TR201815476T4 - Antibodies capable of binding to Coagulation Factor XI and / or its activated form factor XIa and their use. - Google Patents

Abstract

The present invention relates to antibodies capable of binding coagulation Factor XI and / or its activated form factor XIa, and methods of use thereof, in particular methods of use as agents that inhibit platelet aggregation and thereby inhibit thrombus formation.

Description

DESCRIPTION COAGULATION FACTOR xi VENYA ACTIVE FORM OF THIS FACTOR ANTIBODIES AVAILABLE TO XIA AND THEIR USES Field of Invention The present invention uses coagulation Factor X1 and/or its activated form factor Antibodies that can bind Xla and their methods of use, specifically platelet as agents that inhibit its aggregation and thereby inhibit thrombus formation related to usage methods.

Background of the Invention In 1964, Macfarlane and Davie & Ratnoff [Macfarlane RG. Ari enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier. Nature 1964; 202: 498-9.; Davie EW, Ratnoff OD. Waterfall sequence for intrinsic blood clotting. introduced their hypotheses. Since then, our knowledge of the in vivo coagulation function has increasing. In recent years, coagulation, called the extrinsic and intrinsic pathways, which initiates and combines this into a common pathway, ultimately leading to thrombin production and fibrin The theory of two separate pathways leading to its deposition is being reconsidered. Current In the model, initiation of coagulation is plasma protease-activated factor VII Tissue It happens when it comes into contact with the factor (TF) and forms a complex with it.

This Tissue-Factor-FVIIa complex is part of it on zymogen FX. its active form, where it can convert prothrombin (coagulation factor ll) to thrombin (IIa) It can activate to FXa. Thrombin, a key player in coagulation, ultimately the fibrinogen It can catalyze its conversion to fibrin. In addition, thrombin is metabolized by platelets. specific receptors expressed, leading to the activation of subsequent molecules activates. Activated platelets in combination with fibrin for clot formation are essential and therefore key players in normal hemostasis.

The second amplification pathway is generated by the coagulation factor XI (FXI). FXI's, like other members of the coagulation cascade, initiation of blood coagulation in vivo A plasma serine protease that has a key role in causing the phase and amplification phase zymogen [Davie EW, Fujikawa K, Kisiel W. The coagulation cascade: initiation, Kravtsov DV, Matafonov A, Tucker El, Sun MF, Walsh PN, Gruber A, et al. Factor XI 8.3-5]. While the severity of bleeding was weakly correlated with the plasma level of FXI, FXI Deficiency does not generally cause spontaneous bleeding, but is accompanied by haemostatic difficulties. associated with an increased risk of bleeding. Severe FXI deficiency in humans is thrombotic. has certain protective effects from diseases [Salomon O, Steinberg DM, Zucker M, Varon D, Zivelin A, Seligshon U. Patients with severe factor XI deficiency have a Salomon O, Steinberg DM, Koren-Morag N, Tanne D, Seligsohn U. Reduced incidence However, an elevated level of FXI has been associated with thrombotic events [Meijers JC, Tekelenburg WL, Bouma BN, Bertina RM, Rosendaal FR. High levels of coagulation Therefore, the inhibition of FXI is novel to provide an improved benefit-risk ratio. It is recommended as a new approach in the development of anti-thrombotics. Thus, Anti-inflammatory drugs that effectively block intravascular thrombosis without attenuating hemostasis. There is still a high medical need for thrombotic, anti-platelet drugs. Near In the past, binding of antibodies to FXI has been described in the art. For example, binds an epitope on the A3 domain of the chain and activates FXI and FXIa thereby blocking the interaction of FXI with FXII. However, available rabbit and baboon (of important pre-clinical animal models) as described in the invention zymogen FXI, which is cross-reactive to FXIa and binds only FXIa thus without compromising platelet-dependent primary hemostasis. There is still a medical need for Human antibodies that reduce the reservoir.

Brief Description of the Invention In recent years, the development of new anti-thrombotic agents has shown excellent progress; however, undesirable bleeding events from these agents still occur. is a serious problem. Therefore, it will ideally inhibit thrombosis but The optimal anti-thrombotic compound that will preserve hemostasis has not yet been discovered.

Coagulation factor XI (FXI/FXIa) interacts with the platelet receptor apoER2.

The present invention demonstrates that inhibiting FXIa activity under drag flow conditions is pathological. for the first time that it interferes with the process of platelet activation and platelet aggregation. shows. In an ex vivo model of thrombosis, inhibition of FXIa is associated with platelet-like CD62P. A significant decrease of the activation markers is also a collagen surface. downstream micro- leads to a decrease in aggregates. Accordingly, inhibition of FXIa is platelet-dependent. Platelet-dependent primary in a primate model of arterial-type thrombus formation It reduces platelet-storage without compromising hemostasis. Notable anti-platelet primary hemostatic occlusion due to tissue factor of platelets Its initial interaction with extravascular matrix proteins necessary for the formation of is not affected in the sequence. Therefore, inhibition of FXIa activity has a half-life related to bleeding. An ideal pharmacological principle showing anti-thrombotic activity without causing effects Represent.

To clarify: Without compromising hemostasis, the coagulation factor Xla other anti-coagulation compounds and/or anti-platelet compounds It means that it does not cause undesirable and measurable bleeding events even in the presence of income. Similar to that shown for patients with hemophilia C, bleeding is only heavy. occur in the context of surgeries and/or severe injuries.

Antigen-binding or antibodies directed against coagulation factor Xla Inhibiting platelet aggregation of fragments or variants thereof or reducing and thereby producing microaggregates and/or thrombotic clots. to demonstrate that it exhibits anti-platelet activity by inhibiting or reducing compositions and methods are provided.

These anti-coagulation factor Xla antibodies, antigen-binding antibody fragments and Using variants of the antibodies and fragments of the invention prevents platelet aggregation. and thereby inhibits thrombosis without compromising hemostasis.

Administration of anti-coagulation factor Xla antibodies neutralizes the antibodies and of these antibodies, antigen-binding antibody fragments and antibodies of the invention and for variants of fragments to be an anti-coagulant, anti-thrombotic therapy, It is also described herein that it does not lead to an increased risk of adverse bleeding events.

The present invention selectively metabolizes the activated form of plasma factor X1, FXIa. able to bind platelet aggregation and related hemostasis without compromising hemostasis. provides human monoclonal antibodies that can inhibit thrombosis. Compositions, defined anti-coagulation whose epitopes can bind the light chain of coagulation factor XIa Contains factor Xla antibodies. These antibodies are proteolytic of coagulation factor XIa. It shows neutralizing activity by blocking its activity.

A preferred embodiment of the invention is the variable light chain inhibiting human FXIa. SEQ ID NO: 19 for the amino acid sequence for the domain and amino acid for the variable heavy chain domain. It is a human monoclonal antibody capable of binding a fragment.

An additional preferred embodiment is SEQ ID NO: 21 as CDRH1, with CDRH2 as CDRH2. Set SEO ID NO: 22 and SEQ ID NO: 23 as CDRH3 and SEO ID NO: CDRL1. Factor including 24, SEQ ID NO: 25 as CDRL2 and SEQ ID NO: 26 as CDRL3 A human monoclonal capable of binding Xla and its antigen-binding fragment is an antibody.

An additional preferred embodiment of the invention is the amino acid for the variable light chain domain. FXIa according to any one of claims 1 and 2 including NO: 20 and its antigen- It is a human monoclonal antibody capable of binding the binding fragment.

An additional preferred embodiment of the invention is SEQ ID NO: 21 as CDRH1, CDRH2 SEQ ID NO: 22 as CDRH3 and SEQ ID NO: 23 as CDRL1 NO: 24, SEQ ID NO: 25 as CDRL2, and SEQ ID NO: 28 as CDRL3 A human capable of binding FXIa and its antigen-binding fragment according to claims 1 and 4 It is a monoclonal antibody.

In another aspect, the invention is also predominantly derived from species other than humans. From the rabbit, coagulation involves cross-reactivity of antibodies for Xla, thus a It allows in-depth pharmacological and toxicological analysis.

In another aspect, methods reduce the risk of developing anti-drug antibodies. to optimize and reduce the immunogenicity of the compositions of the present invention in order to using for.

Another aspect of the present invention is to additionally compete with one of the antibodies described herein. Contains human antibodies.

In addition, the compounds contain antigen-binding antibody fragments and antibodies of the invention and fragments, cell lines producing these antibodies, and Contains isolated nucleic acids encoding amino acids. The invention also includes a pharmaceutical acceptable carrier and/or anti-coagulation factor Xla in solution antibodies or antigen-binding antibody fragments and antibodies of the invention and It also includes pharmaceutical compositions containing variants of fragments.

The compositions of the methods of this invention described above inhibit platelet aggregation. and thereby inhibiting thrombosis, any other anti- Reducing a required dose of a coagulant or anti-thrombotic agent may result in an acute treating inflammatory reaction or treating cancer or coagulation for the purpose of treating any other disease associated with the activation of the cascade It involves applying to a subject in need. Anti-coagulation FXla antibodies or antigen-binding antibody fragments and antibodies and fragments of the invention Methods for generating variants are also provided.

Brief Description of Figures For the amino acid sequence for the variable light chain domain that inhibits human FXla SEQ ID NO: 19 and SEQ ID for the amino acid sequence for the variable heavy chain domain It includes SEQ ID NO: 22 and SEQ ID NO: 23 as CDRH3. In addition, this antibody SEQ ID NO: 24 as CDRL1, SEQ ID NO: 25 as CDRL2, and CDRL3 as SEQ ID NO: 26. defined in the pan/scan runtime antibody for its ability to inhibit the proteolytic activity of human FXIa. Tested at the concentrations shown. Relevant DNA sequences SEQ ID NO: 1 to It is shown in SEQ ID NO: 8.

Dose-response of anti-FXIa antibody O76D-M007-H04 that inhibits rabbit FXIa curves. The antibody identified in the panning/scan run time, its rabbit demonstrated for its ability to inhibit the proteolytic activity of FXIa concentrations have been tested.

For the amino acid sequence for the variable light chain domain that inhibits human FXIa SEQ ID NO: 27 and SEO ID for amino acid sequence for variable heavy chain domain response curves. The antibody identified in the panning/scan run time demonstrated for its ability to inhibit the proteolytic activity of human FXIa concentrations have been tested.

Anti-FXIa antibody that inhibits rabbit FXIa O76D-M007-H04-CDRL3- The dose-response curves of the N110D. defined in the pan/scan runtime antibody for its ability to inhibit the proteolytic activity of rabbit FXIa Tested at the concentrations shown.

Conversion of human FXI to FXIa by coagulation factor Xlla SEQ ID NO: 29 for the amino acid sequence for the variable light chain domain that inhibits and SEQ ID NO:30 for the amino acid sequence for the variable heavy chain domain. dose-response curves of anti-FXla antibody O76D-M028-H17. This antibody, CDRH1 SEQ ID NO: 31 as CDRH2, SEQ ID NO: 32 as CDRH3 Includes SEQ ID NO: 33. This antibody additionally has SEQ ID NO: 34 as CDRL1, It contains SEQ ID NO: 35 as CDRL2 and SEQ ID NO: 36 as CDRL3.

The antibody identified in the panning/scan runtime is the result of their zymogen FXI for its ability to inhibit its conversion to its activated form FXIa Tested at the concentrations shown. Relevant DNA sequences SEQ ID NO: 11 to SEO ID NO: 18.

Conversion of human FXI to FXla by coagulation factor Ila dose-response curves of the inhibiting anti-FXI antibody 076D-M028-H17.

The antibody identified in the panning/scan runtime is the result of their zymogen FXI for its ability to inhibit its conversion to its activated form FXla Tested at the concentrations shown.

Conversion of rabbit FXI to FXla by coagulation factor Xlla dose-response curves of the inhibiting anti-FXI antibody 076D-M028-H17.

The antibody identified in the panning/scan runtime is the result of their zymogen FXI for its ability to inhibit its conversion to its activated form FXla Tested at the concentrations shown.

Conversion of rabbit FXI to FXla by the coagulation factor Ila dose-response curves of the inhibiting anti-FXI antibody 076D-M028-H17.

The antibody identified in the panning/scan runtime is the result of their zymogen FXI for its ability to inhibit its conversion to its activated form FXla Tested at the concentrations shown.

Activity of binding and blocking the catalytic domain of human FXIa. Human 076D-MO28-H17 inhibits the proteolytic activity of FXIa. shows no activity, indicating that FXIa binds its catalytic domain.

demonstrating that this antibody exhibits a competitive type inhibitory activity. Characterization of the coupling method using the Lineweaver-Burk graph.

Flow cytometric for CD62P expression and platelet microaggregate formation analysis. Single platelets light scattering and FlTC-CD41/CD61 (GPllbllla) fluorescence detected in combination. (A) Fluorescence of FlTC-CD41 and PE-CD62P determination of CD62P expression by dot plot. Before perfusion (left) and then (right) coated platelets are shown. (B) Platelet microaggregate Its occurrence is described by the increased size (pre-scatter) shown in the circle.

Dot plots of samples collected before (left) and after (right) perfusion is displayed.

Platelet CD62P expression is reduced by FXI(a) antibodies. Full The blood was treated with (A) and (B) 076D-M028-H17 and was not recalcified. perfused over the collagen-coated surface immediately afterwards. In parallel, whole blood samples were collected after treatment with the vehicle or inhibitor and Incubate for 5 minutes with or without TRAP6 (10 tig/ml) has been made. Platelet CD62P expression flowed as shown in Figure 11. analyzed by cytometry. Data covered by at least 5 tries as the mean ± SEM percent of CD62P-positive platelets in the population is reported. Maximum CD62P during 5 min perfusion with each treatment expression levels are shown in graphs.

Platelet microaggregate formation was inhibited by FXI(a) antibodies. and perfused over the collagen-coated surface immediately after recalcification has been made. Platelet microaggregates as shown in Figure 13 and represented analyzed by flow cytometry as described. Data at least 5 tries 104 as mean ± SEM aggregate count compared to a single plated platelet is reported. Maximum aggregate during 5 min perfusion in each treatment The counts are shown in graphs.

(a) and ear bleeding time on ferric chloride-induced thrombosis (b) in vivo effect on Thrombus without increasing the time of ear bleeding It can be shown that the weight of 076D-M007-HO4 is decreased in a dose-dependent manner. and (b) in vivo effect on ear bleed time (described in example xxx). It can be shown that it reduces the thrombus weight in a dose-dependent manner.

Effect of 076D-M028-H17 on ferric chloride-induced thrombosis (a) and ear (b) in vivo effect (described in example xxx) on bleeding time. 076D-M028-H17 reduces thrombus weight without increasing ear bleeding time. can be shown to decrease in a dose-dependent manner.

This figure shows Fab 076D-M007-H04 (lower fragment) in complex with FXIa (top piece). piece) shows a cartoon representation.

Figure 18a: This figure shows the Fab's 07öD-MOO7-HO4 (cartoon) FXIa CSOOS binding epitope shows a detailed view. FXIa 05008 is shown as surface representation.

Figure 18b: This figure is a superimposition of the FXIa CSOOS shown as a surface representation. shows Fab 076D-M007-H04 with its peptidic x-ray structure. active ground race highlighted by a red ellipsoid.

Figure 19a: This figure shows that zymogen FXI (odb) with superimposed Fab O76D-M007-H04 input 2F83) shows the crystal structure.

Figure 19b: This figure shows the same view, but the catalytic domain of the zymogenin FXI is Fab O76D- is changed. Catalytic domains of FXI and FXIa C5OOS as surface representations is displayed, all other areas are shown as cartoons. To Fab 076D-M007-H04 Inappropriately aligned folds at the interface are highlighted in Figure 19.

Plasma collected from baboons following administration of O76D-M007-H04 Increase in in vitro aPTT coagulation time determined in samples.

ACT measurements following H04 administration (i.v. bolus). 24 hours.

aPTT measurements following H04 administration (i.v. bolus). 24 hours. 2 mm i.d. Platelet deposition in collagen-coated ePTFE vascular grafts. On collagen-coated ePTFE vascular grafts as described in Example 12 platelet storage. In the venous expansion chamber (and platelet (at the connecting section between the collagen-coated graft and the silicone chamber) storage. TAT measured in baboon plasma following administration of 076D-M007-H04 levels.

Chewable alone or at a concentration of 32 mg/kg Bleeding time in baboons treated with 076D-M007-H04.

Detailed description of the invention Definitions Hemostasis: The term hemostasis is used to stop the flow of blood at the injury sites and Major mechanisms for restoring vascular patency during wound healing Represent. During normal hemostasis and pathological thrombosis, the three mechanisms coexist. becomes activated in a timely manner: the activated platelets interact with the vessel wall. as primary hemostasis, fibrin formation and fibrinolysis, which means their interactions. named process [Arthur A. Sasahara, Joseph Loscalzo(2002) New Therapeutic Agents for Thrombosis and Thrombosis (2nd Edition) Marcel Dekker Inc. New York, Coagulation and Coagulation cascade: The so-called coagulation cascade protein-based system, stabilized clot formed and additionally closed the wound functions. The coagulation pathway is a proteolytic cascade. Each enzyme of the pathway is plasma proteolytic action to release the active factor from the precursor molecule on It is available as a Zimogen that undergoes cleavage (in an inactive form). coagulation cascade, as a series of positive and negative feedback loops that control the activation process shows function. The ultimate goal of the pathway is to subsequently release soluble Fibrinogen, which forms a clot. It produces Thrombin, which can convert to Fibrin. The production process of thrombin is divided into three phases. divisible: production of an active clotting factor: FXa (Activated Factor-X), with Intrinsic and Extrinsic pathways and Thrombin formation that provide alternative pathways for the final Common road [Hoffman M.M. and Monroe DM. (2005) Rethinking the coagulation cascade. Curr Hematol Rep. 4:391-396; Johne J, Blume C, Benz PM, Pozgajova M, Ullrich M, Schuh K, Nieswandt B, Walter U, Renné T. (2006) Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma. biol Platelet aggregation: When a rupture occurs in a blood vessel, normally blood Substances that are not in direct contact with the flow are released. These items (primarily Collagen and von Willebrand factor) platelets on the ruptured surface allows it to stick. When a platelet adheres to the surface, these additional platelets are damaged. releases chemicals that attract the affected area, this is expressed as platelet aggregation. This The two processes are the first responses to stop bleeding.

