutes. Then 40 μL of plasma / antibody mixture was incubated with 40 μL aPTT reagent (SynthASil, #0020006800, InstrumentationLaboratory, Bedford, MA) at 37°C for 3 minutes. After incubation for 3 minutes, 40 μL CaCl2 was added and the clotting time was determined on a KC4 analyzer (TCoag). Each sample was measured in duplicate.

[0473] FXIIa inhibition

[0474] Prepare a solution of 20nM FXIIa (Enzyme Research Labs #HFXIIa3350) (HBS) + 0.1% BSA in HEPES buffered saline. Each humanized antibody as well as mouse monoclonal AB053 and chimeric antibodies were added to the 20nM FXIIa solution, with a final antibody concentration of 10nM. 90μL of each antibody sample was spread in duplicate on a 96-well plate, and 10μL of the chromogenic substrate S-2302 was added to each well. The plate was read at 405nm for 15 minutes at 37°C.

[0475] Calculation of the Humanity Score of the Humanized Chain

[0476] The T20 scores of the parental and humanized antibodies are shown in Table 4.

[0477] Table 4: Humanity scores of all humanized chains

[0478]

[0479] Affinity measurement by ELISA

[0480] Fig.11 The results of affinity measurements by ELISA are shown in Table 5. The calculated EC50 for each antibody is listed in Table 5.

[0481] Table 5: Calculated EC of humanized and chimeric antibodies 50

[0482]

[0483]

[0484] Affinity measurement by Octet

[0485] Affinity measurements of nine humanized antibodies and chimeric antibodies measured by Octet are shown in Table 6.

[0486] Table 6: Kinetic measurements of humanized and chimeric antibodies

[0487]

[0488] KD = equilibrium dissociation constant

[0489] Kon = binding constant

[0490] Koff = dissociation constant

[0491] aPTT screening

[0492] Fig.12 The results of aPTT concentration-response curves in human plasma are shown in .

[0493] Effects of humanized antibodies on FXIIa activity

[0494] Fig.13 The effect of fixed concentrations of 5C12 antibody variants on FXIIa activity is shown in Figure 1. Based on the above data, humanized antibody PP13893 (HC1+LC1) was selected as the lead candidate and used for further development as antibody AB054. Comparison of AB054 and AB053 on aPTT in human and baboon plasma

[0495] The effect of AB054 on aPTT prolongation in human and baboon plasma was compared with that of the murine parent antibody AB053 ( Figures 14A-14B ).

[0496] Summarize

[0497] The results described in this example indicate that AB053 was successfully humanized and PP13893 (hereinafter referred to as "AB054") was selected for further study. Antibody AB054 has a KD of 0.3 mM and was shown to prolong aPTT in human and baboon plasma, similar to the mouse monoclonal AB053. In addition, AB054 inhibited human FXIIa, confirming that binding to FXII and inhibition of FXIIa activity were maintained after humanization.

[0498] Example 6: Evaluation of the Antithrombotic Efficacy of AB054 in a Membrane Lung Oxygenator Perfusion System

[0499] This example describes a study evaluating the antithrombotic effect of AB054 in a surface-induced thrombosis model. The antibody was tested in a surface-induced thrombosis model in baboons. A pediatric membrane oxygenator for extracorporeal membrane oxygenation was inserted in an extension loop of a baboon arteriovenous shunt. Clot formation and growth within the membrane oxygenator was measured after treatment with AB054, heparin, or a combination of both. The data disclosed herein demonstrate that AB054 alone or in combination with heparin reduced thrombosis within the membrane oxygenator device.

[0500] introduce

[0501] Blood-contacting medical devices, including catheters, stents, grafts, filters, and extracorporeal organ support systems, can fail due to accumulation of thrombi in the system and can also trigger device-related thromboembolism. To maintain patency, devices that are perfused for various lengths of time require prophylactic anticoagulation, which can increase the incidence and / or severity of bleeding. Extracorporeal membrane oxygenation (ECMO) has been increasingly used for short-term treatment of acute respiratory failure, such as in complicated influenza situations or in temporary relief of acute heart failure; however, bleeding associated with anticoagulation reduces its benefit. ECMO systems have several components that promote activation of platelets and blood coagulation contact systems, including hollow fibers and membranes exposed to flowing blood. Inhibition of blood coagulation contact activation pathways has been proposed as a safer alternative to anticoagulation.

[0502] aPTT, ACT, PT, and CBC

[0503] The activated partial thromboplastin time (aPTT) was measured in plasma samples anticoagulated with 0.32% sodium citrate. 40 μL of plasma was incubated with 40 μL of aPTT reagent (SynthASil, #0020006800, Instrumentation Laboratory, Bedford, MA) at 37°C for 3 minutes. After incubation, 40 μL of 20 mM CaCl2 was added and the clotting time was measured on a KC4 analyzer (TCoag, Ltd, Ireland).

[0504] The activated clotting time (ACT) was measured in non-anticoagulated blood samples. TM 40 μL of blood was added to 40 μL of LupoTek KCT (r 2 -Diagnostics, South Bend, IN) and recorded the clotting time.

[0505] Prothrombin time (PT) was measured in plasma samples anticoagulated with 0.32% sodium citrate. 40 μL of plasma was incubated at 37°C for 3 minutes. After incubation, 40 μL of (Siemens Healthcare, Marburg, Germany), and clotting time was determined on a KC4 analyzer.

[0506] Complete blood count (CBC) was measured using 0.25 mL of blood collected into K2-EDTA coated vials and analyzed using Hemavet 950FS (Erba Diagnostics, Germany).

[0507] Generation of AB054 antibody

[0508] Experimental AB054 was produced by transient expression in HEK293 cells of LakePharma (Belmont, CA). In short, each DNA expression construct was scaled up to an amount suitable for transfection. Plasmid DNA was electrophoresed on agarose gel for quality assessment and sequence confirmation before transfection. Suspended HEK293 cells were inoculated into shake flasks and amplified using a serum-free chemically defined culture medium. On the day of transfection, the amplified cells were inoculated into a new flask filled with fresh culture medium. Each DNA construct was transiently transfected into HEK293 cells using LakePharma standard operating procedures. Cells were maintained as batch feed cultures (C4541 and C4542, Medna) until the end of the production run.

[0509] Conditioned media was harvested and clarified from transient production runs by centrifugation and filtration. The supernatant was added to a protein A column pre-equilibrated with binding buffer. Wash buffer was passed through the column until the OD280 value (NanoDrop, ThermoScientific) was measured to be zero. The target protein was eluted with a low pH buffer, the fractions were collected, and the OD280 value of each fraction was recorded. The fractions containing the target protein were combined and filtered through a 0.2 μm membrane filter. The OD280 value and the calculated extinction coefficient (1.60 mg / mL) were used to determine the OD280 value of the target protein. -1 cm -1 ) to calculate protein concentration.

[0510] animal

[0511] A total of 9 oxygenator perfusion experiments were performed on two baboons, including 3 treatment groups (Table 7). The experiments were performed on each baboon for a period of two weeks.

[0512] Treatment Group

[0513] - Group 1: Heparin (20 U / kg) 15 minutes before starting membrane oxygenator perfusion (n=3)

[0514] - Group 2: AB054 (5 mg / kg) administered 30 minutes before starting membrane oxygenator perfusion (n=3)

[0515] - Group 3: AB054 (2 mg / kg) was administered 30 minutes before the start of membrane oxygenator perfusion and heparin (20 U / kg) was administered 15 minutes before (n=3). In this group, 2 mg / kg of AB054 was added to the AB054 (5 mg / kg) administered 24 hours before to maintain complete inhibition of FXII.

[0516] Table 7: Summary of treatments for each baboon

[0517]

[0518] Membrane oxygenator perfusion

[0519] Perfusion experiments with non-terminal membrane oxygenators were performed in juvenile male baboons (papio anubis) weighing 9 to 15 kg. Each baboon had a healed, surgically placed, chronic external arteriovenous (AV) shunt connecting the femoral artery and vein. During the perfusion experiment (60 min), the chronic AV shunt was extended to incorporate a saline-treated oxygenator cartridge (Terumo- RX05, coated hollow fiber design, Terumo Cardiovascular Group, Ann Arbor, MI). The perfusion was set to a controlled flow rate of 100 mL / min.

[0520] Experiments were performed on lightly sedated or awake animals restrained in a sitting position. Animals were sedated with acute ketamine sedation (10 mg / kg) or Telazol (5 mg / kg) prior to transport from their cages to the experimental room. For transport back to the cage, animals were lightly sedated with ketamine (1 to 2 mg / kg as needed) or Telazol (1 mg / kg).

[0521] During the experiment, the test article was administered through a silicone rubber extension tube incorporated into the AV shunt, and blood samples were collected from the silicone rubber extension tube. Complete blood counts, including red blood cell counts and hematocrit, were measured daily for each animal, including before and after the experiment, and the blood loss calculated on any experimental day did not exceed 4% of the total blood volume. Each animal was subjected to multiple experiments (4 to 5 times per animal) on different days over a two-week period. In the repeated experiments, only animals with shunts with sufficient unrestricted baseline flow (>250 mL / min) were used.

[0522] Blood sample collection

[0523] At the beginning (0 min) and end (60 min) of each experiment, systemic blood samples (1 mL) were collected proximal to the membrane oxygenator, entering 1 / 10 volume of 3.2% citrate anticoagulant from the midstream of the AV shunt. On days when heparin was administered, additional samples were collected before heparin administration (pre-heparin). To measure ACT, 0.2 mL of whole blood was collected and analyzed immediately. To measure CBC, 0.25 mL of whole blood was collected into a tube coated with K2-EDTA and analyzed on a Hemavet 950FS.

[0524] Hemostasis assessment

[0525] Skin bleeding time test using a standard template ( International Technidyne Corp, Piscataway, NJ) to evaluate the effect of the test substances on initial hemostasis in baboons. All bleeding time tests were performed by the same professional technician 15 minutes after the start of the membrane oxygenator experiment.

[0526] Radiolabeling of platelets and fibrinogen

[0527] To quantify platelet deposition, 1 mCi of 111 In-labeled autologous baboon platelets were then reinfused into the animals and allowed to circulate for at least 1 hour and up to 4 days before experimentation. The accumulation of platelet-associated radioactivity on the oxygenator membrane was measured every 5 minutes using a GE-Brivo NM 615 interfaced with Xeleris 3.1 software (GE Healthcare, Chicago, IL).

[0528] Ten minutes before each study, the same source of 125 I-labeled baboon fibrinogen (5-25 μg, 4 μCi, >90% clottable). 111 After In decay, the incorporation of labeled fibrinogen / fibrin into the thrombus was assessed using a gamma counter (Wizard-3, PerkinElmer, Shelton, CT). 125 I radioactivity was compared with the radioactivity of clottable fibrin(ogen) in plasma samples collected during the experiment.

[0529] Platelet deposition in membrane oxygenators

[0530] Platelet deposition in the oxygenator was measured in real time during the perfusion experiment. Radiometric data were collected every 5 minutes using a gamma camera, and the measured CPM raw data were analyzed to calculate platelet deposition.