Coagulation factor XI and coagulation factor Xla Coagulation Factor XI (FXI) is synthesized in the liver and High Molecular Dimer bound by a disulfide bond complexed with weighted Kininogen circulates in the plasma. Each polypeptide chain of this dimer is approximately 80 kDa.

Factor XI is released through the contact phase of blood coagulation or on the platelet surface. It is converted to its active form, the coagulation factor Xla, via thrombin-mediated activation. (to FXIa) is converted. During this activation of factor XI, an internal peptide bond forms two cleaved in each of the chain, this activated factor Xla is held together by disulfide bonds. results in a serine protease consisting of two heavy and two light chains. This cool protease FXIa activates coagulation Factor IX followed by coagulation Factor X (Xa) which converts to IXa. Xa subsequently to coagulation Factor II/Thrombin may mediate its activation. Defects in this factor (also called hemophilia C) Also known as Rosenthal syndrome, prolonged bleeding from injuries, frequent or heavy with nosebleeds, traces of blood in the urine, and heavy menstrual bleeding in women leads to a characterized blood coagulation abnormality. used here as "coagulation factor XI", "factor XI" or "FXI", the protein expressing means any FX1 from any mammalian species. For example, FXI human, human- non-primates (eg, baboon), mouse, dog, cat, cow, horse, pig, rabbit, and blood coagulation involved in the regulation of flux, coagulation and/or thrombosis It can be any other species that exhibits factor X1.

Cleavage by coagulation factor XIIa for activation of coagulation factor XI site is an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain. human factor XI, a blood coagulation factor with four tandem repeats that are highly each heavy chain (369 amino acids) of factor Xla (denoted as Ai-A4) apple four tandem repeats of 90-91 amino acids, called domains, plus a short link peptide [Fujikawa K, Chung DW, Hendrickson LE, Davie EW. (1986) Amino acids sequence of human factor XI, a blood coagulation factor with four tandem repeats that Zhao M, Gailani D. (1999) Identification of amino acids in the factor XI apple 3 domain The light chains of factor Xla (238 amino acids each), the serine proteases trypsin contains the catalytic portion of the enzyme containing sequences typical for the family [Fujikawa K, Chung DW, Hendrickson LE, Davie EW. (1986) Amino acid sequence of human factor XI, a blood coagulation factor with four tandem repeats that are highly homologous with Activation triggers the intermediate phase of the intrinsic pathway of blood coagulation.

Conservative amino acid variants Polypeptide that preserves the overall molecular structure of an antibody peptide sequence described herein variants can be made. When the properties of the individual amino acids are taken into account, some reasonable substitutions will be noticed by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions", eg polarity, charge, solubility, in terms of hydrophobicity, hydrophilicity and/or amphipathic nature of the included residues. According to the similarity, it can be done. For example, (a) non-polar (hydrophobic) amino acids are alanine, leucine, isoleucine, valine, includes proline, phenylalanine, tryptophan and methionine; (b) polar neutral amino acids, glycine, contains serine, threonine, cysteine, tyrosine, asparagine and glutamine; (c) positively charged (basic) amino acids include arginine, Izine and histidine, and (d) negatively charged (acidic) amino acids, Contains aspartic acid and glutamic acid. Substitutions are typically within groups (a)-(d) can be done. In addition, glycine and proline are classified according to their ability to disrupt oi-helices. can be substituted for the other. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more in d-helices. common, valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine ß- It is more commonly found in folded leaves. Glycine, serine, aspartic acid, asparagine and proline are commonly found in the turns. Some preferred Substitutions may be made between the following groups: (i) S and T; (ii) P and G and (iii) A, V, L and I. Known genetic code and recombinant and synthetic DNA techniques are taken into account When joined, the scientist with the expertise encodes conservative amino acid variants. As used herein, "sequence identity" between two polypeptide sequences, sequences Shows the percentage of amino acids that are identical between "Sequence homology", identical or the percentage of amino acids that represent conservative amino acid substitutions. shows. Preferred polypeptide sequences of the invention are at least 60% in the CDR regions, more preferably at least 70% or 80%, more preferably at least 90% and most preferably at least 95% has a number of identities. Preferred antibodies are also most abundant in the CDR regions. It also has a sequence homology of at least 80%, more preferably 90%, and most preferably 95%.

Invention DNA molecules The present invention also relates to DNA molecules encoding an antibody of the invention. This sequences, but not limited to, the DNA disclosed in SEQ ID NOs: 1 to 18 contains molecules.

The DNA molecules of the invention are not limited to the sequences described herein, but also includes variants thereof. DNA variants of the invention in hybridization can be described with reference to their physical properties. Worker with expertise, nucleic acid By using hybridization techniques, DNA is able to combine its complement and DNA double-stranded since it can be used to identify its equivalent or homolog will notice. In addition, hybridization with complementarity less than 100% It will be realized that it can be realized. However, the choice of appropriate conditions should be taken into account. When added, hybridization techniques are based on their structural relevance to a particular probe. It can be used to distinguish between DNA sequences. Guidance on such conditions for, see Sambrook et al., 1989 [Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA)] and Ausubel et al., 1995 [Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). current Protocols in Molecular Biology. New York: John Wiley and Sons].

Structural similarity between two polynucleotide sequences means that the two sequences will hybridize with each other. can be expressed as a function of the "hardness" of conditions. As used herein, conditions do not strongly favor hybridization, and only structurally the most the molecules involved will hybridize to each other under such conditions. On the contrary, hard- conditions that do not have a lesser degree of structural relevance for molecules supports hybridization. Therefore, the hybridization stringency of the two nucleic acid sequences is directly correlated with their structural relationships. The following relationships hybridization and relevance (where Tm is the melting temperature of a nucleic acid duplex): b. The Tm of a duplex DNA is every 1% in the number of unpaired base pairs. 1°C decreases with increase. where u1 and u2 are the ionic strength of the two solutions.

Hybridization hardness, total DNA concentration, ionic strength, temperature, probe one of many factors, including its size and the presence of hydrogen bonding agents. is the function. Factors promoting hybridization include high DNA concentrations, high ionic strengths, low temperatures, longer probe size and hydrogen bonding includes the absence of disrupting agents. Hybridization is typically performed in two phases: First, in the binding phase, the probe is exposed to the target under conditions that favor hybridization. it connects. Hardness is generally controlled at this stage by changing its temperature. High For stiffness, temperature is generally used unless short (< 20 nt) oligonucleotide probes are used. It is between 65°C and 70°C. A representative hybridization solution 6x SSC, 0.5% SDS, 5x Contains Denhardt's solution and 100 pg of non-specific carrier DNA [see, 15]. Certainly, a number of different but still functionally equivalent buffer conditions are known. When the degree of relevance is lower, a lower temperature can be selected. Low Hardness bonding temperatures range from approximately 25°C to 40°C. Medium hardness, at least is in the range from about 40°C to less than about 65°C. High hardness, at least at about 65°C.

Second, the excess probe is removed by washing. This means that harsher conditions generally It is in this phase that it is applied. Therefore, its relevance through hybridization It is this "washing" phase that is most important in determining the Wash solutions are typically low contains salt concentrations. A comparable medium-hard solution, 2X SSC and 0.1% SDS includes. A high-strength wash solution is combined with a preferred-strength solution. containing about 0.1X SSC, less than about 0.2X SSC (ionic strength in terms of ) contains the equivalent value. Temperatures associated with various hardnesses for "bonding" same as discussed above. The wash solution also typically is changed many times. For example, typical high stringency wash conditions are 30 at 55°C. It includes washing twice for 1 minute and three times for 15 minutes at 60°C.

Accordingly, the subject of the present invention may use the antibody and/or antigen- is an isolated nucleic acid sequence encoding binding fragments. present invention another application is the above-mentioned isolate encoding antibodies of the present invention. is the nucleic acid sequence. In this regard, the present invention provides high-rigidity bonding and nucleic acid molecules that hybridize to the molecules described under the washing conditions. wherein such nucleic acid molecules have properties as described herein. encodes an antibody or functional fragment thereof. (One mRNA perspective) preferred molecules combine the DNA molecules described herein with one at least 75% or 80% (preferably at least 85%, more preferably at least 90% and at least preferably at least 95%) sequence identity. DNA of coagulation factor Xl reducing or inhibiting its conversion to its active form factor Xla and/or molecules that directly block the catalytic activity of the coagulation factor Xla codes.

Recombinant DNA structures and expression The present invention additionally includes one or more of the nucleotide sequences of the present invention. It provides recombinant DNA constructs containing Recombinant constructs of the present invention are included in A vector into which a DNA molecule encoding an antibody of the invention is inserted, for example, used in conjunction with a plasmid, phagemid, phage or viral vector.

The encoded gene is Sambrook et al., 1989 and Ausubel et al., 1989. [Sambrook, J., Fritsch, E.

F. and Maniatis, T. (1989) Molecular Cloning: A Iaboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA; Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols big Molecular Biology. Techniques described in New York: John Wiley and Sons] can be produced with Alternatively, using DNA sequences, eg synthesizers, can be chemically synthesized. See, for example, OLIGONUCLEOTIDE SYNTHESIS Oligonucleotide Synthesis a Practical Approach. Ed. MJ. Gait, IRL Press Oxford UK] techniques described in the source. The expert in the field is the DNA that encodes the variable fields. encoding constant regions of variable human IgG isotypes or their derivatives, mutated or unmutated, with gene fragments can fuse. This person contains binders, for example, three times glycine-glycine-glycine-glycine-serine a fifteen amino acid elongation, using both variable domains as a single chain can apply recombinant DNA technology to fuse in the format. find it recombinant structures, RNA and/or protein products of encoded DNA (DNAs) It is included with expression vectors capable of expressing eKS. Vector addition, open reading Regulatory sequences containing a promoter operatively linked to the framework (ORF) may contain. The vector may additionally contain a selectable marker sequence. Specific starting and bacterial secretory signals are also associated with sequences encoding the inserted target gene. may also be necessary for effective translation.

The present invention additionally includes host cells containing at least one of the DNAs of the present invention. it provides. The host cell is virtually nonexistent for which expression vectors are available. It can be any cell. This is, for example, a higher eukaryotic host cell, for example, a mammalian cell, a lower eukaryotic host cell, for example, a yeast cell, and it may be a prokaryotic cell, eg, a bacterial cell. recombinant administration of the construct into the host cell, calcium phosphate transfection, DEAE, may be affected by dextran-mediated transfection, electroporation, or phage infection.

Bacterial expression Expression vectors useful for bacterial use encode a desired protein. a structural DNA sequence with appropriate translational initiation and termination signals. by insertion into the operable read phase together with a functional promoter makes. The vector maintains one or more phenotypic selectable markers and vectors. to ensure and, if desired, to provide amplification within the host. will contain the origin of replication. Prokaryotic hosts suitable for transformation are E. coli, Bacillus subtilis, Salmone/Ia typhimurium and Pseudomonas, Streptomyces and It includes several species in the Staphylococcus genera.

Bacterial vectors can be, for example, bacteriophage-, plasmid- or phagemid-based. This vectors, a selectable marker, and typically the well-known cloning vector pBR322. Bacterial derived from commercially available plasmids containing the elements (ATCC 37017) may contain the origin of replication. Transformation of a suitable host strain and host strain following growth to a suitable cell density, the selected promoter become unsuppressed by means (eg, temperature shift or chemical induction). is brought in/induced and cells are cultured for an additional period. Cells are typically It is harvested by centrifugation, broken down by physical or chemical means and obtained. The crude extract is stored for further purification.

In bacterial systems, depending on the intended use for the protein being expressed Numerous expression vectors can be advantageously selected. For example, this style when a large amount of a protein is to be produced, for the production of antibodies or a peptide for example, easily purified fusion protein products Vectors that direct the expression of high levels of

It is therefore an object of the present invention to encode novel antibodies of the present invention. is an expression vector containing a nucleic acid sequence.

Mammalian expression and purification of protein Preferred regulatory sequences for mammalian host cell expression viral elements that direct high levels of protein expression in their cells, for example, from cytomegalovirus (CMV) (eg, CMV promoter/accelerator), Simian From virus 40 (SV40) (eg, SV40 promoter/accelerator), adenovirus (for example, adenovirus major late promoter (AdMLP)) and polyoma-derived promoters and/or accelerators. Additional description of viral regulatory elements and their sequences [see U.S. Pat. 5,168,062; Bell et al. by vectors may also contain origins of replication and selectable markers [see Bell by U.S. 4,399,216]. Suitable selectable markers can be inserted into the vector. to drugs, for example, G418, hygromycin on a host cell to which it has been administered or contain genes conferring resistance to methotrexate. For example, dihydrofolate reductase The (DHFR) gene confers resistance to methotrexate and the neo gene confers resistance to 6418.

Transfection of the expression vector into a host cell, standard techniques, for example, electroporation, calcium-phosphate precipitation and DEAE-dextran transfection can be carried out using

Antibodies provided herein, their antigen binding portions or derivatives mammalian host cells suitable for expressing, for example, [Axel et al. by U.S. ,179,017] for use with a DHFR selectable marker [Axel et al. by U.S. Chinese Hamster containing dhfr-CHO cells, described in 4,634,665 . Ovary (CHO cells), NSO myeIOm cells, COS cells, and SP2 cells includes. In some embodiments, the expression vector is the host of the expressed protein. It is designed so that it is secreted into the culture medium in which cells are grown. antibodies, standard protein purification of their antigen-binding fractions or derivatives. It can be recovered from the culture medium using various methods.

Antibodies of the invention or an antigen-binding fragment thereof, limited to ammonium sulfate or ethanol precipitation, acid extraction, Protein A chromatography, Protein G chromatography, anion or cation exchange chromatography, phospho-cellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and Iectin recovered from recombinant cell cultures by well-known methods including chromatography can be recovered and purified. High performance liquid chromatography ("HPCL") can be used for purification [see Urlaub G, Chasin LA. (1980) hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U their antigen-binding fragment, naturally purified products, chemical products of synthetic procedures and, for example, yeast, higher plants, insects and mammals products produced by recombinant techniques from a eukaryotic host containing cells includes. Depending on the host used in a recombinant production procedure, the available The antibody of the invention may be glycosylated or non-glycosylated. This style methods are described in numerous standard laboratory manuals [see Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA); Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995).

Current Protocols in Molecular Biology. New York: John Wiley and Sons, chapters 10, are host cells containing an acid molecule so that the host cell is a higher eukaryotic host cell, eg, mammalian cell, a lower eukaryotic host cell, eg, it may be a yeast cell, and it may be a prokaryotic cell, eg, a bacterial cell.

Another object of the present invention is to produce an antibody and antigen binding fragments. culture of the host cell under appropriate conditions and a method comprising recovering said antibody.

It is therefore another object of the present invention to interact with the host cells of the present invention. 005-C04 antibody produced and purified to at least 95% by weight homogeneity.

affine by measuring the dissociation constant (kd) and calculating kd divided by ka (KD=kd/ka) determines. The term "immunospecific" or "specifically binding" means that the antibody coagulation factor XI and/or its activated form, coagulation factor Xla with an affinity KD below or equal to 10'6 M (single-value affinity) means it's tied. The term "high affinity" refers to the antibody's coagulation factor XI. and/or its activated form, coagulation factor Xla, 10'7 M (single- valence affinity) means KD that binds with a KD affinity below or equal to 107 M income. The antibody is notable for the target antigen compared to other unrelated molecules. may have significantly greater affinity. The antibody is also targeting compared to homologs. Significantly greater for the antigen, for example, compared to the affinity for the target antigen may have greater affinity. Such affinities, using traditional techniques, for example, with balance dialysis; general procedure outlined by the manufacturer with the use of the BlAcore 2000 device using; radioactively affected target by radioimmunoassay using the antigen or known to the person skilled in can be easily determined by another method. Affinity data, for example, [Kaufman RJ, Sharp PA. (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol.159:601-621] It can be analyzed by the method described in the source.

antibodies As used herein, "coagulation FXI and/or its activated form of "antibodies that block coagulation factor Xla", FXI and/or FXIa with antibodies of the present invention, coagulation factor FXI and/or FXIa expressing a blockade that leads to a complete or partial inhibition of its activity is meant. The amino acid sequences include, but are not limited to, SEQ ID NOs: 19 to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), antibody fragments that can bind antigen (eg Fab', F'(ab)2, Fv, single chain antibodies, diabodies), camel-bodies, and these includes recombinant peptides containing the above, as long as Antibodies preferably can move fields (Fc fields) (see below). Effector functions mutations can be applied for modification. The complement protein Ciq at the Fc-domain and Amino acid residues that play a dominant role in interactions with F0 receptors have been identified and mutations affecting effector functions have been described [a for review, see Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001)]. Specifically, lgGi's aglycosylation abrogates antibody-induced cell-mediated cytotoxicity (ADCC) reported asparagine alanine or asparagine at amino acid position 297 can be achieved by mutating glutamine [Labrijn AF, Aalberse RC, Schuurman J. (2008) When binding is enough: nonactivating antibody formats. Curr Opin Immunol. position while leading to removal of complement-mediated cytotoxicity (CDC) Simultaneous switching of the two Ioins at 234 and 235 with the domains of ADCC and CDC [Sazinsky SL, Ott RG, Silver NW, Tidor B, Ravetch JV, Wittrup KD. (2008) Aglycosylated immunoglobulin G1 variants productively engage activating Fc To apply IgG4 isotypes as bivalent therapeutics in vivo, a modification is for example, the change from serine to proline in the 'core hinge region' [Schuurman J, Van Ree R, Perdok GJ, Van Doorn HR, Tan KY, Aalberse RC. (1999) normal human immunoglobulin G4 is bispecific: it has two different antigen-combining sites. One of the cysteines at positions 127, 232, and 233 tends to form dimers. can be avoided by changing to serie [Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G, Sims P, Hong K, Shields RL, Damico LA, Rancatore P, Yansura DG. (2002) Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient an alternative format with effector function is four IgG4-specific amino acid residues it may be the IgG2m4-derived format that carries the change [Hezareh M, Hessell AJ, Jensen RC, van de Winkel JG, Parren PW. (2001) Effector function activities of a panel of mutants of a broadly neutralizing antibodies against human immunodefioiency virus or by enzymatic or chemical cleavage of the same antibodies and additionally described. Non-limiting examples of monoclonal antibodies are each Murine, chimeric, humanized, human, and Human EngineeredT'V' immunoglobulins, antibodies to sequences derived from immunoglobulins chimeric fusion proteins or muteins or derivatives thereof.

Identical molecules and/or multimers or aggregates of fragments are contemplated.

As used herein, the term "monoclonal antibody" refers to a remarkably homogeneous refers to an antibody obtained from a population of antibodies, i.e. population possible naturally occurring antibodies that may be present in minor amounts identical except for mutations. Monoclonal antibodies against a single antigenic site It is highly specific as directed. In addition, typically different determinants conventional (polyclonal) antibody containing different antibodies directed against (epitopes) In contrast to the preparations, each monoclonal antibody has only a single antibody on the antigen. directed against the determinant. In addition to their specificity, monoclonal antibodies by other immunoglobulins with different specificities and characteristics. It is advantageous in that it is synthesized by an uncontaminated, homogeneous culture.

The modifying "monoclonal" means that the antibodies are remarkably homogeneous. shows its character as obtained from a population of It is not meant to be produced by a special method. For example, monoclonal antibodies to be used were first described by Kohler et al. [Allen MJ, Guo A, Martinez T, Han M, Flynn GC, Wypych J, Liu YD, Shen WD, Dillon TM, Vezina C, Balland A. (2009) Interchain disulfide bonding in human IgGZ antibodies probed by or by recombinant DNA methods [see An 2, Forrest G, Moore R, Cukan M, Haytko P, Huang L, Vitelli S, Zhao JZ, Lu P, Hua J, Gibson CR, Harvey BR, Montgomery D, Zaller D, Wang F, Strohl W. (2009) lgG2m4, ari engineered antibody isotype with reduced Fc function. MAbs. 1:572-579].

Human Engineered T'V' or antibody fragments can also be.

An "immunoglobulin" or "native antibody" is a tetrameric glycoprotein. a natural In the formed immunoglobulin, each tetramer is a "light" (approximately 25 kDa) of each pair. and a "heavy" chain (two identical polypeptide chains of which it has approximately is created from the pair. The amino-terminal portion of each chain is primarily non-antigen-recognised. a "variable" region of about 100 to 110 or more amino acids that is responsible includes. The carboxy-terminal portion of each chain is primarily responsible for effector function. defines a fixed region. Immunoglobulins, their heavy chains Depending on the amino acid sequence of alanine, it can be assigned to different classes. Are heavy chains They are classified as (u), delta (A), gamma (v), alpha (ci), and epsilon (5) and determine the isotype of the antibody. defines it as IgM, IgD, IgG, IgA and IgE, respectively. A few of these additionally to classes or isotypes, for example, . lgG1, lgG2, IgG3, lgG4, lgA1 and IgA2 can be cleaved.

Different isotypes have different effector functions; for example, IgG1 and IgG3 Isotypes It often has ADCC activity. Human light chains kappa (K) and lambda (A) light classified as chains. Variable and fixed regions in light and heavy chains, with the heavy chain also containing a "D" region of about 10 more amino acids, joined by a "J" region of approximately 12 or more amino acids [see general as Köhler G, Milstein C. (1975) Continuous cultures of fused cells secreting A "functional fragment" or "antigen-binding" of an antibody/immunoglobulin "antibody fragment" herein refers to the antigen-binding of an antibody/immunoglobulin. as a fragment (for example, a variable region of an IgG) that retains the is defined. An "antigen-binding site" of an antibody is typically defined as one or more of an antibody. in more than one hypervariable region(s), ie ', CDR-1, -2 and/or -3 are found in the regions; however, variable "frame" regions can also be It may also play an important role in antigen binding, by providing a scaffold for CDRs.

Preferably, the "antigen-binding site" is at least 4 to 103 of the variable light (VL) chain and amino acid residues 5 to 109 of the variable heavy (VH) chain, more preferably It contains amino acid residues 3 to 107 of VL and 4 to 111 of VH, and the complete VL and VH chains [numbering of amino acid positions are available in the Kabat database [U.S. amino acid positions] are particularly preferred. For use in existing invention A preferred class of immunoglobulins is IgG. denotes amino acid residues of the VH and VL variable domains of the functional fragment.

Hypervariable region, [Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)], determining a "complementarity" amino acid residues [i.e., in the light chain variable domain] and in the heavy chain variable domain residues between] and/or a hypervariable loop residues [i.e., in the light chain variable domain (in LCDR1)) 91-96 and in the heavy chain variable area (in HCDR1) 96-101 residues].

Non-limiting examples of antibody fragments, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, diabodies, triabodies, tetrabodies, minibodies, linear antibodies [Johnson G, Wu TT. (2000) Kabat database and its chelating recombinant antibodies, tribodies or bibdies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), an antigen-binding- alanine immunoglobulin fusion protein, a camelliated antibody, a VHH antibody or muteins or derivatives thereof, and the antibody An immunoglobulin is at least polypeptide-specific as long as it retains its activity. containing a portion, eg, a CDR sequence, sufficient to induce antigen binding. Contains polypeptides and multispecific antibodies formed from antibody fragments immunoglobulins. J Mol Biol. 196z901-917; Zapata G, Ridgway JB, Mordenti J, Osaka G, Wong WL, Bennett GL, Carter P. (1995) Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity. Protein Eng. 8:1057-1062]. An antibody other than a "bispecific" or "bifunctional" antibody it is understood that each one is identical and that it has attachment points. to F(ab')2 or inter-molecular disulfide interactions between the CH1 and CL domains to the Fab engineering can be applied to minimize or completely remove it.

Papain digestion of antibodies, each with a single antigen-binding site "Fab" two identical antigen-binding fragments, termed fragments, and produces a residual "FC" fragment that reflects its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment having two "Fv" fragments. A "Fv" trailer, It is the minimum antibody fragment containing one complete antigen recognition and binding site. This region One of a heavy- and a light-chain variable domain in tight, non-covalent coupling It consists of dimer. This is the surface of the VH-VL dimer of the three CDRs of each variable domain. interacting to identify an antigen binding site on is in the configuration. Collectively, the six CDRs provide antigen-binding specificity to the antibody.

However, a single variable domain (or only three CDRs specific to an antigen) even half of an Fv containing Fv) is capable of recognizing and binding the antigen. contains domains, where these domains are contained in a single polypeptide chain.

Preferably, the Fv polypeptide is additionally for antigen binding of Fv between the VH and VL domains. contains a polypeptide linker that allows it to form the desired structure. st's For a review, see [0. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press].

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain. (CH1) also contains. Fab fragments are derived from the Fab' fragments of the heavy chain CH1 alanine. several cells containing one or more cysteines from the antibody hinge region to the carboxy terminus. differs with the addition of the residue. Fab'-SH is the cysteine residue of constant domains. Here is the notation for Fab' (residues) containing a free thiol group. F(ab')2 antibody fragments originally had hinge cysteines in between It was produced as pairs of Fab' fragments. are residues. the desired binding affinity or biological activity of the mutein or variant. An antibody can be found in or within the variable region, provided that it is retained. substitution, deletion or insertion of at least one amino acid in the value part refers to the polypeptide sequence that contains it.

Muteins are remarkably homologous or remarkably identical to the parent antibody. it could be. with radionuclides or various enzymes) labeling, covalent polymer attachment, for example, pegylation (derivatization with polyethylene glycol) and non-natural amino acids by techniques such as insertion or substitution by chemical synthesis refers to covalently modified antibodies.

A "human" antibody or functional human antibody fragment herein is chimeric or is defined as a non-molecule. A human antibody or functional antibody fragment may be derived from a human or be a synthetic human antibody. a "synthetic "human antibody" herein refers, in whole or in part, to known human antibody sequences. An antibody having an in silico-derived sequence from synthetic sequences based on analysis is defined as. In sil/co-design of a human antibody sequence or fragment thereof for example, analyze a database of human antibody or antibody fragment sequences. and by arranging a polypeptide sequence using the data obtained therefrom, can be printed. Another human antibody or functional antibody fragment example, from a library of antibody sequences of human origin (ie, a human native from such a library (according to antibodies from the source) It is a molecule encoded by a nucleic acid. Examples of human antibodies, Laboratory Manual), Springer Verlag; Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269- antibodies for diagnostic use and therapy. Expert Rev Mol Diagn. 1:102-108] Contains n-CoDeR-based antibodies as described in the source.

A "humanized antibody" or functional humanized antibody fragment (I) from a non-human source (for example, a heterologous immune system transgenic mouse), where this antibody is based on a human germ-line sequence, or (ll) CDR-grafted, where one or more frames of the variable domain are human origin and (if any) fixed area is of human origin, while CDRs of variable area are human origin. is of non-existent origin, it is defined as a molecule.

As used herein, the expression "chimeric antibody" typically originates from different species. refers to an antibody containing sequence derived from two different antibodies. Most typically, chimeric antibodies, human and murine antibody fragments, generally human fixed and mouse includes variable regions.

An antibody of the invention may be derived from a recombinant antibody gene library.

Technologies for making repertoires of recombinant human antibody genes development and filamentous bacteriophage surface of encoded antibody fragments Displayed on can also be attributed to humanized, chimeric, murine or mutein antibodies. a recombinant for directly making and selecting human antibodies that can be applied the vehicle is intact. Antigen-binding antibodies produced by phage technology fragments - generally Fv or Fab fragments - and therefore It lacks effector functions. Effector functions with either of the two strategies applicable: Fragments into whole antibodies for expression in mammalian cells or bispecific antibody with a second binding site capable of triggering an effector function can be engineered into fragments. Typically, the Fd fragment of antibodies (VH- CH1) and its light chain (VL-CL) are cloned separately by PCR and subsequently identified a specific antigen. randomly selected combinatorial phage display libraries for binding is reassembled. Fab fragments are expressed on the phage surface, that is, they It is physically linked to the coding genes. Thus, the Fab's antigen binding selection then co-selects sequences encoding Fab that can be amplified. Antigen several rounds of coupling and re-amplification are performed in a process called panning. The procedure is enriched with antigen-specific Fab and finally isolated.

Various procedures are described for deriving human antibodies from phage-display libraries. has been made. Such libraries, [U.S. Patent No. 6,989,250; BASE. Patent No: 4,816,567], various in Viva-formed (i.e., human-generated) built on a single mainframe with allowed CDR reassembly can be done. Alternatively, such an antibody library can be designed in silico and amino acid encoded by synthetically created nucleic acids can be based on series. In silico design of an antibody sequence, for example, a sequence of human sequences analyzing the database and using the data obtained from it to create a polypeptide With sequence editing, it can be achieved. designing and obtaining in silico-formed arrays methods for doing so are described; see, for example, [Carlsson R, Söderlind E. (2001) n- CoDeR concept: unique types of antibodies for diagnostic use and therapy. Expert Rev. P, Fischer M, Wellnhofer G, Hoess A, Wölle J, Plückthun A, Virnekas B. (2000) Fully synthetic human combinatorial antibodies Iibraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol. 29657- 86]. For a review of phage display techniques, see [Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao M, Dreier T, Fischer D, Höss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Söhlemann P, Winter J, Wölle J, Kretzschmar T. (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods. 254:67-84].

Alternatively, an antibody of this invention may come from animals. This type of antibody Dreier T, Fischer D, Höss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Söhlemann P, Winter J, Wölle J, Kretzschmar T. (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods. 254:67-84] may be Humanized or Human Engineering outlined in the source; this kind of antibody may come from transgenic animals [see Krebs B, Rauchenberger R, Reiffert S, Rothe C, Tesar M, Thomassen E, Cao M, Dreier T, Fischer D, Höss A, Inge L, Knappik A, Marget M, Pack P, Meng XQ, Schier R, Söhlemann P, Winter J, Wölle J, Kretzschmar T. (2001) High-throughput generation and engineering of recombinant human antibodies. J Immunol Methods. 254:67-84].

As used herein, antigen, for example, coagulation factor X1 and/or The different 'forms' of the coagulation factor Xla are presented here in different translational and post- translational modifications, for example, but not limited to, primary From differences in splicing of the FXI transcript to differences in glycosylation different protein resulting from differences in post-translational proteolytic cleavage As used herein, the term "epitope" refers to an immunoglobulin or T-cell includes any protein marker that can specifically bind its receptor. epitopic determinants are generally chemically active surface groupings of molecules, consists of, for example, amino acids or sugar side chains and is generally specific three-dimensional structural characteristics as well as specific load characteristics. has. One antibody competes with the second antibody in a competitive binding assay. by any of the methods well known to those skilled in the art. and preferably all amino acids of the epitope are detected by the two antibodies When bound, the two antibodies are said to 'bind the same epitope'. mature Fab variants have a given antigen, for example, FXI, stronger and/or an antibody showing improved binding—that is, binding with increased affinity—or Contains derivatives of the antibody fragment. maturation of an antibody or antibody few mutations within the six CDRs of the fragment that lead to this increased affinity is the process of identification. The maturation process is used to introduce mutations to the antibody. molecular biology methods and screening to identify improved binders combination.

The present invention may also be used alone or with at least one other agent, eg solubilisation. The compound may contain, in combination with, FXI/FXla antibodies, limited to Any sterile product containing saline, buffered saline, dextrose and water, provided that It also relates to pharmaceutical compositions that can be administered in a biocompatible pharmaceutical carrier.

Any of these molecules herein as an excipient (excipients) or pharmaceutical alone in pharmaceutical compositions, mixed with acceptable carriers or in combination with other agents, drugs, or hormones. applicable. In one embodiment of the present invention, pharmaceutically acceptable The carrier is pharmaceutically inert.

The present invention also relates to the administration of pharmaceutical compositions. This style Administration is done parenterally. Parenteral delivery methods topical, intra-arterial (direct to tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, includes intravenous, intraperitoneal, intrauterine, or intranasal administration. Active In addition to substances, these pharmaceutical compositions are pharmaceutically active compounds. excipients and ancillaries that facilitate processing into the preparations that can be used may contain suitable pharmaceutically acceptable carriers containing substances.

Additional details on techniques for formulation and administration, Remington's Latest source of Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.) can be found in print.

Pharmaceutical formulations for parenteral administration consist of aqueous solutions of active compounds. includes. For injection, the pharmaceutical compositions of the invention are preferably in aqueous solutions. physiologically compatible buffers, eg Hank's solution, Ringer's solution or may be formulated in physiologically buffered saline. 8qu injection suspensions, substances that increase the viscosity of the suspension, eg sodium may contain marboxymethyl cellulose, sorbitol or dextran. In addition, active compounds suspensions can be prepared as suitable oily injection suspensions. Appropriate synthetic fatty acid esters, for example, ethyl oleate or triglycerides or liposomes includes. Optionally, the suspension can also be combined with suitable stabilizers or highly concentrated the solubility of the compounds to allow the preparation of diluted solutions. may also contain potentiating agents.

Appropriate penetration of the specific barrier to be penetrated, for topical or nasal application stimulants are used in the formulation. Such penetrants are generally known in the art.

Parenteral administration also includes intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal and intraventricular, intravenous, intraperitoneal, intrauterine, vaginal or includes parenteral delivery methods, including intranasal administration.

kits In addition, the invention includes any of the ingredients of the above-mentioned compositions of the invention. pharmaceutical containing one or more containers filled with one or more relates to packages and kits. This type of container(s) of product for human application reflecting the approval of the production, use or sale by the administration, regulating the manufacture, use or sale of pharmaceuticals or biological products A statement may be attached in the form prescribed by a government agency.

In another embodiment, the kits may contain DNA sequences encoding antibodies of the invention.

Preferably, the DNA sequences encoding these antibodies are transfected into a host cell or provided in a plasmid suitable for expression by a host cell.

The plasmid is a promoter to regulate the expression of DNA in the host cell. (often an inducible promoter). The plasmid also contains other DNA sequences. to facilitate insertion into plasmid to produce various antibodies It may also contain suitable restriction sites. Plasmids also contain encoded proteins. Numerous other elements are also included to facilitate its cloning and expression. may contain. Such elements are well known to those skilled in the art and for example, selectable markers, start codons, end codons, and includes similar. Production and storage Pharmaceutical compositions of the present invention are known in the art, for example, conventional mixing, dissolving, granulating, dragee-making, surface smoothing, It can be produced by emulsifying, encapsulating, trapping or lyophilizing.

The pharmaceutical composition has a pH of 4.5 to 5.5, which is combined with the buffer before use. in the range of 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol in a lyophilized It can be supplied as a powder.

Pharmaceutical containing a compound of the invention formulated in an acceptable carrier Once the compositions have been prepared, they can be placed in a suitable container and can be labeled for the treatment of the ailment shown. Anti-coagulation factor XI and/or For the administration of anti-coagulation factor XIa antibodies, such labeling It will include the amount, frequency and method of administration.

According to the FDA, the IC50 is a compound for 50% inhibition of a given biological process. represents the required concentration. Antibodies of the present invention, 100 µM. more preferably IC50 values of 0.0001 pM.

Therapeutically effective dose Pharmaceutical compositions suitable for use in the present invention are its purpose, that is, characterized by coagulation factor XI and/or coagulation factor Xla. in an amount effective to provide the treatment of a particular disease state contains the compositions. Determination of an effective dose is for those skilled in the art. is within its capacity.

A therapeutically effective dose is a protein or protein that relieves symptoms or discomfort. refers to the amount of antibodies, antagonists or inhibitors thereof. This style therapeutic efficacy and toxicity of compounds in cell cultures or experimental standard pharmaceutical procedures in animals, for example, ED 50 (in 50% of the population) therapeutically effective dose) and LD50 (fatal dose for 50% of the population), with can be determined. The dose ratio between therapeutic and toxic effects is the therapeutic index. It can be expressed as the ED50/LD50 ratio. Pharmaceutical showing large therapeutic indices compositions are preferred. Obtained from cell culture analyzes and animal studies The data obtained are used to formulate a range of dosage for human use. This style The dosage of the compounds preferably exceeds EDgo with little or no toxicity. It lies within a range of circulating concentrations containing dosage, dosage used within this range depending on the form, the sensitivity of the patient and the route of administration. shows diversity.

The exact dosage must be determined by the individual physician, taking into account the patient to be treated. is selected. Dosage and administration should be used to provide adequate levels of the active ingredient or as desired. adjusted to maintain the effect. Additional factors that can be taken into account severity, patient's age, weight, and gender; the duration of the diet and frequency, drug combination(s), reaction sensitivities, and includes tolerance/response to therapy. Long-acting pharmaceutical compositions every 3 to 4 days, weekly depending on the half-life of the formulation and the rate of clearance It can be applied once or twice a week or once a month.