[0531] Fibrinogen content

[0532] At the end of the experiment, store the oxygenator device with the thrombus until 111 In radioactivity decays (over 30 days). At this point, the only radioactivity remaining in the graft is from the labeled fibrinogen. 125 I.

[0533] result

[0534] To evaluate the antithrombotic efficacy of AB054, an extracorporeal membrane oxygenator inserted into an arteriovenous shunt in baboons was used to simulate surface-initiated thrombosis.

[0535] The anticoagulation effect during the experiment was measured using aPTT, ACT, and PT at three time points during the experiment ( Figures 15A-15C ). Plasma samples were collected at the beginning (0 min) and end (60 min) of the experiment. If heparin was given, plasma was also collected before heparin administration.

[0536] In each experiment, the skin template bleeding time was performed 15 minutes after the start of perfusion to assess hemostasis ( Figures 16A-16C ). Bleeding time, volume, and rate did not appear to vary between treatments.

[0537] To measure platelet accumulation in the membrane oxygenator, blood from the shunt femoral artery was passed through the device for 60 minutes and returned to the animal's femoral vein. The number of platelets retained in the device and the growth rate of the thrombus were calculated ( Figures 17A-17B The combination of AB054 and heparin appeared to reduce platelet deposition within the oxygenator compared to heparin or AB054 treatment alone.

[0538] Terminal platelet deposition and fibrinogen ( Figures 18A-18B The combination of AB054 and heparin reduced platelet and fibrin accumulation measured at the end of the experiment compared to heparin or AB054 treatment alone.

[0539] in conclusion

[0540] Results from this study showed that AB054 reduced platelet-dependent thrombus formation in an extracorporeal oxygenator device without significant changes in prothrombin or template bleeding times. The combination of AB054 and heparin improved antithrombotic efficacy compared to heparin alone.

[0541] Example 7: Evaluation of the antithrombotic efficacy of AB054 in the baboon vascular graft thrombosis model

[0542] This example describes a study evaluating the antithrombotic effect of AB054 in a baboon thrombosis model. Synthetic vascular graft devices were coated with collagen or tissue factor to create a thrombogenic surface. The data disclosed herein demonstrate that AB054 limited the accumulation of collagen-induced thrombi compared to controls, while having no antithrombotic activity when thrombi were induced by tissue factor. These data are consistent with the mechanism of action of AB054 as a contact pathway inhibitor.

[0543] introduce

[0544] Blood-contacting medical devices, including catheters, stents, and grafts, can fail due to thrombus accumulation and can also trigger device-related thromboembolism. To maintain patency, devices that are perfused for various lengths of time require prophylactic anticoagulation, which can increase the incidence and / or severity of bleeding. Inhibition of contact activation pathways of blood coagulation has been proposed as a safer alternative to anticoagulation.

[0545] Reagents and measurements

[0546] aPTT, ACT, PT and CBC were measured as described in Example 6. Experimental grade AB054 was produced as described in Example 6.

[0547] animal

[0548] A total of 72 vascular graft thrombosis experiments were performed on a single baboon ( Fig.19 ). The experiment was conducted for a period of 7 weeks.

[0549] Treatment Group

[0550] A total of 72 graft thrombosis experiments were performed. On 18 study days, 2 perfusion experiments were performed, in which 1 collagen and 1 tissue factor coated graft were inserted into an extended shunt ring, including 4 graft experiments per day - 2 collagen coated and 2 tissue factor coated grafts. In weeks 2, 3 and 4, animals received three weekly injections of 9 mg / kg AB054, and experiments were performed on the same day (Day 1) and the following two days - 24 hours after injection (Day 2) and 48 hours after injection (Day 3). After the last injection, experiments were performed in the 1st and 2nd weeks of the clearing period (clear out peroid). Each week included 3 days of experiments. Saline controls were performed in the 1st and 7th weeks after the removal of antibodies. All groups N=6.

[0551] Vascular graft perfusion

[0552] Non-terminal graft perfusion experiments were performed in 10 kg juvenile male baboons (Papio anubis) with a healed surgically placed chronic externalized arteriovenous (AV) shunt connecting the femoral artery and vein.

[0553] For sustained perfusion experiments (60 min), chronic AV shunts were extended to introduce 4-mm ID and 20-mm long saline-treated ePTFE (expanded polytetrafluoroethylene, Gore-Tex; WL Gore and Associates, Flagstaff, AZ) grafts coated with either equine type I collagen (Chrono-Log Corporation, Haverton, PA) or tissue factor ( Siemens Healthcare, Marburg, Germany).

[0554] Blood flow through the shunt in non-anticoagulated baboons consistently triggered acute thrombosis in the collagen-coated ePTFE graft segments. During each experiment, the maximum blood flow rate through the graft (usually about 250 ml / min) was limited to 100 ml / min by distal clamping, resulting in 265 s in the 4 mm graft. -1 The average initial wall shear rate of . Flow rate was continuously monitored using an ultrasonic flowmeter (Transonics Systems, Ithaca, NY). During thrombosis, the 4mm graft was not blocked and the pulsatile flow rate was maintained at 100ml / min. The graft segment (and thrombus) was removed from the shunt at 60 minutes, and permanent shunt was restored after each experiment. Because thrombosis typically extends downstream from the collagen surface over time, platelet accumulation was also measured in a 10 cm long area of ​​the AV shunt immediately distal to the graft. In this model, thrombi grow on the proximal collagen or tissue factor surface ("graft" thrombi), and thrombi spread to the distal end of the collagen portion (forming a thrombus "tail").