Normal dosage amounts range from 0.1 to 100,000 micrograms, depending on the route of administration. may vary over a total dose range of about 2 g. Private transfer Guidance on dosages and methods is provided in the literature [see U.S. Pat. Patent No: 6,300,064]. Persons skilled in the art can look for proteins or inhibitors thereof. will use different formulations for polynucleotides compared to what is available. Similarly, transfer of polynucleotides or polypeptides to specific cells, ailments, The specific activities reported can range from 0.1 to 10 mCi/mg of protein Frattarelli M, Lazzari S, Riva N, Giuliani G, Casi M, Saiti G, Guiducci G, Giorgetti G, Gentile R, Santimaria M, Jermann E, Maeke HR. (1999) Loco-regional radioimmunotherapy of high-grade malignant gliomas using specific monoclonal 328031 The present invention is further described by the following examples. Examples are specific It is provided to describe the invention with reference to applications. These examples are specific to the invention. when describing specific aspects of the invention, it does not describe limitations or does not limit its scope.

All examples are provided to those skilled in the art, except where the contrary is described in detail. using standard techniques well known and routine to the person has been executed. The routine molecular biology techniques of the following examples are standard. laboratory manuals, for example [Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA] as described.

Coagulation factor XI and or coagulation factor Xla in buffer measurement of inhibition To determine factor Xa inhibition of the substance listed above, a factor Xla The substrate was used to determine the enzymatic activity of human Factor Xla. biological test system. Determinations are made on microtiter plates.

Determination of anti-coagulatory activity Anti-coagulation activity of test substances in human plasma and/or rabbit determined in vitro in plasma and/or rat plasma. For this purpose, as a buyer, a A 1/9th sodium citrate/blood mix using 0.11 molar sodium citrate blood is taken. Immediately after the blood is drawn, this is mixed well and approximately 4000 Centrifuge for 15 minutes at g. The supernatant is pipetted and discarded.

Prothrombin time (PT, synonyms: thromboplastin time, quick test) is a commercial test kit (Neoplastin® from Roche (formerly Boehringer Mannheim) or of varying concentrations of the substance or the corresponding solvent. determined in existence. Test compounds are incubated with plasma for 3 minutes at 37°C.

Coagulation is then initiated by the addition of thromboplastin, and coagulation determined when it happens. Influenced a doubling of prothrombin time test substance concentration is determined.

Thrombin time (TT) using a commercial assay (thrombin reagent from Roche) varying concentrations of the test substance or the corresponding solvent. determined in existence. Test compounds are incubated with plasma for 3 minutes at 37°C.

Coagulation is then initiated by the addition of thrombin reagent and the coagulation determined when it happens. Test affecting a doubling of thrombin time substance concentration is determined.

Activated partial thromboplastin time (aPTT) is a commercial test kit (Roche). of the test substance or the corresponding solvent using the PTT reagent from determined in the presence of varying concentrations. Test compounds 3 at 37°C It is incubated with plasma and PTT reagent (cephalin, kaolin) for minutes. coagulation It is then started with the addition of 25 mM calcium chloride and coagulation takes place. time is determined. Test substance affecting a doubling of aPTT concentration is determined. Concentrations of antibodies of the present invention, 100 µM preferably at a concentration of 1 µM, more preferably 0.1 At a concentration of pM, more preferably 0.1 µM, preferably at a concentration of 0.001 pM, more preferably 0.0001 µM at a concentration, more preferably 0.00001 pM, of aPTT causes it to rise.

Therapeutic Use Anti-coagulation factor XI antibodies of thisIUS and/or anti-coagulation factor Xla antibodies to inhibition of the coagulation cascade and platelet aggregation to any subject for whom inhibition and inhibition of thrombosis would be beneficial. applicable.

Therefore, anti-coagulation factor XI antibodies of the invention and/or anti- Coagulation factor Xla antibodies can be found in humans as well as animals. It is suitable for the treatment and/or prophylaxis of coagulation-related disease. or non-STEMI) myocardial infarction (MI) or acute myocardial infarction (AMI), stable Angina Pectoris is also known as unstable Angina Pectoris, angioplasty or Re-occlusion and re-occlusion following coronary intervention such as coronary artery bypass graft (CABG) stenosis, peripheral artery occlusive disease (PAOD), pulmonary embolism (PE), deep vein thrombosis (DVT) also known as renal vein thrombosis, transient ischemic attack (TIA), thrombotic It includes diseases such as stroke and thromboembolic stroke.

These antibodies are also associated with cerebral ischemia, apoplectic stroke, as well as systemic treatment and prevention of cardiogenic thromboembolism such as thromboembolism, irregular patients with a heartbeat or abnormal heart rhythm, for example, those with atrial fibrillation for patients with artificial heart valves, for patients with valvular heart disease, for treatment is also useful. In the future, these antibodies will be used in patients with disseminated intravascular coagulation (DlC). It can be helpful in the treatment of patients.

Thromboembolic complications arise from atherosclerotic lesions of the vessel wall, endothelial function, which can lead to acute thrombotic occlusions. may result from deterioration. Atherosclerosis, too many cardiovascular risks It is a multi-factorial disorder that depends on the factor. Clinical studies, anti-coagulants and prophylaxis does not definitively affect the course of arterial vascular disorder. has shown. Therefore, the risk associated with an anti-thrombotic therapy Targeted treatment of these factors is advantageous. Coronary, peripheral and cerebral vascular Risk factors for disorders are, for example: elevated serum cholesterol levels, arterial hypertension, smoking and diabetes mellitus. preventive medicine principles are based on the elimination of these risk factors. A change in life as well as pharmacological measures, eg, anti-hypertensive therapy, lipid- reducing drugs or thrombosis prophylaxis are also included. In addition, the coronary combination with therapeutic agents, where pre-existing coronary heart disease when it is suitable for treatment.

Thromboembolic complications, micro-angiopathic hemolytic anemia (MAHA), including extra-corporeal blood circulation such as hemodialysis and aortic valve replacement In addition, antibodies of this invention may be found in rheumatoid arthritis (RA) or Alzheimer's disease. for the treatment of inflammatory diseases such as neurological diseases such as AD, or useful for prophylaxis. In the future, these antibodies will prevent cancer and metastasis, thrombotic microangiopathy (TMA), grief-related macular degeneration, diabetic retinopathies, diabetic nephropathies as well as other microvascular diseases may be useful for treatment.

Antibodies of this invention are also useful in tumor patients or in a chemo- and/or radiotherapy Treatment of thromboembolic complications following surgery of tumor patients receiving It is also useful for

Antibodies of this invention are also useful in the treatment and/or prophylaxis of Dialysis patients, especially It is also useful for the prevention of centr thrombosis Cimino-fistula in hemodialysis.

Hemodialysis native arteriovenous fistulas, synthetic fold grafts, wide-hole central using venous catheters or other Devices consisting of artificial surfaces realizable. Administration of antibodies of this invention during dialysis and after After a short time, clot formation in the fistula (and embolization in the pulmonary arteries) will prevent the spread of the dead clot).

Antibodies of this invention can also be recovered from cardiopulmonary bypass surgeries (eg, ECMO: After extra-corporeal membrane oxygenation), intracardiac and for the treatment and/or prophylaxis of patients with intrapulmonary thrombosis. it is useful.

Under the requirement of systemic anti-coagulation and the mechanical stability and process of the device, Major limitations of ventricular assist devices are the high incidence of thromboembolic events. incidence. Therefore, the antibodies of this invention also provide a left ventricular assist It is also useful for the treatment and/or prophylaxis of patients receiving the device.

Here, anti-inflammatory drugs are used in dialysis patients without increasing the risk of undesirable bleeding events. there is a high need for coagulation and here in this population venous thromboembolism (VTE) and atrial fibrillation (for example, in hemodialysis patients) Stage renal disease) incidence is high. Antibodies of this invention are also useful in such patients. It is also useful for treatment and/or prophylaxis.

Antibodies of this invention have also been affected by idiopathic thrombocytopenic purpura (IPT). It is also useful for the treatment and/or prophylaxis of patients. These patients belong to the general population has an increased thrombotic risk compared to Coagulation factor Xla concentration is significantly higher in ITP patients compared to controls and The aPTT is significantly longer in ITP patients.

Antibodies of this invention are also used in the treatment and/or prophylaxis of pulmonary hypertension. It is also useful for

Pulmonary Hypertension, Venice 2003) for example, pulmonary arterial hypertension caused by left ventricular disease from pulmonary hypertension, lung diseases and/or hypoxia, blood clots, from arterial constriction and chronic thromboembolic pulmonary hypertension (CTEPH) pulmonary hypertension caused by other diseases such as pulmonary sant vitia, HIV infections or thyroid disease of certain drugs, Glycogen storage disease (GSD), Morbus Gaucher, hereditary hemorrhagic telangiectasia, combination with diseases such as hemoglobinopathy and/or myeloproliferative disorders associated pulmonary arterial hypertension (APAH), which may be associated with includes diseases such as

Antibodies of this invention, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatous (PCH) as well as neonatal persistent pulmonary hypertension It is also useful for the treatment and/or prophylaxis of diseases. interstitial lung disease (ILD), sleep apnea. alveolar hyperventilation, altitude The disease also includes diseases such as constitutional dysplasia.

Diseases resulting from chronic thromboembolic pulmonary hypertension (CTEPH) with proximal and/or distal pulmonary artery obstruction or with cancer, parasites or may be associated with non-thrombotic pulmonary embolism, such as contaminants.

In the future, antibodies of this invention will be developed from sarcoidosis, histiocytosis X, and lymphangiomatosis. It can be used for the treatment and/or prophylaxis of pulmonary hypertension caused by

In addition, antibodies of this invention are used in the treatment of pulmonary and/or hepatic fibrosis. and/or prophylaxis.

Antibodies of this invention are also used in sepsis, systemic inflammatory syndrome (SIRS), organ dysfunction, multiple organ dysfunction syndrome (MODS) acute respiratory distress syndrome (ARDS), acute lung injury (ALI) also for the treatment and/or prophylaxis of disseminated intravascular coagulation (DlC). it is useful.

(SIRS) describes its occurrence. SIRS is predominantly caused by infections. induced but also lesion, burn, shock, operations, ischemia, pancreatitis, It can also occur following reanimation or tumor disposition. In the course of a sepsis, coagulation cascade, disseminated intravascular coagulation, and DIC for short A process called a process can be activated. This is due to the formation of microthrombosis and secondary In addition, sepsis or SIRS can lead to endothelial dysfunction, which leads to an increase in vascular permeability. In the course of sepsis or SLRS, several combined failure of the organ, for example, kidney failure, liver failure, lung failure, failure of the cardiovascular system, may occur.

Pathogenic organism that induces sepsis or SIRS, gram-positive and gram-negative are bacteria, fungi, viruses and/or eukaryotic pathogens.

DIC and/or SIRS may occur in conjunction with a sepsis but also a from operation, tumor diseases, burns or other types of injury. can be caused.

During DIC, an activation of the coagulation cascade causes damaged veins or occurs on the surface of other tissue types. This is small on the part of them can lead to the formation of micro-thromboses leading to occlusion of the vessels.

In one embodiment, anti-coagulation factor XI antibodies and/or anti-coagulation factor Xla antibodies treatment and/or prophylaxis of the diseases already mentioned It is used in combination with other drugs for

Below are examples of suitable combinations and therefore preferably mentioned: - lipid lowering compounds, especially Lovastatin (Mevacor; US 4,231,938), Simvastatin (Zocor; US 4,444,784), Pravastatin (Pravachoh US 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors such as US 5,273,995) combination with.

- Compounds suitable for the treatment of coronary disease and/or compounds showing vasodilating activities, especially Captopril, Lisinopril, Enalapril, Ramipril, Cilazapril, Benazepril, Fosinopril, Quinapril and Angiotensin converting enzyme inhibitors such as perindopril or Embusartan (US 5,863,930), Losartan, Valsartan, Irbesartan, Angiotensin II receptor such as Candesartan, Eprosartan, and Temisarta antagonists or Carvedilol, Alprenolol, Bisoprolol. acebutolol, Atenolol, Betaxolol, Carteolol, Metoprolol, Nadolol, Penbutolol, Pindolol, ß-adrenergic receptor antagonists such as propanolol and Timolol, with combination or such as Prazosin, Bunazosin, Doxazosin and Terazosin Combination with alpha1 adrenergic receptor antagonists.

- Diuretics Hydrochlorothiazide, Furosemide, Bumetanide, Pyretanide, Combination with Torsemide, Amiloride and Dihydralazine.

- Calcium channel inhibitors such as Verapamil and Diltiazem, Nifedipine (Adalat), Nitrendipine (Bayotensin), Izosorbid-5-m0nonitrate, Izosorbid- combination with dihydropyridine derivatives such as dinitrate and Glyceroltrinitrate. - Cyclic guanosine monophosphate such as soluble Guanylate cyclase stimulators with compounds that cause an increase in the concentration of CGMP - Other inhibitors of the coagulation cascade, such as plasminogen activators (thrombolytics/Fibrinolytics) also known as Thrombolysis and/or Fibrinolysis enhancing compounds or inhibitors of plasminogen activator or tissue such as plasminogen activator (t-PA), Streptokinase, Reteplase and Urokinase Thrombin-activated Fibrinolysis-Inhibitors (TAFI-Inhibitors) - Unfractionated heparins, low molecular weight Heparins, Anti-coagulants such as Heparinoid, Hirudin, Bivalirudin and/or Argatroban combination with. In addition, combination therapies may be associated with anti-coagulation factor XI antibodies and/or An antibiotic therapy of anti-coagulation factor Xla antibodies, anti-fungal co-administration with therapeutics and anti-viral therapeutics.

In addition, anti-coagulation factor XI antibodies and/or anti-coagulation factor Xla antibodies - Vasopressors such as Norepinephrine, Dopamine and Vasopressin - inotropic therapies, eg Dobutamine - corticosteroids such as hydrocortisone or fludrocortisone - recombinantly expressed activated protein C - fresh frozen plasma, erythrocyte concentrates and/or platelet concentrates Combinations with blood products such as

Another application of this invention is coagulation factor XI and/or coagulation factor Snow probes, snow guards, other plasma products or biological samples containing Xla Anti-coagulation factor XI antibodies and/or anti-coagulant It is the use of coagulation factor Xla antibodies. These examples show that antibodies are an effective such that its concentration is added to prevent in vitro coagulation. is characterized.

Anti-coagulation factor XI antibodies and/or anti-coagulation factor of this invention Xla antibodies also contain coagulation factor XI and/or coagulation factor Xla, preparation of blood catheters or other medical additives or devices For ex vivo inhibition of coagulation, the same for coating artificial surfaces in vivo. medical additives, devices, or other biological specimens used ex vivo can also be used for Example 1: Identification of Antibodies Tools used for phage selections: The proteins of the present invention used for the isolation of human antibodies are listed in Table 1. an approximately 2-fold molar composition of biotin-LC-NHS (Pierce; Cat. No: 21347) It is biotinylated by using excess and Zeba desalination columns (Pierce; Cat. No: 89889), desalinated using

Table 1: List of proteins used in phage selections and screening: Protein Origin Supplier (Cat. No:) hFX human Haematologic Technologies Inc. (HCX-OOSO) Protein Origin hFXa human erX rabbit erXa rabbit hPrekaIIkrein human hKallikrein human Aprotinin bovine Phage Selections: Supplier (Cat. No:) Haematologic Technologies Inc. (HCXA-0060) In-House In-house Enzyme Research Laboratories Enzyme Research Laboratories Sigma (A1153) of human antibodies of the present invention or antigen-binding fragments thereof. DYAX human, a Fab library whose isolation combines natural and synthetic diversity Fab antibody library FAB-310 (DYAX Corp., Cambridge, MA; Hoet et al., Nat. has been carried out. Tables 2 to 6 to select antibodies covering multiple epitopes summarizes the different strategies that have been used.

Selection round: Table 2: Selection strategy 1: Before each round of selection, biotinylated A depletion step is included above Kallikrein/pre-Kallikrein.

Strategy IA Strategy IB 500 nM biotinylated hFXI 200 nM biotinylated hFXI 200 nM biotinylated erXI 100 nM biotinylated hFXI 200 nM biotinylated hFXl 100 nM biotinylated erXl Table 3: Selection strategy II: As described for Strategy I, each of the selection A depletion on biotinylated Kallikrein/pre-Kallikrein ( name is included. In addition, selections include complex hFXIa (/ carried out in the presence of aprotinin (25 pM).

Selection round: Strategy IIA Strategy "B" 1 500 nM biotinylated hFXI 2 200 nM biotinylated hFXI 200 nM biotinylated erXl 3 100 nM biotinylated hFXI 200 nM biotinylated hFXI 4 100 nM biotinylated erXI Table 4: Selection strategy III: Before each round of selection, biotinylated A depletion step is included on Kallikrein/pre-Kallikrein.

Selection round: Strategy IIIA Strategy IIIB 1 500 nM biotinylated hFXIa 2 200 nM biotinylated hFXIa 200 nM biotinylated erXla 3 100 nM biotinylated hFXIa 200 nM biotinylated hFXIa 4 100 nM biotinylated erXIa Table 5: Selection strategy IV: Each of the selections as described for Strategy III before a round of biotinylated Kallikrein/pre-Kallikrein ( depletion step is included. In addition, selections include complex hFXIa (500 nM) / aprotinin (25 µM).

Selection round: Strategy IVA Strategy IVB 1 500 nM biotinylated hFXIa 2 200 nM biotinylated hFXIa 200 nM biotinylated erXla 3 100 nM biotinylated hFXIa 200 nM biotinylated hFXIa 4 100 nM biotinylated erXIa Table 6: Selection strategy V: Before each round of selection. biotinylated Depletion on Kallikrein/pre-Kallikrein ( names are included.

Selection round: Strategy V 1 500 nM biotinylated hFXIa Selection round: Strategy V 2 200 nM biotinylated erXIa 3 200 nM biotinylated hFXla 4 100 nM biotinylated erXIa The standard buffers used in this example are: 1x PBS: from Sigma (D5652-50I) Blocking buffer: PBST fortified with 3% BSA (Sigma A4503) Precipitation buffer: Cell panning-buffer: 3% FBS (GIBCO, 10082) and 0.01% NaNa (Sigma, 71289) supplemented with PBS.

The general method used for library selection, Hoet et al., (Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Neer NC, Nastri HG, Rondon IJ, Leeds JA, Hufton SE, Huang L, Kashin l, Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, Hoogenboom HR, Ladner RC. (2005) Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity.

Nat Biotechnol. 23:344-348). In summary, the Fab antibody library on ice after adding 1/5 volume of precipitation buffer. with one hour of incubation and one centrifugation step (1h at 5500 rpm) precipitated. The precipitated library was then reconstituted in 1 ml of blocking buffer. suspended and incubated for 30 minutes at room temperature.

Also, aliquots of streptavidin-coated Dynabeads M It is prepared by washing 3 times with PBST. After that, as shown in Tables 2 to 6 Some aliquots, such as biotinylated Kallikrein/pre-KaIlikrein (or biotinylated mixed with hFXI ( while the rest is with biotinylated target protein mixed. Mixtures were incubated overnight on an end-to-end rotator at 4°C. and then washed 5 times in 1 ml of PBST. coated balls end by resuspension in 1 ml of blocking buffer, 5 tubes collecting the balls and removing the supernatant. has followed. As shown in the sequential depletion step, blocked Dynabeads coated with biotinylated Kallikrein/pre-Kallikrein ( room when inserting and rotating the (described above) blocked library This was done by incubation at temperature for 10 minutes. on a magnetic shelf After collecting the swatches, the supernatant was clarified by centrifugation. and mixed with blocked Dynabeads coated with the target protein. from one end After 30 minutes of incubation on the tip rotator, the samples were placed in blocking buffer. was washed 3 times with PBST, followed by 9 washes with PBST. Enriched phages Half of the resuspended balls containing preparation of new phage stocks used in the next round of selection according to strategies to infect E. coli TG1 (from Stratagene), which grows exponentially for used. In more detail, 6 ml of TG1-culture without shaking at 37°C for 30 were infected with 500 µl of dynabead/phage suspension for 1 minute. From now on, Aliquots were taken for output titration. Remaining culture at 5000 rpm for 15 min. centrifuged and the resulting pellet resuspended in 2 ml of 2xYT and plated on if plates (2xYT, 100 µg/ml ampicillin, 2% glucose). at 37°C After overnight incubation, cells were scraped into 5 ml of 2xYT and a 0.05 To inoculate a fresh 20 ml culture of 2xYT (100 pg/ml Amp) at OD600 and glycerol used for the preparation of stocks. Fresh liquid culture ODGOO 0.5 to 0.8 It was shaken for about 2 hours at 37°C until it reached the with an infection (MOI) grade of M13 co-fa] M mixed. After 30 minutes of slow shaking at 37°C, the pre-chilled swayed all night. The next morning, the supernatant was centrifuged at 6000 rpm. harvested and clear by filtration through Steriflip ( has been made. Following this, the phages were precipitated as described above and 1 ml of blocking buffer (or cell) for use in the next round of selection resuspended in panning buffer). Aliquots of the input titer was used for the determination.

Enzyme-linked immunity assay (ELISA): Phage ELISA: After different rounds of selection, phage pools biotinylated target proteins. analyzed for specific binder enrichment by ELISA on Summary aliquots from glycerol stocks on 2xYT (100 mg/ml ampicillin, 1% glucose) Plated overnight at 37°C. Single colonies 100 µl medium (2xYT, 100 µg/ml ampicillin, 1% glucose) were collected in MTP wells and stored overnight at 37°C. swayed. Phage expression helper-phage M123KO7 culture overnight in 10 µl and incubation at 37°C.

96-well ELISA-plates pre-coated with streptavidin (Pierce, 15500) 1ug/ml coated with biotinylated target protein overnight at 4°C. The next day, the plates They were washed 7 times with PBST, treated with blocking reagent and they were washed again with PBST 3 times. At the same time, overnight phage cultures were pl was mixed with blocking buffer. After that, 100 µl of blocked phages were added to each transferred to a well and incubated for 1 hour at room temperature. PBST After washing 7 times with anti-M13 antibody coupled to HRP (GE Healthcare, added, 1 hour at room temperature It was incubated throughout and the eyes were washed 7 times again. Color reaction 100 |JI recorded at 450 nm on a reader (Tecan).

Table 7: Hit rates of pools from different strategies in Fab/Phage ELISA: the numbers represent % of hit rate in human / rabbit / both targets (cross-reactive), respectively tu 3 7 4 3 3 Recloning of sFabs for sFab screening with RelIl-removal To generate soluble Fab fragments (sFabs), from the selection rounds 2, Phagemid DNA from 3 and 4 was isolated according to the supplier's instructions and The restriction enzymes were digested with Mlul (New England Biloabs, R0198L). Gen III To remove the containing fragment, vectors were gel-extracted and Ndel (New England Biolabs, RO111S) to a kill-cut. EtOH- After precipitation, the resulting fragment is re-linked and structures chemically transformed into complete E. coli Top10 using standard methods. has been made. Example 2: Anti-coagulation factor XI antibodies and/or anti-coagulation factor Xla antibodies Antibodies, antigen-binding antibody fragments and antibodies of the invention and variants of the fragments include a light chain variable region and a heavy chain variable region. region is included. of antibodies or antigen-binding fragments designed in the invention. coagulation factor XI of the antibody or antigen-binding antibody fragment The binding activity for the velor coagulation factor Xla is maintained. are molecules.

The present invention provides antibodies or antigen-binding fragments I so that the amino acid sequences of the variable heavy and light regions are variable light chain for the domain to SEQ ID NO: 1 for the DNA sequence and SEO ID NO for the amino acid sequence: SEO ID NO: is identical to 2 and 20 for amino acid sequence, or Thus, for matured forms of these antibodies, variable heavy chain and light chain alanine amino acid sequences corresponding to at least 60%, more preferably 70%, more preferably 80% or 90% or even more preferably 95% identical. so the amino acid sequences of the CDRs are the SEQ ID for the DNA sequence for the heavy chain domain. NO: 3, 4, and 5, and for the amino acid sequence, SEQ ID NO: 21, 22, and 23, and for the variable light chain domain to SEQ ID NOs: 6, 7, and 8 for the DNA sequence, and for amino acid sequence at least 60% to SEQ ID NOs: 24, 25 and 26, more preferably is identical.

The present invention additionally provides antibodies or antigen-binding fragments. thus the amino acid sequences of the variable heavy and light regions are variable in the light chain. to SEQ ID NO: 9 for the DNA sequence and SEQ ID NO: 27 for the amino acid sequence. at least 60%, more preferably 70%, more preferably 80% or 90% or even more preferably 95% identical and SEQ ID for DNA sequence for variable heavy chain domain is identical to NO: 2 and 20 for the amino acid sequence, or Thus, for matured forms of these antibodies, variable heavy chain and light chain alanine amino acid sequences corresponding to at least 60%, more preferably 70%, more preferably 80% or 90% or even more preferably 95% identical. thus the amino acid sequences of the CDRs are the DNA sequence for the variable light chain domain. at least 60% to SEQ ID NO: 10 for amino acid sequence and SEQ ID NO: 28 for amino acid sequence, more preferably 70%, more preferably 80%, more preferably 90% or even more preferably 95% identical.

The present invention also provides antibodies or antigen-binding fragments. thus the amino acid sequences of the variable heavy and light regions are variable in the light chain. For alani, refer to SEQ ID NO: 11 for the DNA sequence and SEQ ID NO: 11 for the amino acid sequence. is identical to SEQ ID NO: 12 and 30 for amino acid sequence, or Thus, for matured forms of these antibodies, variable heavy chain and light chain alanine amino acid sequences corresponding to at least 60%, more preferably 70%, more preferably 80% or 90% or even more preferably 95% identical. - so that the amino acid sequences of the CDRs are SEO ID for the DNA sequence for the heavy chain domain for the variable light chain domain to SEQ ID NOs: 16, 17, and 18 for the DNA sequence preferably 70%, more preferably 80%, more preferably 90% or even more preferably Example 3: Using activated partial thromboplastin time (aPTT) analysis determination of anti-coagulation activity Analysis of partial thromboplastin time (aPTT) with activated anti-coagulation activity tested using

Required for doubling aPTT in human and rabbit plasma Values for concentrations are given in Table 8: Table 8: Requirements for doubling the aPTT of human and rabbit plasma antibody concentrations. 2xaPTT human [pM] 2xaPTT rabbit [pM] Table 9: Shows examples and sequences of antibodies of the present invention.