[0555] Experiments were performed while the animals were lightly sedated and restrained in a sitting position. Animals were sedated with acute ketamine sedation (10 mg / kg) or Telazol (5 mg / kg) prior to transport from their cages to the experimental room. For transport back to the cage, animals were lightly sedated with ketamine (1 to 2 mg / kg as needed) or Telazol (1 mg / kg).

[0556] During the experiment, AB054 (9 mg / kg) was administered through a silicone extension tube incorporated into the AV shunt, and blood samples were collected from the silicone extension tube. Complete blood counts, including red blood cell count and hematocrit, were measured daily for each animal, including before and after the experiment, and the calculated blood loss on any experimental day did not exceed 4% of the total blood volume.

[0557] Blood sample collection

[0558] At the beginning (0 min) and end (60 min) of each experiment, systemic blood samples (1 mL) were collected proximal to the implant device, entering 1 / 10 volume of 3.2% citrate anticoagulant from the midstream of the AV shunt. Additional samples were collected before dosing with AB054 at weeks 3 and 4. To measure ACT, 0.2 mL of whole blood was collected and analyzed immediately. To measure CBC, 0.25 mL of whole blood was collected into tubes coated with K2-EDTA and analyzed on the Hemavet 950FS.

[0559] Hemostasis assessment

[0560] Skin bleeding time test using a standard template ( International Technidyne Corp, Piscataway, NJ) to evaluate the effect of the test substances on initial hemostasis in baboons. All bleeding time tests were performed by the same professional technician 15 minutes after the start of the perfusion experiment.

[0561] Graft coating and assembly

[0562] Horse type I collagen (Chrono-Log Corporation, Haverton, PA) or tissue factor ( The ePTFE vascular graft material with an inner diameter of 4 mm was coated with Siemens Healthcare, Marburg, Germany).

[0563] Radiolabeling of platelets and fibrinogen

[0564] To quantify platelet deposition, 1 mCi of 111 In labeled autologous baboon platelets, which were then reinjected into the animals and allowed to circulate for at least 1 hour and up to 4 days before the experiment was performed. The accumulation of platelet-associated radioactivity on the graft surface and in the distal shunt segment was measured every 1 minute using a GE-Brivo NM 615 interfaced with Xeleris 3.1 software (GE Healthcare, Chicago, IL). From this data, platelet accumulation was calculated at 5-minute intervals.

[0565] Ten minutes before each study, the same source of 125 I-labeled baboon fibrinogen (5-25 μg, 4 μCi, >90% clottable). Allow at least 30 days after the experiment to allow the platelets to adhere to the fibrinogen. 111 After In decay, the incorporation of labeled fibrinogen / fibrin into the thrombus was assessed using a gamma counter (Wizard-3, PerkinElmer, Shelton, CT). 125 I radioactivity was compared with the radioactivity of clottable fibrin(ogen) in plasma samples collected during the experiment.

[0566] Platelet deposition in grafts

[0567] Platelet deposition in the graft was measured in real time during the perfusion experiment. Radiometric data were collected every 1 minute with a gamma camera, and the measured CPM raw data were analyzed to calculate platelet deposition. Cumulative platelet deposition was calculated over 5-minute intervals.

[0568] Fibrinogen content

[0569] At the end of the experiment, the graft and tail segments with thrombus were cut away from the shunt ring and stored until 111 In radioactivity decays (over 30 days). At this point, the only radioactivity remaining in the graft is from the labeled fibrinogen. 125 I.

[0570] Statistical analysis

[0571] Real-time platelet deposition of complete thrombi of collagen-coated and tissue factor-coated grafts was analyzed using two-way analysis of variance (ANOVA) for time and treatment factors, and compared with controls using Bonferroni post hoc analysis. All other measurements (aPTT, ACT, terminal platelet aggregation, terminal fibrin content, bleeding time, PT) were analyzed using one-way analysis of variance, and compared with controls using Dunnett post hoc analysis. All analyses were performed using GraphPadPrism 5.

[0572] result

[0573] To evaluate the antithrombotic potential of AB054 on thrombogenic surfaces, synthetic vascular graft devices were coated with either collagen (an activator of the contact pathway) or tissue factor (an initiator of the extrinsic pathway), and these devices were deployed into arteriovenous shunts in baboons.

[0574] The experiment was repeated on a single baboon that received three weekly injections of AB054 (9 mg / kg) as Fig.19As shown. aPTT and ACT were used to measure anticoagulation during the experiment. Plasma samples were collected at the beginning (0 minutes) and end (60 minutes) of the perfusion experiment. The timeline in Figure 1 shows the aPTT data derived from the measurements obtained at the beginning of each experiment over a 7-week period. Figures 20A-20D Shown are aPTT and ACT measurements grouped by time point or treatment group at the beginning and end of each experiment.

[0575] In each experiment, hemostasis was assessed by PT measurement and skin template bleeding time. PT ( Figures 21A-21B A template skin bleeding test (Surgicutt) was performed 15 minutes after the start of the perfusion experiment ( Fig. 22 ).

[0576] To assess thrombus accumulation on the thrombogenic surface, blood from the bypassed femoral artery was passed through the graft for 60 min. Figures 23A-23D ) and tissue factor-coated ( Figures 24A-24D ) The number of platelets retained by the graft. Platelet accumulation in the growing thrombus was analyzed for both the graft and tail portions as well as the combined total thrombus.

[0577] For collagen-coated ( Figures 25A-25F ) and tissue factor-coated ( Figures 26A-26F ) grafts, and the terminal platelet deposition and fibrin content of the thrombi formed at 60 minutes were assessed. Grafts and tails were assessed individually and together as described above. Complete blood counts ( Figures 27A-27E ).