Açiklama Tipi Dizisi H04-VI 1 DNA Açiklama H04-Vh H04 CDR H1 HO4 CDR H2 HO4 CDR H3 H04 CDR L1 HO4 CDR L2 H04 CDR L3 N11OD-VI GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA GGCTTTACCTTTAGCCAGTATGGCATGGAT GGCATTGGCCCGAGCGGCGGCAGCACCGTG GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT Açiklama Tipi Dizisi GATATTCAGATGACCCAGAGCCCGAGCAGCGTGAGC GCGAGCCAGGGCATTAGCAGCTGGCTGGCGTGGTAT CAGCAGCGCCCGGGCAAAGCGCCGAAACTGCTGATT CCTATTATTGCCAGCAGGCGGATAGCTTTCCGATTGC GTTTGGCCAGGGCACCCGCCTGGAAATTAAA GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCGATTATGAAATGGCGTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCAGCATTGTGCCGAGCGGCGGCTGGACCCTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCG CGACCTGGGGCGATA GCTGGGGCTTTGATTTTTGGG H17-Vh 12 DNA DIQMTQSPSS LSASVGDRVTITCOASQD|SNYLNWYOQK H04-VI aa 19 PRT PG KAPKLLlYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQANSFPVTFGGGTKVEIK Açiklama H04-Vh aa H04 CDR H1 H04 CDR H2 H04 CDR H3 HO4 CDR L1 H04 CDR L2 HO4 CDR L3 N11OD-VI aa N110D-CDRL3 H17 -VI aa H17-Vh aa EVOLLESGGGLVQPGGSLRLSCAASGFTFSQYGMDWV ROAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLOMNSLRAEDTAVYYCTRGGPYYYYGMDVWGO GFTFSQYGMD GIGPSGGSTV TRGGPYYYYGMDV QASQDISNYLN DASNLET QQANSFP DIOMTQSPSSLSASVGDRVTlTCQASQDlSNYLNWYQQK PG KAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTlSSLQ PEDIATYYCOQADSFPVTFGGGTKVEIK QQADSFP DIOMTOSPSSVSASVGDRVTlTCRASQGlSSW LAWYQQ RPG KAP KLLlYDASTLQSGVPSRFSGSGSGT DFTLTINSL OPENFATYYCQQADSFPlAFGOGTRLEIK Açiklama Tipi Dizisi EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYEMAWV RQAPGKGLEWVSSIVPSGGWTLYADSVKGRFTISRDNS KNTLYLOMNSLRAEDTAVYYCATWG DSWGFDFWGQGT H17 CDR H1 31 PRT GFTFSDYEMA H17 CDR H2 32 PRT SIVPSGGWTL H17 CDR H3 33 PRT ATWGDSWGFDF H17 CDR L1 34 PRT RASQGISSWLA H17 CDR L2 PRT DASTLQS H17 CDR L3 36 PR T QQADSFPIAFG GAGCGGCTTTACCTTTAGCCGCTATATTATGCATTGG MOO 9-G02-Vh 37 DNA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC CGCGCGAATTTGAAAACGCGTATCATTATTATTATTAT M009-G02-Vl 38 DNA Açiklama G16-Vh G16-VI G11-Vh GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC CGCTTTAGCGGCAGCGGCAGCGGCACCGAATTTACC CTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGA CTTTGGCCAGGGCACCAAACTGGAAATTCGC GAGCGGCTTTACCTTTAGCTGGTATCCGATGCAGTGG GATATTCAGATGACCCAGAGCCCGGCGACCCTGAGC CTGAGCGCGGGCGAACGCGCGACCCTGAGCTGCCG GCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAC CCTTTGGCCCGGGCACCAAAGTGGATATTAAA Açiklama G11-VI MO14-G02-Vh M014- G02-Vl MO13-J04-Vh ACCTATTATTGCCAGCAGAGCTATAGCAACCTGGTGA CCTTTGGCCAGGGCACCCGCCTGGAAATTAAA CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCTGTATTATATGAAATGGG GATATTCAGATGACCCAGAGCCCGAGCAGCGTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGC GCGAGCCAGGATATTAACATTTGGCTGGCGTGGTATC GCGCGGCGAGCACCGTGCAGAGCGGCGTGCCGAGC CCTATTATTGCCAGCAGGCGGCGAGCTTTCCGCTGAC CTTTGGCGGCGGCACCAAAGTGGAAATGAAA ATGGCATGGATGTGTGGGGCCAGGGCACCACCGTGA Açiklama MO13-J04-VI A1 0-Vh A1 O-VI M1 O-Vh TCAGCAGCGCCTGGGCCAGAGCCCGCGCCTGCTGAT GCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAC CCTTTGGCCAGGGCACCCGCCTGGAAATTAAA GAGCGGCTTTACCTTTAGCTGGTATCCGATGCAGTGG CCTTTGGCCCGGGCACCAAAGTGGATATTAAA Açiklama M1 O-VI H15-Vh H15-VI GAGCGGCTTTACCTTTAGCTGGTATCCGATGCAGTGG CCTGACCATTAGCAGCCTGGAACCGGAAGATTTTGCG CCTTTGGCCCGGGCACCAAAGTGGATATTAAA ATGGCATGGATGTGTGGGGCCAGGGCACCACCGTGA CCTTTGGCCAGGGCACCCGCCTGGAAATTAAA Açiklama F11-Vh F11-Vl K12-Vh K12-VI GAGCGGCTTTACCTTTAGCAACTATATGATGACCTGG GAGCGGCATTTATCCGAGCGGCGGCTTTACCCAGTAT GCGAGCGTGGGCGATCGCGTGGCGATTACCTGCCGC ATTATTGCCAGCAGTTTGATGATCTGCCGCTGACCTTT GAGCGGCTTTACCTTTAGCCGCTATATTATGCATTGG CGCGCGAATTTGAAAACGCGTATCATTATTATTATTAT GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGC CGCTTTAGCGGCAGCGGCAGCGGCACCGAATTTACC CTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGA CTTTGGCCAGGGCACCAAACTGGAAATTCGC Açiklama O15-Vh 01 5-VI A08-Vh A08-VI GAGCGGCTTTACCTTTAGCCGCTATATTATGCATTGG CGCGCGAATTTGAAAACGCGTATCATTATTATTATTAT GATATTCAGATGACCCAGAGCCCGAGCAG CCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGC CGCTTTAGCGGCAGCGGCAGCGGCACCGAATTTACC CTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGA CTTTGGCCAGGGCACCAAACTGGAAATTCGC GAGCGGCTTTACCTTTAGCGAATATGGCATGATTTGG GAGCTTTATTAGCCCGAGCGGCGGCACCACCTTTTAT GATAACTTTAAAAACACCCTGTATCTGCAGATGAACAG Açiklama E12-Vh E12-Vl Y111W-Vh GCGAGCCAGGCGATTCGCGATGATTTTGGCTGGTATC CTATTATTGCCAGCAGAGCTATAGCACCCCGCTGACC ATGGCATGGATGTGTGGGGCCAGGGCACCACCGTGA GATATTCAGATGACCCAGAGCCCGGCGACCCTGAGC CTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCG CGCGAGCCAGAGCGTGAGCAGCTATCTGGCGTGGTA TCAGCAGCGCCTGGGCCAGAGCCCGCGCCTGCTGAT GCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAC CCTTTGGCCAGGGCACCCGCCTGGAAATTAAA CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATTATTGGGGCATGGATGT A iklama Ti i Y111W-VI 64 DNA N110D- 65 DNA S111N-Vh N110D- S111N-VI Y109W-Vh 67 DNA GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTAT GGCATGGATTGG GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGGATAACCTGCCGGTGACCTT GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTGGTATTATGGCATGGATGT GTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Açiklama Y109W-VI Y1 1OS-Vh Y1 1OS-VI S111N-F11 2L-Vh GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA G CCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA GTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Açiklama S111N-F112L- P107G-Vh P107G-VI Y1 10R-Vh GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATCG CTATGGCATGGATG TGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Açiklama Tipi Dizisi GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA Y1 10R-VI 76 DNA TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATTGGTATGGCATGGATGT Y11OW-Vh 77 DNA GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA Y11OW_V| 78 DNA TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA Y110N-Vh 79 DNA Açiklama Tipi Dizisi GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATTATCAGGGCATGGATGT Y111Q-Vh 81 DNA GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA Y1 1 1Q-VI 82 DNA TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATTATAAAGGCATGGATGT GTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Y111K-Vh 83 DNA Açiklama Y111K-VI Y111V-Vh Y111V-VI Y1 1OA-Vh GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATTATGTGGGCATGGATGT GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGA TTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCAGTATGGCATGGATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCGGCATTGGCCCGAGCGGCGGCAGCACCGTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCA CCCGCGGCGGCCCGTATTATGCGTATGGCATGGATG TGTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC Açiklama Tipi Dizisi GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC AGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA Y1 1OA-VI 88 DNA TGATGCGAGCAACCTGGAAACCGGCGTGCCGAGCCG CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGACCT ATTATTGCCAGCAGGCGAACAGCTTTCCGGTGACCTT MOO1-G16-Vh 89 DNA GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAG GCGAGCCAGGATATTAGCAACTATCTGAACTGGTATC CTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTT ACCATTAGCAGCCTGCAGCCGGAAGATATTGCGAC CT GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCACCTATGAAATGAACTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCTGGATTGGCCCGAGCGGCGGCTTTACCTTTTAT MOO1-J11-Vh 91 DNA GCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC CGAAAGATAAAGCGGTGGCGGGCATGGGCGAAGCGT TTGATATTTGGGGCCAGGGCACCATGGTGACCGTGA Açiklama M028-H17-Vh M028-H17-VI M067-FO4-Vh ATTATTGCCAGCAGTTTTATAACCTGCCGCTGACCTTT CCTATTATTGCCAGCAGGCGGATAGCTTTCCGATTGC GTTTGGCCAGGGCACCCGCCTGGAAATTAAA CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGC GAGCGGCTTTACCTTTAGCCCGTATGATATGTATTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCTATATTTGGAGCAGCGGCGGCATTACCCAGTAT GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCG Açiklama M067-F04-Vl M067-CO4 -Vh M067-CO4-VI MO71-F17-Vh GCGAGCCAGAGCATTAGCAGCTATGTGAACTGGTATC TGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGAC CAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGCGCGGC GTGCAAAGGCCTTGAATGCGGCAAAGGCCTTGAATGCGCGAGGCCTGGAATGCGCGAGGCCTTGAATGCGCGAGGCCTGGAATGCGCGAGGCCTGGAATGCGCGAGGCTGAGATTCCCGCGCGCGAGATTCCCGCGCGTGGATGGACGCGCGGATGGATGCGCGCGAGATTCCCGCGCGAGATTCCCGCGCGAGATT TTTTGCGACCT ATTATTGCCAGCAGTATCATACCCTGCCGCCGCTGAC GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTG GAGCAGCATTTATAGCAGCGGCGGCTGGACCGATTAT GATAACAGCAAAAACACCCTGTATCTGCAGATGAACA GCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCG Açiklama M071-F17-VI H17-R47 K-Vh H17-R47 K-VI H1 7-T698-Vh GCGAGCCAGAGCATTAGCAGCTATCTGAACTGGTATC CTATTATTGCCAGCAGAGCTATAGCACCCCGCCGTGG GAGCGGCTTTACCTTTAGCGATTATGAAATGGCGTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGT GAGCAGCATTGTGCCGAGCGGCGGCTGGACCCTGTA TGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATATTCAGATGACCCAGAGCCCGAGCAGCGTGAGC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGC CAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTT GCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCC TGACCATTAACAGCCTGCAGCCGGAAAACTTTGCGAC TTTGGCCAGGGCACCCGCCTGGAAATTAAA Açiklama H17-T69S-VI H17- N100D- H17-N100D-VI H17-A115T-Vh CCTATTATTGCCAGCAGGCGGATAGCTTTCCGATTGC GTTTGGCCAGGGCACCCGCCCTGGAAATTAAA GAGCGGCTTTACCTTTAGCGATTGAAATGGCGTGG GTGCGAAGCAGGCGCCGGGCGGATAGCTTGGAATGGGCTCGCTGGAGCTGCTGAGCTGCTGCTGGACG1GCTGGAGCTGAGCTGAGGAG 1 -VI H17-R47 K-Vh H17-R47 K-VI CCTATTATTGCCAGCAGGCGGATAGCTTTCCGATTAC GCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGC CAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTT TTTGGCCAGGGCACCCGCCTGGAAATTAAA EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYI MHWVR QAPGKGLEWVSS lSPSGGLTSYAD SVKGRFT ISRDNS K NT LYLQMNS LRAEDTAVYYCARE FENAYHYYYYGM DV WGQGTTVTVSS Açiklama G16-Vh G16-VI G11-Vh G11-VI M014-G02-Vh M014 -G02-Vl MO13-J04-Vh DIQ MTQS PSS LSASVGDRVTITCRASGDI GNALGWYQQ KPGKAP RLLISDASTLQSGVPLRFSGSGSGTEFTLTISSL Q PE D FATYYC LQGYNYP RTFGQGTKLElR EVOLLESGGGLVQPGGSLRLS CAASGFTFSWYPMQWV RQAPG KGLEWVS G ISSSG GGTYYADSVKG RFTIS RDN S KNTLYLQM N S LRAEDTAWYCARDWGYSNWMD LGLD YWGOGTLVTVSS Dl OMTQS PATLSLSAG ERATLS CRASQTVSSSLAWYQ H KPGQAPRLLIYETSNRATGIPARFSGSGSGTDFTLTISSL EPE DFAVYYCQHRS NWPPTFGPGTKVDIK EVQLLESGGG LVQPGGSLRLSCAASG FTFSTYSMGWV RQAPG KG LEWVSSISPSGGDTDYADSVKG RFTIS R DNS KNTLYLQMNSLRAEDTAVYYCARERTMVRDPRYYGMD VWGQGTTVTVSS DlQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ RLGQSPRLLIYDASSRATGIPARYFSGGSCQTDQFTGLT LEI K EVQLL ESGGGLVOPGGS LRLSCAASGFTFS LYYM KWVR QAPG KG LEWVSSISPSGGFTSYADSVKGRFTISRDNSK NTLY LQMNS LRAEDTAVYYCAR E FE NAYHYYYYGMDV WGQGTTVTVSS DIQMTQS PSSVSASVGDRVTITC RASQDl NIWLAWYQQK PG KAPKLLISAASTVQSGVPSRFSGS GS GTD FTLTINTLQ PDDFATYYCQQAASFPLTFGGGTKVEMK Açiklama MO13-J04-VI A1 O-Vh A1 O-Vl M1 O-Vh M1 O-Vl H15 -Vh EVQLLESGGG LVQPGGSLRLSCAASG FTFSTYSMGWV RQAPG KG LEWVSSISPSGGDTDYADSVKG RFTlS R DNS KNTLYLQMNSLRAEDTAVYYCARERTMVRDPRYYGMD VWGQGTTVTVSS DlQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ RLGQSPRLLIYDASSRATGI PARFSGSGSGTDFTLTI SSL QP KDFATWCQQSYS N LVTFGQGTRLEI K EVOLLESGGGLVQPGGSLRLS CAASGFTFSWYPMOWV RQAPG KGLEVWS G ISSSG GGTYYADSVKG RFTIS RDN S KNTLYLQM N S LRAEDTAVYYCARDWGYSNYVMD LGLD YWGOGTLVTVSS Dl QMTQS PATLSLSAG ERATLS CRASQTVSSSLAWYQ H KPGQAPRLLIY ETS N RATG l PARFSGSGSGTDFTLTISSL EPE DFAVYYCQHRS NWPPTFGPGTKVDIK EVQLLESGGGLVQPGGSLRLS CAASGFTFSWYPMQWV RQAPG KGLEWVS G lSSSG GGTYYADSVKG RFTIS RDN S KNTLYLO M N S LRAEDTAWYCARDWGYSNWATLS CLDAGQSS PLS RASQTVSSSLAWYQ H KPGQAPRLLIYETSNRATGlPARFSGSGSGTDFTLTISSL EPE DFAVYYCOHRS NWPPTFGPGTKVDIK EVQLLESGGG LVQPGGSLRLSCAASG FTFSTYSMGWV RQAPG KG LEWVSSISPSGGDTDYADSVKG RFTIS R DNS KNTLYLQMNSLRAEDTAWYCARERTMVRDPRYYGMD VWGQGTTVTVSS Açiklama H15-VI F11-Vh F11-Vl K12-Vh K12-VI 015-Vh O1 5-VI DlQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ RLGQSPRLLIYDASSRATGIPARFSGSGSGTDFTLTISSL QPEDFATYYCQQSYSN LVTFGQGTRLEI K EVQLLESGGGLVQP GGS LRLSCAAS GFTFSNYM MTWV RQAP G KGLEWVSGIYPS GG FTQYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTATYYCARDASDVWLRFRGGGAF DIWGQGTMVTVSS DIOMTQSPTSLSASVG DRVAITCRASQS l DTYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQFDDLPLTFGPGTRVDIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYl MHWVR QAPGKGLEWVSSlSPSGGLTSYADSVKGRFTISRDNSK NT LYLQMNS LRAEDTAVYYCARE FENAYHYYYYGM DV WGQGTTVTVSS DIQ MTQS PSS LSASVGDRVTITCRASGDI GNALGWYQQ KPGKAP RLLISDASTLQSGVPLRFSGSGSGTEFTLTISSL Q PE D FATYYC LQGYNYP RTFGQGTKLEIR EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYl MHWVR QAPGKGLEWVSSlSPSGGLTSYADSVKGRFTISRDNSK NT LYLQ MNS LRAEDTAVYY CARE FENAYHYYYYGM DV WGQGTTVTVSS DIQMTQSPSSLSASVGDRVTITCRASGDI GNALGWYQQ KPGKAPRLL|SDASTLQSGVPLRFSGSGSGTEFTLTISSL QPEDFATYYCLQGYNYP RTFGQGTKLEIR Açiklama A08-Vh A08-VI E12-Vh E12-Vl Y111W-Vh Y111W-VI N110D- S111N-Vh EVQLLESGGGLVQPGGSLRLSCAASGFTFSEYGMIWVR QAPGKGLEWVSFlSPSGGTTFYADSVKGRFTISRDN FKN TLYLOMNSLRAE DTAVYYCARGGGNWNHRRALNDAFDI WGQGTMVTVSS DlQMTQSPSSLSASVGDRITITCRASQAIRDDFGWYQQK PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQSYSTPLTFGGGTKVEIK EVQLLESGGG LVQPGGSLRLSCAASG FTFSTYSMGWV RQAPG KG LEWVSSISPSGGDTDYADSVKG RFTIS R DNS KNTLYLQMNSLRAEDTAVYYCARERTMVRDPRYYGMD VWGQGTTVTVSS DIQMTQS PAT LSLS PG ERATLSCRASQSVSSYLAWYQQ RLGQSPRLLIYDASSRATGl PARFSGSGSGTDFTLTI SSL QP EDFATYYCOQSYS N LVTFGQGTRLEI K EVQ LLESGGG LVQPGGSLRLS CAASGFTFSQYGMDWV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTIS RDNS KNTLY LQMN SLRAE DTAVYYCTRG GPYYYWGMDVWG DIQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWYQQK PGKAPKLLIYDASN LETGVPS RFSGSGSGTDFTFTISSLQ EVOLLESGGGLVQPGGSLRLSCAASGFTFSQYGM DWV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYYYGMDVWGQ Açiklama N110D- S111N-VI Y109W-Vh Y109W-VI Y1 1OS-Vh Y1 1OS-VI S111N-F112L- S111N-F112L- DlQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWY QQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQADNL PVTFGGGTKV El K EVO LLESGGG LVQPGGSLRLS CAASGFTFSQYGMDWV RQAPGKGLEWVSGlGPSGGSTVYADSVKGRFTlS RDNS KNTLYLQMN SLRAE DTAVYYCTRG GPYWYYGMDVWG DIQMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQANSFPVTFGGGTKVEIK EVO LLESGGGLVQ PGGSLRLS CAASGFTFSQYG M DWV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYSYGMDVWGQ DIQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWYQQK PGKAPKLLIYDASN LETGVPS RFSGSGSGTDFTFTISSLO PEDIATYYCQQANS F PVTFGGGTKV EIK EVQLLESGGGLVOPGGSLRLSCAASGFTFSQYGMDWV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYYYGMDVWGQ DIQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWYQQK PGKAPKLLIYDASN LETGVPS RFSGSGSGTDFTFTISSLO Açiklama P107G-Vh P107G-VI Y1 10R -Vh Y110R-VI Y1 1OW-Vh Y1 1 OW-Vl Y110N-Vh EV Q LLESGGGLVQPGGSLRLSCAASGFTFSQYGMDWV RQAPGKGLEWVSGI GPSG GSTVYADSVKG RFTISRDNS KNTLYLQMN S LRAE DTAVYYCTRGGGYYYYGM DVWGQ DlQMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQANSFPVTFGGGTKVEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYGMDWV RQAPGKGLEWVSGI G PSG GSTVYA DSVKG RFTIS RDN S KNTLYLQMNSLRAEDTAVYYCTRGGPYYRYGMDVWGQ DIOMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLO PEDIATYYCQQANSFPVTFGGGTKVEIK EVO LLESGGG LVQPGGSLRLS CAASGFTFSQYGMDWV RQAPG KGLEWVSG I GPSGGSTVYADSVKGRFTIS RDNS KNTLY LQMN SLRAE DTAVYYCTRG GPYYWYGMDVWG DIQMTQSPSS LSASVGDRVTITCOASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLQ PEDIATWCQQANS F PVTFGGGTKV El K Açiklama Tipi Dizisi EVQLLESGGGLVOPGGSLRLSCAASGFTFSQYGMDWV RQAPG KG LE WVSGI G PSG GSTVYADSVKG RFTIS RDN S KNTLYLOMNSLRAEDTAVYYCTRGGPYYNYGMDVWGQ DIQMTQSPSS LSASVGDRVTlTCQASQDISNYLNWYQQK Y110N-VI 154 PRT PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATWCQQANSFPVTFGGGTKVEIK EVQLLESGGGLVQPGGSLRLSCA ASGFTFSQYGMDWV RQAPGKGLEWVSGI GPSG GSTVYADSVKGR FTISRDNS KNTLYLQMN S LRAEDTAVYYCTRGG PYYYQGM DVWGQ Y111Q-Vh 155 PRT DIQMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK Y111Q-VI 156 PRT PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTlSSLQ PEDIATYYCQQANSFPVTFGGGTKVEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYGMDWV ROAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYYKGMDVWGQ Y111K-Vh 157 PRT DlQMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK Y111K-VI 158 PRT PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQANSFPVTFGGGTKVEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYGMDVVV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYYVGMDVWGO Y111V-Vh 159 PRT Açiklama Y111V-Vl Y1 10A-Vh Y1 10A-VI DlQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWYQQK PGKAPKLLIYDASN LETGVPS RFSGSGSGTDFTFTISSLQ PEDIATYYCQQANS F PVTFGGGTKV El K EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYGM DWV RQAPGKGLEWVSGIGPSGGSTVYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCTRGGPYYAYGMDVWGQ DlQMTQS PSS LSASVGDRVT] TCQASQD l SNYLNWYQQK PGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLO P EDIATYYCQQANS F PVTFGGGTKV El K EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYWMTWV RQAPGKG LEWVSSIWSSG GWTLYADSVKGRFTIS RDNS KNTLYLQMNSLRAEDTAVYYCAREVGAAGFAFDIWGQG DIQMTQSPSS LSASVGDRVTITCQASQDISNYLNWYQQK PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQSSSTPLTFGGGTKMEIK EVOLLESGGGLVOPGGSLRLSCAASGFTFSTYEMNWV RQAPGKGLEWVSWI G PSGGFTFYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCAKDKAVAGMGEAFDlWG QGTMVTVSS DlQMTOSPSSLSASVGDRVTlTCQASQDßlYLNWYQQK PGKAPKLLIYDASNVETGVPSRFSGSGSGTDFTFTISSLQ PEDlATYYCQOFYNLPLTFGGGTKVEIK Açiklama M028-H17-Vh M028-H17-Vl M067-F04-Vh M067-F04-VI M067-CO4-Vh M067-C04-Vl MO71-F17-Vh EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYEMAWV RQAPGKGLEWVSSIVPSGGWTLYADSVKGRFTISRDNS KNTLYLOMNSLRAEDTAVYYCATWGDSWGFDFWGQGT Dl Q MTTCQSSGVT | SSWLAWYQQ RPG KAP KLL lYDASTLQSGVPS RFSGSGSGTDFTLTl NS L OPEN FATWCQQADSFPIAFGQGTRLEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYDMYWV RQAPG KG LEWVSYIWSSGGITQYA DSVKGR FTIS RDNS KNTLYLQM NSLRAEDTAVYYCARHASYYDSSGRP DAF D DIQMTQS PSS LSASVGDRVTITC RASQS | SSYVNWYQQK PGKAPNLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQ PE DFATWCQQSYSTPYTFGQGTKL DlK EVO LL ESGGGLVQPG GS L RLSCAASG FTFSHYSMQWV RQAPGKGLEWVSSISPSGGYTMYADSVKGRFTISRDNS KNTLYLQMNS LRAEDTAMYYCAREKAS DLSGTYSEAL D YWGQGTLVTVSS DlQMTQS PSSLSASVGDRVTITCQASQDIDYYLNWYQQ QPGKAPQLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL HPEDFATYYCOQYHTLPPLTFGGGTKVDlK EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYWMHWV RQAPGKGLEWVSSIYSSGGWTDYADSVKGRFTISRDNS KNTLYLQM NSLRAEDTAVYYCAREGVAGTN DAFD lWGQ Açiklama M071-F17-VI H17-R47 K-Vh H17-R47 K-VI H1 7-T698 -Vh H17-T69S-Vl H17-N100D- H17-N1OOD-VI DlQMTQSPLSLSASVGDRVTlTCRASQSlSSYLNWYQQK PGKAPKLLlYAASSLQSGVPSRFSGSGSGTDFTLTlSSLQ PEDFATYYCQQSYSTPPWTFGQGTKVEI EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYEMAWV RQAPGKGLEWVSSlVPSGGWTLYADSVKGRFTlSRDNS KNTLYLQMNSLRAEDTAVYYCATWGDSWGFDFWGQGT DIQ MTQSPSSVSASVGDRVTlTCRASQGISSWLAWYQQ KPG KAP KLLlYDASTLQSGVPSRFSGSGSGTDFTLTI NS L OPEN FATYYCQQADSFPIAFGQGTRLEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYEMAWV RQAPGKGLEWVSSlVPSGGWTLYADSVKGRFTISRDNS KNTLY LQMNS LRAEDTAVYYCATWGDSWG F DFWGQGT Dl QMTQS PSSVSASVGD RVTlTCRASQGISSWLAWYQO RPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTI NSL OPEN FATYYCQQADSFP lAFGQGTRLEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYEMAWV RQAPGKGLEWVSSlVPSGGWTLYADSVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCATWGDSWGFDFWGQGT Dl Q MTOSPSSVSASVG DRVTlTCRASQG | SSWLAWYQQ RPG KAP KLLlYDASTLQSGVPS RFSGSGSGTDFTLTINSL OPEDFATYYCOQADSFPIAFGQGTRLEIK Açiklama Tipi Dizisi EVQLLESGGGLVQPGGSLRLSCAASGFTFS DYEMAWV RQAP G KGLEWVSSIVPSGGWTLYA DSVKGRFTISR DNS KNTLY LQIVINS LRAEDTAVYYCATWGDSWG FDFWG QGT H17-A115T-Vh 181 PRT DlQMTQSPSSVSASVGDRVTITCRASQG|SSWLAWYQQ H17-A115T-VI 182 PRT RPG KAPKLLIYDASTLQSGVPS RFSGSG SGTDFTLTINSL OPENFATYYCQQADSFPITFGQGTRLEIK EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYEMAWV RQAPGKGLEWVSSlVPSGGWTLYADSVKGRFTlSRDNS KNTLYLQMNSLRAEDTAVYYCATWGDSWGFDFWGQGT H17-R47K- Vh 183 PRT DIQMTQSPSSVSASVGDRVTlTCRASQG[SSWLAWYQQ H17-R47K-VI 184 PRT KPGKAPKLLlYDASTLOSGVPSRFSGSGSGTDFTLTINSL OPEDFATYYCQQADSFPIAFGQGTRLEIK Örnek 4: FXla antikorunun anti-agregatör aktivitesinin belirlenmesi Akis kosullari altinda platelet aktivasyonunun ölçümü için, cam Iamlar (Menzel-Glaser SUPERFROST 76 X 26 mm; Gerhard Menzel GmbH, Braunschweig , Germany) was coated with collagen (150 µg/ml) overnight at 4°C, followed by a Zeiss Axiovert 135 microscope (Carl Zeiss, Göttingen, Germany). a) followed by blocking with BSA (5 mg/ml) before being incorporated into a flow system on the object table. Citrated whole blood was incubated at 37°C for 10 minutes with GPRP (final 3 mM) and vehicle or FXI antibodies. After addition of CaCl2 (5 mM), blood was immediately perfused over collagen-coated tubes at an initial drag rate of 1000 S' 1 for 5 min. After perfusion of whole blood as described above, post-compartment whole blood was pooled every 1 minute in sodium citrate (1:10 v/v). treated for 5 minutes with or without it.

Pre- and post-compartment blood samples Cell Wash (BD Biosciences, Heidelberg, Germany) and incubated with antibodies for 20 minutes at 4°C.

The antibody reaction was stopped by the addition of ice-cold-Cell Wash and all Samples were kept on ice until measurement. 10000 single platelets, FITC-conjugated platelet marker (CD41a or CD61a) positivity and flow cytometry (FACSCaIibur; BD Biosoiences, Heidelberg, Germany) with characteristic light scattering determined by patterns. For CD62P expression, single platelets, unlabeled with a PE-CD62P fluorescence threshold validated with control samples. is captured by the scatterplot. Activation of the platelet population above the threshold value They are considered to have been quantified and these have been quantified. Platelet microaggregates It is defined by arbitrary threshold-values for Front Scatter (FSC) and FITC-fluorescence. Example 5: Time of FeCl2-induced thrombosis and ear hemorrhage in rabbits. its antithrombotic activity was determined in an arterial thrombosis model. An i.v. bolus mg/kg) administration, a combination with ferric chloride was observed in thrombosed rabbits 15 minutes after administration. induced by chemical damage to the carotid artery. To male rabbits (CrI:KBL (NZW)BR, Charles River) i.m. xylazine and ketamine given by injection (Rompun, Bayer 5 mg/kg and Ketavet Pharmacia & Upjohn GmbH, 40 mg/kg body weight) has been euthanized. Supplementary anesthesia anesthetic mixture into the marginal vein of the right ear administered by infusion. After exposure of the right common carotid artery, vascular A Parafilm® is placed under the right common carotid artery without affecting the damaged blood flow. A piece of blotting paper (10 mm x 10 mm) on the (25 mm x 12 mm) strip produced by insertion. Blotting paper 100 µl FeCl2 (Iron(II) chloride tetrahydrate), A. 13% in dest, Sigma). After 5 minutes, filter paper was removed and the vessel was rinsed twice with 0.9% NaCl. 30 without injury minutes later, the carotid artery was removed, the thrombus was withdrawn and immediately weighed. -7 animals were used for each group. Ear bleeding time, FeClz- determined 2 minutes after injury. The left ear is shaved and a standardized incision (3 mm in length), parallel to the long axis of the ear with a surgical blade (number 10-150-10, Martin, Tuttlingen, Germany) has been made. Care was taken to avoid damage to visible blood vessels. incision sites were blotted with filter paper at 30-minute intervals, during which time they were removed from contact with the wound. has been carefully avoided. At the time of bleeding, the blood from the incision is now removed from the filter paper. It was determined by measuring the time until no dyeing. Example 6: Time of FeClz-induced thrombosis and ear bleeding in rabbits determination. It reduces the amount of ear bleeding and does not prolong the time of ear bleeding. Figure 15, ear bleeding no elongation is shown. Example 7: Crystal Formation, Crystallization and Fab O76D-M007-H04:FXla Determination of the X-ray Structure of the complex.

Complex formation and crystallization.

The solution was stored on ice for 18 hours to allow for complex formation.

The complex solution was loaded onto a Superdex 200 HR 16/60 column and additionally the pH To a final concentration of 20 mg/ml in 20 mM Tris/HCl and 75 mM NaCl at 7.5 crystals of the complex were grown at 20°C using the sit-drop method and of protein complex solution and eye solution as precipitator (100mM TRIS pH 8.25, A rosette-like crystal was seen after about five days.

Data Collection and Processing.

The crystal was snap-frozen in liquid nitrogen without the use of cryo-buffer. crystalline BL burn data on a MAR CCD detector collected in its beam path. Data is indexed and with POINTLESS Orthorhombic space with a Fab 076D-M007-HO4:FXIa CSOOS complex in asymmetric unit belongs to group P2(1)22(1).

Identification and improvement of the structure.

The complex-structure of FXIa and the Fab 076D-M007-H04 monoclonal antibody, different solved by molecular displacement in steps. First, the H-chain, the research model using the 3GJE pdb code as BALBES (F.L0ng, A.Vagin, P.Y0ung and Subsequently, FXIa 05008 is MolRep with an internal FXIa crystal structure as the research model. added using the program. REFMACS.5 (G.N. Murshudov et al., (1997) Acta has been concluded. Finally, the H-chain is used as the research model to use the L- of the 3IDX pdb input. chain and fixation of the first cured H-chain and FXIa CSOOS solution. located using coordinates. Iterative of model building with COOT the maximum possible improvement model has been completed. Dataset and optimization statistics are summarized in Table 10.

Wavelength 0.91823 Ä Tamlika 998% (99.15%) Rbmesmea'b 0.12 (0.43) Space group P2(1)22(1) Unit cell parameters a 61.94Ä b 87.68Ä c 185.89Ä erisiaic 0.228 Rfreed 0.305 Wilson temperature factor 51.7 Ä2 RMSD bond lengthe 0.022 Ä RMSD bond angles 1.95° protein atoms 5042 Water molecules and solvent molecules 34 ' Values in parentheses are for high resolution sheath. where Ihki is the density of right reflection and first” is the concentration of a large number of observations to the medium. ° eristai = Z |Feye-Faccount| / Z Feye, where Feye and Feye, respectively, observed and are the calculated structure factor widths. d 5% test set e RMSD, root mean square deviation from parameter set for ideal stereochemistry EXAMPLE 8: X-ray Structure-Based Epitope Mapping.

Fab O76D-M for each asymmetric unit crystallized as a copy of the complex. In contact with FXla 05008 (epitope) Residues of Fab 076D-M007-H04 (paratope) have been identified and are in Table Xa and Xb. is listed. Embedded surface CCP4 program AREAIMOL (P.J. Briggs (2000) CCP4 Newsletter No. 38) and residues, bound Ile and bound Fab, respectively. when calculated without shows the total area difference.

Table 11a: Residues of FXIa in contact with Fab 076D-M007-HO4 Residue Number Area Differences Table 11b: Residues of Fab O76D-M007-H04 in contact with FXIa Paratogue: Residual Number Area Differences -4.50 -15.90 -87.20 -21.80 -18.30 -38.10 -33.50 -12.50 -36.80 -1 1.80 -41.30 -27.80 -60.20 -19:00 -15.00 -3.90 -5.60 -7.20 -10.20 -13.30 -3.00 Paratogue: Residue Number Area Differences In summary, the FXIa 05008 epitope is generated by the following residues: Fab 076D-M007-H04 acts as an allosteric competitive inhibitor. This FXIa is active it does not block its location but connects it adjacent to it. This contiguous binding is the active site of FXIa. triggers a re-sequence of these natural substrates to activate FXIa (Figure 18).

In contrast, Fab 076D-M007-H04 does not bind Zimogen FXI. Reported x-ray of Zimogen FXl the various folds that make it are not arranged properly. Specifically, the epitope region Fab O76D- M007-H042FXIa is well constructed in the C500$ complex. Example 9: Hydrogen/Deuterium-Exchange Mass Spectrometry-based Epitope mapping.

A different analysis of epitope mapping, contract research organization arExSAR USA]. In this case, FXla C5OOS (amino acids 388 - 625; Purchased from Proteros Biostructures) and 076D-MOO7- respectively analyzed by hydrogen/deuterium exchange mass spectrometry method [review for Percy AJ, Rey M, Burns KM, Schriemer DC. (2012) Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry-a review. Anal Chim Acta. ?21:7-21]. Thus, deuterium level differences of more than 10%, indicates a strong protection of the corresponding antigen by Fab. 5% to 10% Values between indicate weak binding, below 5% deuterium level differences indicate no protection at all.

Summarizes residues of Fab 076D-MO49-O15.

Table 12a: Residues of Fab O76D-M007-H04 in contact with FXIa Residue Number mean deuterium level difference (%) Residue Number mean deuterium level difference (%) Residue Number mean deuterium level difference (%) Residue Number mean deuterium level difference (%) Residue Number mean deuterium level difference (%) Residue Number mean deuterium level difference (%) These data contain an epitope of 200 amino acids in FXIa (408 to 609 of FXIa CSOOS). to an inhibition of the FXIa proteolytic activity of the coating. clearly shows that it does. Example 10: Functional neutralization of FXIa by antibodies of this invention Human FXIa (Haematologic Technologies, Inc., catalog number HCX1A-0160) activity of a specific, fluorogenically labeled substrate (I-1575, Bachem, final concentration is determined by measuring 25 µM) cleavage and fluorescence is determined by a SpectraFIuorplus It is continuously monitored at 3601465 nm using the Reader (Tecan). Test for inhibitory activity antibodies containing 50mM TrIs/HCl, 100 mM NaCl, 5 mM CaCl2 and 0.1% BSA. 60 minutes at 37°C with a final concentration of 10 nM of FXIa in a buffer pre-incubated throughout. For this incubation step, substrate I-1575 is added and signals from the reaction are measured. Data in figure 1, figure 2, figure 3 and figure 4 analyzed using the GraphPadPrism software as shown. Data, mean ± The SEM is given as n=4. For some experiments, full-length human FXIa (Haematologic Technologies, Inc., catalog number HCXIA-0160), the isolated FXIa C5008 decomposed catalytic domain (amino acids 388 - 625; purchased from Proteros Biostructures taken) is used. All other conditions are as described above. Example 11: Conversion of FXI to its active form, FXIa, functional neutralization by antibodies.

FXI (Haematologic Technologies, lnc., catalog number HCXIA-0150) its conversion to FXIa by FXIIa or Thrombin (Thrombin is Ila) To test inhibition of antibodies, 10 nM human FXI was used for 1 hour at 37°C. with different concentrations of 50 mM Tris/HCl, 100 mM NaCl, 5 mM CaCl2 and 0.1% BSA incubated in. In the next step, human FXIIa (Enzyme Research, catalog number HFXIIa 1212a) has a final concentration of 10 nM or a 1 unit/mg final concentration of Thrombin (Enzyme Research, catalog number HT 1002a) is added and incubated at 37°C for 24 hours. Then at a final concentration of 200 nM Labeled as Corn Trypsin Inhibitor (Enzyme Research, CTI) and fluorogenic substrate (I-1575, Bachem, final concentration 25 µM) is added. fluorescence, a It is continuously monitored at 360/465 nm using the SpectraFIuorplus Reader (Tecan).

The data is shown in figure 5 for FXI's FXIIa-mediated conversion to its activated form FXIa. and in figure 7 and for the Thrombin-induced conversion of FXI to its active form, FXIa. Analysis using GraphPadPrism software as shown in figure 6 and figure 8 is done. Data are given as mean ± SEM, n= 4. Example 12: Anti-FXla antibody in an experimental thrombosis in an in vivo model in primates Evaluation of the anti-thrombotic activity of O76D-M007-H04.

Experimental Procedures Experiments were performed on awake juvenile baboons weighing 9 -11 kg, which were not anti-coagulated. has been made. These animals are elsewhere (Hanson et al., (1993) Journal of Clinical He has implanted chronically displaced arterio-venous (AV) shunts. baseline Cental blood flow exceeded 250 ml/min in all study animals. low anxiety dose was administered with ketamine (< 2mg/kg/hr). Complete blood cell counts from experiment measured daily before and after. Calculated blood loss, any experimental did not exceed 4% of the total blood volume per day.

Thrombus formation has been described previously (Hanson et al., [1993] J. Clin. Invest. The baboon is launched within the AV centi. Consistently monitor platelet-dependent thrombus formation. The clinical graft segment was coated with immobilized collagen to trigger 2 or Twenty-mm-long grafts with internal diameters (i.d.) of 4 mm were treated for 15 minutes. with equine type I collagen (1 mg/ml; Nycomed Arzenmittel, Munich, Germany) filled and then dried overnight under a flow of sterile air. This The method consists of a single layer in the graft lumen as determined by scanning electron microscopy. produced a flat collagen coating. In addition, in some applications, the average A 200 with 9 mm i.d. was used to model the venous and arterial drag velocities, respectively. 20 mm long graft with 4 mm i.d. thrombogenic The collagen-coated grafts are then joined between segments of silicone rubber tubes and It is deployed on AV systems. Grafts are exposed to blood for a maximum of 60 minutes. has been abandoned. During each experiment, the blood flow rate through the graft was determined by the proximal silicone rubber core segment clamping, thus initial MWSR on 2mm grafts 2120/sec vs. an average wall drag speed of 265/sec on 4mm grafts (MWSR) generation is limited to 100 mI/min. Flow rates of an ultrasonic flow meters (Transonics Systems, Ithaca, N.Y.). 4 mm grafts are not occluded and pulsatile flow rates, thrombogenic graft segments 60 remained at 100 ml/min until removed per minute. Baseline blood flow After the experiment, it was restored by permanent shunt. 2 mm diameter grafts, Flow rates decreased progressively due to thrombus formation. grafts, flow rate, From 100 ml/min to less than 20 ml min, signaling possible occlusion When it fell, it was removed from the AV shunts. From the initiation of blood flow, the graft The time until its removal (<20 ml/min blood flow) is defined as the occlusion time. has been taken.

For visualization of platelet deposition, autologous baboon patelets were pre-assembled. labeled with auxin. Labeled platelets have been infused and studies have been They were allowed to circulate for at least 1 hour before being carried out.

Deposition of labeled platelets on thrombogenic grafts and silicone chambers Measured at 5-minute intervals using a gamma scintillation camera. Homologous Baboon fibrinogen labeled with 125I (4 uCi, >90% coagulated) 10 from each study infused minutes ago and incorporation of labeled fibrin into the thrombus 111In using a gamma counter > 30 days late to allow for degradation has been evaluated. Stored radioactivity (cpm), taken at the original runtime divided by the coagulating fibrin(ogen) radioactivity (cpm/mg) of the samples.

Occlusion studies, high initial wall drag rates (at 100 mI/min) 2120/sec clamped blood flow) 20 mm long, collagen-coated with 2 mm i.d. performed using devices. Thrombogenic 2 mm of labeled platelets 3-minute deposition on grafts using a gamma scintillation camera measured at intervals. Flow is proximal for as long as possible. maintained at 100 mI/min by clamping, and subsequently the proliferating thrombus device It is allowed to fall as it starts to clog. A fully occlusive thrombi and device The absence of snow flow throughout the entire length causes centn occlusion and no significant blood flow for animals. A final blood flow rate of 20 mI/min is a prerequisite for Occlusion, as used as cut-off value.

Blood sample analysis. Blood cell counts with a micro-60 automated cell counter (Horiba-ABX Diagnostics). Blood samples 032% sodium collected into a final concentration of citrate. 12,900 in all samples 5 minutes out of 9 were centrifuged throughout and plasmas were collected and stored at minus 80°C. Cross-reacting ELISA analyzes reveal thrombin-antithrombin complexes (TAT, Enzygnost-TAT, Dade-Behring; LCD: 2 ng/mL). This All ELISA test kits used for the studies were pre-loaded with baboon markers. has demonstrated sensitivity.

In interruption studies (collagen-coated graft with 4 mm i.d. only), O76D-M007-H04, this to determine whether the antibody can interrupt acute thrombus proliferation. A bolus of 30 minutes was administered to the study (0.5 mg/kg i.v. for 10 seconds). bolus). In occlusion studies (only in collagen-coated graft with 2mm i.d.). O76D- M007-H04 was administered as a bolus 3 hours before the experiment (i.v. a 0.5 mg/kg dose). 0.5 mg/kg or 2 mg/kg for 24 hours following the dose). In prevention work (4mm i.d. administered as a bolus one hour prior (24 hours following an i.v. dose of 0.5 mg/kg) 0.5 mg/kg or 2 mg/kg).

Hemostatic evaluation. Primary hemostasis of FXIa inhibition in baboons effects on the standard template skin bleeding time test (Surgicutt®, International Evaluated using Technidyne Corp. Experimentally, this and similar tests (for example, Simplate bleeding times) in humans and non-human therapeutic anti-coagulants, anti-platelet agents and coagulation in primates They have been shown to be sensitive to the effects of abnormalities (Gruber et al., [2007] Blood carried out by the technician. For indirect assessment of hemostasis, aPTT (activated partial thromboplastin time; SynthASil, HemosIL; Instrumentation Laboratory Company, Bedford, MA) and ACT (activated clotting time, LupoTek KCT; r2 Diagnostics, South Bend, IN) measurements were also made before the experiment. performed at various time-points during and after.

In vitro aPTT. Various concentrations of O76D-M007-H04 were used for aPTT analysis. It was incubated in plasma 10 minutes before starting. in figure 20 As shown, aPTT coagulation times are at the "front" time-point, that is, any in plasma samples collected from baboons before a treatment is administered. has not been determined. The results show that O76D-M007-H04 was used in the 6th thrombosis studies. shows anti-coagulant in plasma from all experimental baboons.

In vivo coagulation Studies: Three baboons were used in these studies: two baboons 32 then dosed with a 2 mg/kg dose (i.v. bolus). ACT (figures 21 and 22) and aPTT (fig. 23 and fig. 24) were measured at various time-points following administration.

Platelet deposition during sent experiments.