[0578] in conclusion

[0579] Results of this study demonstrate that AB054 significantly reduced thrombus development induced by blood exposure to collagen-coated vascular graft surfaces in baboons. The antithrombotic activity of AB054 persisted for at least 2 weeks after the last dose. Consistent with the mechanism of action of AB054, tissue factor-triggered thrombus development was not inhibited. No significant changes in prothrombin or template bleeding times were observed.

[0580] In view of the fact that the principles of the present disclosure can be applied to many possible embodiments, it should be recognized that the illustrated embodiments are only preferred examples of the present disclosure and should not be considered to limit the scope of the present invention. On the contrary, the scope of the present invention is defined by the appended claims. Therefore, we claim protection for all that falls within the scope and spirit of these claims as our invention. Sequence Listing <110> Oregon Health & Science University & Aronola, Inc. <120> Therapeutic Factor XII Antibodies <130> 899-101321-02 <150> US 62 / 772,235 <151> 2018-11-28 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> 439 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Asn Leu Glu Trp 35 40 45 Met Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu His Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 115 120 125 Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 130 135 140 Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 145 150 155 160 Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 165 170 175 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180 185 190 Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 195 200 205 Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys 210 215 220 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 225 230 235 240 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val 245 250 255 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 260 265 270 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 275 280 285 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 290 295 300 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 305 310 315 320 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 325 330 335 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 340 345 350 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 355 360 365 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 370 375 380 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 385 390 395 400 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 405 410 415 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 420 425 430 Leu Ser His Ser Pro Gly Lys 435 <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 2 Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Asn Leu Glu Trp 35 40 45 Met Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu His Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 <210> 3 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 3 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Gly 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Ile Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 100 105 110 Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125 Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140 Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 145 150 155 160 Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 165 170 175 Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190 Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205 Asn Arg Asn Glu Cys 210 <210> 4 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Gly 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Ile Leu Glu Ile Lys 100 105 <210> 5 <211> 1317 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Nucleic Acid <400> 5 gatgtgcagc ttcaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc 60 acctgcactg tcactggcta ctccatcacc agtggttata gctggcactg gatccggcag 120 tttccaggaa acaatctgga atggatgggc tacatacagt acagtggtaa cactaattcc 180 aacccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgcatttga attctgtgac tactgaggac acagccacat attattgtgc aagatggggg 300 tcctttgact actggggcca aggcaccact ctcacagtct cctcagccaa aacgacaccc 360 ccatctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg 420 ggatgcctgg tcaagggcta tttccctgag ccagtgacag tgacctggaa ctctggatcc 480 ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt ctgaccta cactctgagc 540 agctcagtga ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc 600 cacccggcca gcagcaccaa gttggacaag aaaattgtgc ccagggattg tggttgtaag 660 ccttgcatat gtacagtccc agaggtatca tctgtcttca tcttcccccc aaagcccaag 720 gatgtgctca ccattactct gactcctaag gtcacgtgtg ttgtggtaga catcagcaag 780 gatgatcccg aggtccagtt cagctggttt gtagatgatg tggaggtgca cacagctcag 840 acgcaacccc gggaggagca gttcaacagc actttccgct cagtcagtga acttcccatc 900 atgcaccagg actggctcaa tggcaaggag ttcaaatgca gggtcaacag tgcagctttc 960 cctgccccca tcgagaaaac catctccaaa accaaaggca gaccgaaggc tccacaggtg 1020 tacaccattc cacctcccaa ggagcagatg gccaaggata aagtcagtct gacctgcatg 1080 ataacagact tcttccctga agacattact gtggagtggc agtggaatgg gcagccagcg 1140 gagaactaca agaacactca gcccatcatg gacacagatg gctcttactt cgtctacagc 1200 aagctcaatg tgcagaagag caactgggag gcaggaaata ctttcacctg ctctgtgtta 1260 catgagggcc tgcacaacca ccatactgag aagagcctct cccactctcc tggtaaa 1317 <210> 6 <211> 639 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Nucleic Acid <400> 6 caaattgttc tcacccagtc tccagctatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaaat tacatgcact ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt ccctgctcgc 180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgga 240 gatgctgcca cttattactg ccagcagtgg agtggtaacc caccgacgtt cggtggaggc 300 accatactgg aaatcaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360 agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420 aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480 agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 540 accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600 acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639 <210> 7 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 7 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 <210> 8 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 9 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 10 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 11 <211> 1329 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 11 caggtgcagc tccaggagag cggacccggt ctggtgaagc ccagccagac cctgagcctg 60 acctgcaccg tgagcggcta ctcaatcacc tctggctaca gctggcactg gatcaggcag 120 caccccggca agggcctgga gtggattggc tatatccagt acagcggcaa caccaacagc 180 aaccccagcc tcaagagcag ggtgaccatc agcagggata caagcaagaa ccagttcagc 240 ctgaagctga gcagcgtgac cgccgctgac accgccgtgt actactgcgc caggtggggc 300 agcttcgact actggggcca gggcaccctg gtgaccgtgt cttctgctag caccaagggc 360 cccagcgtgt ttcctctcgc tccctgcagc cggagcacat ccgagagcac cgctgctctg 420 ggctgtctcg tgaaggacta cttccctgaa cccgtcaccg tcagctggaa tagcggcgcc 480 ctgacatccg gcgtccacac attccccgct gtcctgcaga gcagcggcct gtacagcctg 540 agctccgtgg tcaccgtgcc tagcagcagc ctgggaacaa agacctacac ctgcaacgtg 600 gaccataagc cctccaacac caaggtggac aagcgggtgg aatccaagta tggacccccc 660 tgtcctcctt gccctgctcc tgaatttctc ggaggcccct ccgtcttcct gtttcccccc 720 aagcccaagg acaccctgat gatctcccgg acacccgaag tcacctgcgt cgtggtggat 780 gtcagccagg aagatcccga ggtgcagttc aactggtacg tggacggagt ggaggtgcat 840 aacgccaaaa ccaagcccag ggaagagcag ttcaacagca cctatcgggt cgtgtccgtg 900 ctcaccgtcc tgcatcagga ttggctcaac ggcaaggagt acaagtgcaa ggtgtccaac 960 aagggcctgc cctcctccat cgagaagacc atctccaagg ctaagggcca acctcgggag 1020 ccccaagtgt ataccctccc tcccagccag gaggagatga ccaagaatca agtgagcctg 1080 acctgcctcg tgaagggatt ttacccctcc gacatcgctg tggaatggga aagcaatggc 1140 caacctgaga acaactacaa gaccacaccc cccgtgctgg actccgatgg ctccttcttc 1200 ctgtacagca ggctgaccgt ggacaaatcc cggtggcaag agggaaacgt gttcagctgc 1260 tccgtgatgc acgaggctct ccacaaccac tacacccaga agagcctctc cctgagcctc 1320 ggctagtaa 1329 <210> 12 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Nucleic Acid <400> 12 gagatcgtgc tgacccagag cccagcaacc ctgagcttga gccccggtga gagggccacc 60 ctgtcatgca gggccagcag cagcgtgaac tacctgcact ggtatcagca gaagcccggt 120 caagccccca ggaggctgat ctacgacacc agcaagctgg ccaccggcat ccccgccagg 180 ttttccggca gcgggtcagg caccgactac accctcacca taagcagcct ggagcccgag 240 gacttcgccg tgtactactg tcagcagtgg agcggcaacc cacctacctt tggcggaggc 300 actaaggtgg agatcaagcg gaccgtggcc gcccccagcg tgttcatctt ccctcccagc 360 gacgagcagc tgaagtctgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420 cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggactc caaggacagc acctacagcc tgagcagcac cctgaccctg 540 agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggactg 600 tctagccccg tgaccaagag cttcaaccgg ggcgagtgct aa 642 <210> 13 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 13 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 <210> 14 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 14 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 15 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 15 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 405 410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 <210> 16 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 16 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Gln Tyr Ser Gly Asn Thr Asn Ser Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 18 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 19 Asp Ile Val Leu Thr Gln Thr Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 20 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Asp Ile Val Leu Thr Gln Thr Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Gly Asn Pro Pro Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> twenty one <211> 1329 <212> DNA <213> Artificial sequence <220> <223> Synthetic nucleic acids <400> 21 gacgtgcagc tccaggagag cggacccggt ctggtgaagc ccagccagac cctgagcctg 60 acctgcaccg tgaccggcta ctcaatcacc tctggctaca gctggcactg gatcaggcag 120 caccccggca agggcctgga gtggatgggc tatatccagt acagcggcaa caccaacagc 180 aaccccagcc tcaagagcag ggtgaccatc agcagggata caagcaagaa ccagttcagc 240 ctgaagctga gcagcgtgac cgccgctgac accgccgtgt actactgcgc caggtggggc 300 agcttcgact actggggcca gggcaccctg gtgaccgtgt cttctgctag caccaagggc 360 cccagcgtgt ttcctctcgc tccctgcagc cggagcacat ccgagagcac cgctgctctg 420 ggctgtctcg tgaaggacta cttccctgaa cccgtcaccg tcagctggaa tagcggcgcc 480 ctgacatccg gcgtccacac attccccgct gtcctgcaga gcagcggcct gtacagcctg 540 agctccgtgg tcaccgtgcc tagcagcagc ctgggaacaa agacctacac ctgcaacgtg 600 gaccataagc cctccaacac caaggtggac aagcgggtgg aatccaagta tggacccccc 660 tgtcctcctt gccctgctcc tgaatttctc ggaggcccct ccgtcttcct gtttcccccc 720 aagcccaagg acaccctgat gatctcccgg acacccgaag tcacctgcgt cgtggtggat 780 aagcccaagg acaccctgat gatctcccgg acacccgaag tcacctgcgt cgtggtggat 780 gtcagccagg aagatcccga ggtgcagttc aactggtacg tggacggagt ggaggtgcat 840 gtcagccagg aagatcccga ggtgcagttc aactggtacg tggacggagt ggaggtgcat 840 aacgccaaaa ccaagcccag ggaagagcag ttcaacagca cctatcgggt cgtgtccgtg 900 aacgccaaaa ccaagcccag ggaagagcag ttcaacagca cctatcgggt cgtgtccgtg 900 ctcaccgtcc tgcatcagga ttggctcaac ggcaaggagt acaagtgcaa ggtgtccaac 960 ctcaccgtcc tgcatcagga ttggctcaac ggcaaggagt acaagtgcaa ggtgtccaac 960 aagggcctgc cctcctccat cgagaagacc atctccaagg ctaagggcca acctcgggag 1020 aagggcctgc cctcctccat cgagaagacc atctccaagg ctaagggcca acctcgggag 1020 ccccaagtgt ataccctccc tcccagccag gaggagatga ccaagaatca agtgagcctg 1080 ccccaagtgt ataccctccc tcccagccag gaggagatga ccaagaatca agtgagcctg 1080 acctgcctcg tgaagggatt ttacccctcc gacatcgctg tggaatggga aagcaatggc 1140 acctgcctcg tgaagggatt ttacccctcc gacatcgctg tggaatggga aagcaatggc 1140 caacctgaga acaactacaa gaccacaccc cccgtgctgg actccgatgg ctccttcttc 1200 caacctgaga acaactacaa gaccacaccc cccgtgctgg actccgatgg ctccttcttc 1200 ctgtacagca ggctgaccgt ggacaaatcc cggtggcaag agggaaacgt gttcagctgc 1260 ctgtacagca ggctgaccgt ggacaaatcc cggtggcaag agggaaacgt gttcagctgc 1260 tccgtgatgc acgaggctct ccacaaccac tacacccaga agagcctctc cctgagcctc 1320 tccgtgatgc acgaggctct ccacaaccac tacacccaga agagcctctc cctgagcctc 1320 ggctagtaa 1329 ggctagtaa 1329 <210> 22<210> 22 <211> 642<211> 642 <212> DNA<212> DNA <213> 人工序列<213> Artificial sequence <220> <220> <223> 合成核酸 <223> Synthetic nucleic acid <400> 22 <400> 22 gagatcgtgc tgacccagag cccagcaacc ctgagcttga gccccggtga gagggccacc 60 ctgtcatgca gggccagcag cagcgtgaac tacctgcact ggtatcagca gaagcccggt 120 caagccccca agaggtggat ctacgacacc agcaagctgg ccaccggcat ccccgccagg 180 ttttccggca gcgggtcagg caccgactac accctcacca taagcagcct ggagcccgag 240 gacttcgccg tgtactactg tcagcagtgg agcggcaacc cacctacctt tggcggaggc 300 actaaggtgg agatcaagcg gaccgtggcc gcccccagcg tgttcatctt ccctcccagc 360 gacgagcagc tgaagtctgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420 cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggactc caaggacagc acctacagcc tgagcagcac cctgaccctg 540 agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggactg 600 tctagccccg tgaccaagag cttcaaccgg ggcgagtgct aa 642 <210> 23 <211> 1329 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 23 gacgtgcagc tccaggagag cggacccggt ctggtgaagc ccagcgagac cctgagcctg 60 acctgcaccg tgagcggcta ctcaatcacc tctggctaca gctggcactg gatcaggcag 120 ccacccggca agggcctgga gtggattggc tatatccagt acagcggcaa caccaacagc 180 aaccccagcc tcaagagcag ggtgaccatc agcagggata caagcaagaa ccagttcagc 240 ctgaggctga gcagcgtgac cgccgctgac accgccgtgt actactgcgc caggtggggc 300 agcttcgact actggggcca gggcaccatg gtgaccgtgt cttctgctag caccaagggc 360 cccagcgtgt ttcctctcgc tccctgcagc cggagcacat ccgagagcac cgctgctctg 420 ggctgtctcg tgaaggacta cttccctgaa cccgtcaccg tcagctggaa tagcggcgcc 480 ctgacatccg gcgtccacac attccccgct gtcctgcaga gcagcggcct gtacagcctg 540 agctccgtgg tcaccgtgcc tagcagcagc ctgggaacaa agacctacac ctgcaacgtg 600 gaccataagc cctccaacac caaggtggac aagcgggtgg aatccaagta tggacccccc 660 tgtcctcctt gccctgctcc tgaatttctc ggaggcccct ccgtcttcct gtttcccccc 720 aagcccaagg acaccctgat gatctcccgg acacccgaag tcacctgcgt cgtggtggat 780 gtcagccagg aagatcccga ggtgcagttc aactggtacg tggacggagt ggaggtgcat 840 aacgccaaaa ccaagcccag ggaagagcag ttcaacagca cctatcgggt cgtgtccgtg 900 ctcaccgtcc tgcatcagga ttggctcaac ggcaaggagt acaagtgcaa ggtgtccaac 960 aagggcctgc cctcctccat cgagaagacc atctccaagg ctaagggcca acctcggggag 1020 ccccaagtgt ataccctccc tcccagccag gaggagatga ccaagaatca agtgagcctg 1080 acctgcctcg tgaagggatt ttacccctcc gacatcgctg tggaatggga aagcaatggc 1140 caacctgaga acaactacaa gaccacaccc cccgtgctgg actccgatgg ctccttcttc 1200 ctgtacagca ggctgaccgt ggacaaatcc cggtggcaag agggaaacgt gttcagctgc 1260 tccgtgatgc acgaggctct ccacaaccac tacacccaga agagcctctc cctgagcctc 1320 ggctagtaa 1329 <210> 24 <211> 642 <212> DNA <213> Artificial Sequence <220> <223> Synthetic nucleic acid <400> 24 gacatcgtgc tgacccagac cccagcaacc ctgagcttga gccccggtga gagggccacc 60 ctgtcatgca gggccagcag cagcgtgaac tacctgcact ggtatcagca gaagcccggt 120 caagccccca agaggctgat ctacgacacc agcaagctgg ccaccggcat ccccgccagg 180 ttttccggca gcgggtcagg caccgactac accctcacca taagcagcct ggagcccgag 240 gacttcgccg tgtactactg tcagcagtgg agcggcaacc cacctacctt tggccagggc 300 actaggttgg agatcaagcg gaccgtggcc gcccccagcg tgttcatctt ccctcccagc 360 gacgagcagc tgaagtctgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420 cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggactc caaggacagc acctacagcc tgagcagcac cctgaccctg 540 agcaaggccg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca ccagggactg 600 tctagccccg tgaccaagag cttcaaccgg ggcgagtgct aa 642

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that binds to the blood protein factor XII (FXII), comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: The VH domain comprises a complementarity determining region 1 (CDR1), CDR2, and CDR3 sequence of SEQ ID NO: 2, wherein the VH domain CDR1, CDR2, and CDR3 sequences are represented by residues 26-34, 52-58, and 97-104 of SEQ ID NO: 2, respectively; and The VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 4, wherein the VL domain CDR1, CDR2 and CDR3 sequences are shown as residues 27-31, 49-51 and 88-96 of SEQ ID NO: 4, respectively.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein: The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14 or SEQ ID NO: 16; and The amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 18 or SEQ ID NO:

20.

3. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 2, wherein: The amino acid sequence of the VH domain comprises SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14 or SEQ ID NO: 16; and The amino acid sequence of the VL domain includes SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 18 or SEQ ID NO:

20.

4. The monoclonal antibody or antigen-binding fragment of claim 3, wherein the amino acid sequence of the VH domain comprises SEQ ID NO: 8, and the amino acid sequence of the VL domain comprises SEQ ID NO:

10.

5. The monoclonal antibody according to any one of claims 1-2, comprising a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is at least 90% identical to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 15; and The amino acid sequence of the light chain is at least 90% identical to SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO:

19.

6. The monoclonal antibody according to claim 5, wherein: The amino acid sequence of the heavy chain comprises SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13 or SEQ ID NO: 15; and The amino acid sequence of the light chain includes SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO:

19.

7. The monoclonal antibody of claim 5, wherein the amino acid sequence of the heavy chain comprises SEQ ID NO: 7, and the amino acid sequence of the light chain comprises SEQ ID NO:

9.

8. The antigen binding fragment according to any one of claims 1-2, wherein the antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain variable fragment (scFv) or a disulfide-stabilized variable fragment (dsFv).

9. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 2, which is a humanized antibody or antigen-binding fragment.

10. A fusion protein comprising the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 9 and a heterologous protein.

11. An antibody conjugate comprising the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 9 and a detectable label.

12. The antibody conjugate of claim 11, wherein the detectable label comprises a fluorophore, an enzyme or a radioisotope.

13. A composition comprising a pharmaceutically acceptable carrier and the monoclonal antibody or antigen-binding fragment of any one of claims 1-9.

14. A nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment of any one of claims 1-9.

15. The nucleic acid molecule according to claim 14, comprising the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and / or SEQ ID NO:

24.

16. The nucleic acid molecule according to claim 15, comprising the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12 or both.

17. The nucleic acid molecule according to any one of claims 14 to 16, operably linked to a promoter.

18. A vector comprising the nucleic acid molecule according to any one of claims 14 to 17.

19. An isolated cell comprising the nucleic acid molecule of any one of claims 14-17 or the vector of claim 18.

20. A non-diagnostic method for detecting FXII in a sample, comprising contacting the sample with the monoclonal antibody or antigen-binding fragment of any one of claims 1-9; and The binding of the antibody to the sample is detected, thereby detecting FXII in the sample.

21. The non-diagnostic method of claim 20, wherein the monoclonal antibody or antigen-binding fragment is directly labeled.

22. The non-diagnostic method of claim 20, further comprising: contacting the monoclonal antibody or antigen-binding fragment with a secondary antibody, and The binding of the secondary antibody to the monoclonal antibody or antigen-binding fragment is detected, thereby detecting FXII in the sample.

23. An in vitro method of inhibiting the activation and / or activity of FXII in a sample comprising FXII, the method comprising contacting the sample with the antibody or antigen-binding fragment of any one of claims 1-9.

24. The method of claim 23, wherein the sample comprises a blood sample.

25. Use of the antibody or antigen-binding fragment of any one of claims 1 to 9 in the preparation of a medicament for treating microvascular thrombosis, macrovascular thrombosis, thromboembolism or disseminated intravascular coagulation in a subject.

26. Use of the antibody or antigen-binding fragment of any one of claims 1-9 in the preparation of a medicament for treating pathological hypercoagulation involving activation of FXII and / or activity of activated FXII (FXIIa) in a subject.

27. Use of the antibody or antigen-binding fragment of any one of claims 1 to 9 for the preparation of a medicament for inhibiting thrombosis involving activation of FXII and / or activity of FXIIa in a subject.

28. The use of claim 26 or claim 27, wherein the subject has or is at risk of developing a bacterial infection, a fungal infection, a viral infection, a parasitic infection, an ischemic organ disease, microvascular thrombosis, macrovascular thrombosis, thromboembolism, disseminated intravascular coagulation, severe systemic inflammatory response syndrome, an allergic or inflammatory reaction involving FXIIa activity, acute respiratory distress syndrome, cancer, amniotic fluid embolism, trauma, transplant rejection, sickle cell disease, an autoimmune disease, or has received a medical device implant.

29. The use according to claim 28, wherein the ischemic organ disease is myocardial infarction or ischemic stroke.

30. The use according to claim 28, wherein the cancer is a non-metastatic solid tumor cancer, a metastatic solid tumor cancer, or a leukemia.

31. The use according to claim 28, wherein the medical device implant is an implantation of a catheter, a heart valve, a stent or a graft.

32. The use according to any one of claims 25-27, further comprising administering to the subject an effective amount of a second anticoagulant therapy, or an antithrombotic or thrombolytic therapy.

33. The use according to any one of claims 25-27, wherein the monoclonal antibody or antigen-binding fragment is administered by parenteral administration.

34. The use according to any one of claims 25-27, wherein the monoclonal antibody or antigen-binding fragment is administered at a dose of about 0.1 mg / kg to about 2 g / kg.

35. A kit comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-9, the fusion protein of claim 10, the antibody conjugate of claim 11 or claim 12, the composition of claim 13, the nucleic acid molecule of any one of claims 14-17, the vector of claim 18, the isolated cell of claim 19, or any combination thereof.

Images