Thrombosis occlusion experiments. Treatment of 076D-MOO7-HO4 treatment of a small cari Whether it can prevent occlusion or the time to occlusion Thrombogenic device used to assess whether it can prolong a 2 It consists of a 20 mm long collagen-coated graft with mm i.d. in figure 25 platelet deposition for 60 minutes or graft occlusion as shown The time is displayed until applicable. 12/14 controller in 60 minutes mg/kg) device is blocked. Data. means ± SEM.

Thrombosis prevention experiments. Thrombus initiation and proliferation of 076D-M007-H04 Thrombogenic device with a 4 mm i.d. 20 mm with a 9 mm i.d. followed by a mm-long collagen-coated ePTFE graft long silicone rubber section. As shown in Figure 26 The slope of platelet deposition is an indicator of anti-platelet activity. Both 076D- platelet deposition at various times from the onset of formation It showed efficacy as found with a decrease in their velocities. Data between experiments normalized to explain the cause of platelet count variations. Data, means ± SEM. followed by 2mg/kg after 24 hours), varying from the onset of thrombus formation evidenced by the intense reduction in the rates of platelet deposition in the silicon chamber over time. has shown activity. Data are means ± SEM.

Thrombin anti-thrombin complexes. Inhibition of FXI is both thrombin-mediated in vivo By limiting platelet activation and fibrin formation and/or increasing thrombolysis Thrombin anti-thrombin (TAT) levels are commercially available as it can reduce thrombus formation. Measured using a commercially available ELISA kit. As shown in Figure 28, baboons treatment prevents the increase in TAT levels, this is thrombin in the absence of FXIa activity. shows a conspicuous dream in its production.

Bleeding Times: Primary hemostasis FDA for use in children and adults adult Surgicutt device approved by (http://www.itcmed/com/products/surgicutt-bleeding-time-device) has been evaluated. Bleeding time (CT) was recorded manually. wound 30 minutes observed for re-bleeding throughout and the next day skin bruising, petechiae, evaluated for hematomas and sufusions. These hemostasis assessments one or more of nearly all marketed anti-thrombotic agents (anti- sensitive to the anti-hemostatic effects of platelet drugs, anti-coagulants, thrombolytics) and it has been shown to be their predictor.

Baboons are treated with or without chewable aspirin (ASA, 32 mg/kg). There is no increase in bleeding time with either one. Treatment of 076D-M007-H04 with aspirin Bleeding compared to administration of aspirin alone It was not seen that it increased the time additionally.

SERIES LIST <110> Bayer Pharma Aktiengesellschaft <120> Antibodies that can bind to Coagulation Factor XI and/or its activated form factor Xlaly and their uses <130> BHC121014 <160> 184 <170> Patent version 3.5 <210> 1 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 1 <210> 2 < 211> 360 < 212> DNA < 213› Homo Sapiens <400> 2 <210> 3 < 211› 30 < 212> DNA < 213> Homo Sapiens <400> 3 <210> 4 < 211› 30 < 212> DNA < 213› Homo Sapiens <400> 4 <210> 5 < 211› 39 < 212> DNA < 213> Homo Sapiens <400> 5 <210> 6 < 211> 33 < 212> DNA < 213› Homo Sapiens <400> 6 <210> 7 < 211> 21 < 212> DNA < 213> Homo Sapiens <400> 7 <210> 8 < 211› 21 < 212> DNA < 213> Homo Sapiens <400> 8 <210>9 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 9 <210>1O <211>21 < 212> DNA < 213› Homo Sapiens <400> 10 <210> 11 <211>321 < 212> DNA < 213› Homo Sapiens <400> 11 <210> 12 < 211> 354 tgacCcagaq < 212› DNA < 213› Homo Sapiens gcqtgggCga <400> 12 <210>13 < 211> 30 cqachoctc < 212> DNA < 213› Homo Sapiens <400> 13 <210> 14 <211> 30 < 212> DNA < 213› Homo Sapiens <400> 14 <210> 15 <211> 33 < 212› DNA < 213› Homo Sapiens <400> 15 <210> 16 < 211> 33 < 212› DNA < 213› Homo Sapiens <400> 16 accgcgqigt <210> 17 < 211> 21 < 212› DNA < 213› Homo Sapiens <400> 17 <210> 18 < 211› 33 < 212› DNA < 213 › Homo Sapiens <400> 18 <210> 19 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 19 Asp Iie Gin Met Thr Gin Sex Pro Ser Ser Leu Asp Arq Val Thr Ile Thr Cya Gln Ala Ser Gln Leu Asn Trp Tyr Gin Gin Lys Pro Giy Lya Ala Tyr Asp Ala Ser Asn Leu Giu Thr Gly Vai Pro Ser Gly Ser Gly Thr Asp Phe Tht Phe Thr Ile 65 70 75 Giu Asp Ile Ala Thr Tyr Tyr Cya Gin Gin Ala Aan Sar Phe Pro Vai Thr Phe Wear Wear Wear Thr Lya Val Giu Ile Lys 100 105 <210> 20 < 211› 120 < 212› PRT < 213› Homo Sapiens <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly GLy Leu Val Gin Ser Leu arg Leu Ser Cys Ala Ala Set Gly Phe Thr ? be Giy Met Asp Trp Vai Axg Gin Ala Pro Gly Lys Giy Leu 40 45 Ser Gly With Gly Pro Ser Gly Gly Ser Thr Val Tyr Ala Lys Gly Arq Phe Thr With Ser Arq Asp Asn Ser Lys Asn 65 70 75 Lou Gln Met Asn Ser Leu Arq Ala Glu Asp Thr Ala Val Thr Arq Gly Gly Pro Tyr Tyr Tyr Tyr Gly Met Asp Val 100 105 Gly Thr Thr Val Thr Val Sir Sir 115 120 <210> 21 < 211› 10 < 212› PRT < 213› Homo Sapiens <400> 21 Gly Phe Thr Phe Ser Gin Tyr Gly Met Rep <210> 22 < 211› 10 < 212› PRT < 213› Homo Sapiens <400> 22 Gly Ile Gly Pro Ser Gly Gly Ser Thr Val <210> 23 < 211› 13 < 212› PRT < 213› Homo Sapiens <400> 23 Thr Arg Gly Gly Pro Ty: Tyr Tyr Tyr Gly Met Asp Val <210> 24 < 211› 11 < 212› PRT < 213› Homo Sapiens <400> 24 Gln Ala Ser Gln Asp Ile Ser Ann Ty:: ben Asn <210> 25 < 211› 7 < 212› PRT < 213› Homo Sapiens <400> 25 <210> 26 < 211› 7 < 212› PRT < 213› Homo Sapiens <400> 26 <210> 27 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 27 Asp Ile Gln Met. Thr Gin Se: Pro Ser Ser Leu Ser Ala Set Vai Wear 1 5 10 15 Asp Arg Val Thr With Thz Cys Gln Ala Ser Gln Asp With Ser Asn Tyr 25 30 <210> 28 < 211› 7 < 212› PRT < 213› Homo Sapiens <400> 28 <210> 29 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 29 Gly Phe Thr Phe Ser Asp Tyr Glu Met Ala <210> 30 < 211› 118 < 212> PRT < 213> Homo Sapiens <400> 30 <210> 31 < 211› 10 < 212> PRT < 213› Homo Sapiens <400> 31 <210> 32 < 211> 10 < 212> PRT < 213› Homo Sapiens <400> 32 Ser Ile Val Pro Ser Gly Gly Trp Thr Leu <210> 33 <211>11 < 212> PRT < 213> Homo Sapiens <400> 33 Ala rh: Trp Gly Asp Ser Trp Gly Phe Asp Phe <210> 34 < 211> 11 < 212› PRT < 213> Homo Sapiens <400> 34 Arg Ala Ser Gln Wear With Ser Ser Trp Leu Ala <210> 35 <211>7 < 212> PRT < 213› Homo Sapiens <400> 35 <210> 36 <211>11 < 212> PRT < 213› Homo Sapiens <400> 36 Gln Gln Ala Asp Ser Phe Pro With Ala Phe Gly <210> 37 < 211> 372 < 212> DNA < 213› Homo Sapiens <400> 37 ctgcaqntga <210> 38 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 38 <210 > 39 <211> 372 < 212> DNA < 213› Homo Sapiens <400> 39 agctchcgq <210> 40 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 40 99CC599C9C <210> 41 < 211> 372 < 212> DNA < 213> Homo Sapiens <400> 41 OCQÇBCGQCQ <210> 42 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 42 <210> 43 < 211> 372 < 212> DNA < 213› Homo Sapiens 9C99C99593 <400> 43 <210> 44 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 44 <210> 45 < 211> 372 < 212› DNA < 213› Homo Sapiens <400> 45 accatqçtgc <210> 46 < 211> 321 < 212> DNA < 213› Homo Sapiens accgcgqlgt UCVQCVUCtt 9Cq199939a <400> 46 <210> 47 < 211> 372 < 212> DNA < 213> Homo Sapiens <400> 47 OCQÇBCGQCQ <210> 48 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 48 <210> 49 < 211> 372 < 212> DNA < 213› Homo Sapiens gcqCqaCng 9C959ng93 cLLtqgccag qcntaaaccg <400> 49 <210> 50 < 211› 321 < 212> DNA < 213› Homo Sapiens <400> 50 cgcittach <210> 51 < 211> 372 < 212> DNA < 213› Homo Sapiens <400> 51 aqCCQcchq <210> 52 < 211› 321 399C99C99C Lttaccaiga 9C9C999C93 caccuccqtg < 212> DNA < 213› Homo Sapiens <400> 52 <210> 53 < 211> 378 < 212› DNA < 213› Homo Sapiens <400> 53 agcçaigtgi <210> 54 < 211› 321 cgagcqgctL < 212› DNA < 213› Homo Sapiens <400> 54 <210> 55 < 211> 372 < 212› DNA < 213› Homo Sapiens <400> 55 <210> 56 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 56 <210 > 57 <211> 372 < 212› DNA < 213› Homo Sapiens <400> 57 <210> 58 < 211> 321 ttnttatgqc < 212> DNA < 213› Homo Sapiens <400> 58 <210> 59 < 211> 378 < 212› DNA < 213› Homo Sapiens <400> 59 <210> 60 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 60 <210> 61 < 211> 372 Ctgqtgcagc UCÇtQQUqu 9C9CC399C9 < 212> DNA < 213 › Homo Sapiens <400> 61 <210> 62 < 211› 321 < 212> DNA < 213› Homo Sapiens <400> 62 <210> 63 <211> 360 < 212> DNA < 213› Homo Sapiens <400> 63 < 210> 64 < 211> 321 < 212› DNA < 213› Homo Sapiens acctgqigac passecnqch ctitqgccag Cetgcqcctg <400> 64 <210> 65 < 211> 360 < 212> DNA < 213> Homo Sapiens <400> 65 <210> 66 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 66 Cgctttagcq <210 > 67 <211> 360 < 212› DNA < 213› Homo Sapiens accgcgqigt 9Gqtgqchß <400> 67 <210> 68 < 211› 321 < 212> DNA < 213> Homo Sapiens <400> 68 <210> 69 <211> 360 < 212› DNA < 213› Homo Sapiens <400> 69 <210> 70 < 211> 321 < 212> DNA < 213› Homo Sapiens qvtqaocqvc <400> 70 <210> 71 < 211> 360 < 212> DNA < 213> Homo Sapiens <400> 71 <210> 72 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 72 <210> 73 <211> 360 < 212› DNA < 213› Homo Sapiens accgcgqigt <400> 73 gchatLatL <210> 74 < 211> 321 cqachoctc < 212> DNA < 213> Homo Sapiens <400> 74 <210> 75 <211> 360 < 212› DNA < 213› Homo Sapiens <400> 75 <210> 76 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 76 <210> 77 < 211> 360 < 212> DNA < 213> Homo Sapiens <400> 77 <210> 78 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 78 <210> 79 <211> 360 < 212› DNA < 213› Homo Sapiens C99C99C99C C999C99C39 959C5399C9 <400> 79 ocwatach <210> 80 < 21 1> 321 cgagcqgcti < 212> DNA < 213› Homo Sapiens <400> 80 <210> 81 < 211> 360 < 212> DNA < 213› Homo Sapiens < 400> 81 <210> 82 < 211> 321 < 212> DNA < 213› Homo Sapiens accqcgqtgi 9C99C99C39 <400> 82 <210> 83 < 211> 360 < 212> DNA < 213> Homo Sapiens <400> 83 <210> 84 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 84 <210> 85 <211> 360 < 212› DNA < 213› Homo Sapiens <400> 85 <210> 86 < 211> 321 < 212> DNA < 213> Homo Sapiens <400> 86 <210> 87 <211> 360 < 212› DNA < 213› Homo Sapiens <400> 87 <210> 88 < 211> 321 < 212› DNA < 213› Homo Sapiens vcqqcwcao 9C9CC599C9 <400> 88 <210> 89 < 211> 360 < 212> DNA < 213> Homo Sapiens <400> 89 gcqgatach <210> 90 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 90 <210> 91 < 211> 366 < 212> DNA < 213› Homo Sapiens accgcgqigt Cggchqcaq <400> 91 9C99t99C99 <210> 92 < 211> 321 < 212> DNA < 213› Homo Sapiens <400> 92 <210> 93 < 211> 354 < 212› DNA < 213› Homo Sapiens <400> 93 <210> 94 < 211› 321 < 212> DNA < 213› Homo Sapiens tCUCCOQCGQ tLggqgccag C999C9938q 99193CC9L9 <400> 94 <210> 95 < 211> 375 < 212> DNA < 213> Homo Sapiens <400> 95 agciatiatq <210> 96 < 211 > 321 tqaaaÇQCCq < 212› DNA < 213› Homo Sapiens <400> 96 <210> 97 < 211> 375 < 212> DNA < 213› Homo Sapiens tgcagagcqç <400> 97 OCQQötEQCQ <210> 98 < 211› 324 < 212> DNA < 213› Homo Sapiens <400> 98 <210> 99 < 211> 383 < 212› DNA < 213› Homo Sapiens <400> 99 9t99C999C5 <210> 100 < 211> 324 < 212> DNA < 213› Homo Sapiens Lgcagtggqt gcqtqqcha <400> 100 <210> 101 < 211> 354 < 212> DNA < 213> Homo Sapiens <400> 101 gatagciggq <210> 102 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 102 <210 > 103 < 211> 354 < 212> DNA < 213> Homo Sapiens QcqtÇQUCQa tgcagagcqç Cgtqccgaqc <400> 103 <210> 104 < 211> 321 cqachoctc < 212> DNA < 213> Homo Sapiens <400> 104 <210> 105 <211 > 354 < 212› DNA < 213› Homo Sapiens <400> 105 <210> 106 < 211> 321 < 212> DNA < 213› Homo Sapiens Laccittagc ttoavoccaq 9C99C99Ct9 cgthCgagc <400> 106 <210> 107 < 211> 354 < 212 > DNA < 213> Homo Sapiens <400> 107 qcggatach 9atagciggq <210> 108 < 211> 321 < 212› DNA < 213› Homo Sapiens <400> 108 <210> 109 < 211> 354 < 212› DNA < 2 13› Homo Sapiens cLLtqgccag <400> 109 <210> 110 < 211> 321 cqachoctc < 212> DNA < 213> Homo Sapiens <400> 110 <210> 111 < 211> 124 < 212› PRT < 213› Homo Sapiens <400 > 111 acctoqcwq cqthCgagc Leu Gin Met Aan Ser Leu Arg Ala Giu Asp Thr Aia Vai Tyr Tyr Cya BS 90 95 Ala Arg Giu Phe Giu Asn Ala Ty: His Tyr Tyr Tyr Ty: Giy Mel Asp 100 105 110 Vnl Trp Gly Gln Gly . Thr Val Thr Val Sir Sir 115 120 <210> 112 < 211› 107 < 212> PRT < 213> Homo Sapiens <400> 112 Gin Met With Asp Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Vai Wear 1 5 10 IS Asp Arg Vai Thr Ile Thr Cys Arg Aia Ser Giy Asp Iie Wear Asp Iie Wear Asu Ala 25 30 Lou Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arc Leu Leu 110 40 45 Ser Asp Ala Ser Thr Leu Gin Set Gly Val Pro Leu Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr With Ser Ser Leu Gln Pro 65 70 75 80 Giu Asp Phe Ala Thr Ty: Ty: Cys Leu GLn Wear Ty: Asn Ty: Pro Axg 85 90 95 Thr Ph. Gly Gln Gly Thr Lys Lou Glu Ile Arg 100 105 <210> 113 < 211> 124 < 212> PRT < 213› Homo Sapiens <400> 113 Giu Vai Gin Leu Giu Ser Wear GLy Leu Vai Gin Pro Wear 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Type Tyr 25 30 <210> 114 < 211› 107 < 212> PRT < 213› Homo Sapiens <400> 114 1.060 <210> 115 < 211> 124 < 212> PRT < 213> Homo Sapiens <400> 115 <210> 116 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 116 <210> 117 < 211› 124 < 212› PRT < 213> Homo Sapiens <400> 117 <210> 118 < 211› 107 < 212› PRT < 213› Homo Sapiens < 400> 118 Asp With Gin Met Thr Gin Ser Pro Asp Arq Val Thr Ili Thr Cys Rrg Liu Ala Trp Ty: Gln Gln Lys Pro <210> 119 < 211› 124 < 212› PRT < 213> Homo Sapiens <400> 119 <210> 120 <211> 107 <212> PRT < 213› Homo Sapiens <400> 120 <210> 121 < 211› 124 < 212› PRT < 213› Homo Sapiens <400> 121 <210> 122 < 211› 107 < 212› PRT < 213> Homo Sapiens <400> 122 1.91.] <210> 123 < 211› 124 < 212› PRT < 213> Homo Sapiens <400> 123 <210> 124 < 211> 107 < 212> PRT < 213› Homo Sapiens <400> 124 <210> 125 < 211› 124 < 212› PRT < 213› Homo Sapiens <400> 125 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly < 210> 126 <211> 107 <212> PRT < 213› Homo Sapiens <400> 126 <210> 127 < 211> 126 < 212› PRT < 213› Homo Sapiens <400> 127 <210> 128 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 128 65 70 75 BU Glu Asp With Ala Thr Tyr Tyr Cys Gln GLn Phe Asp Asp Lou Pro Lev 85 90 95 Thr Phi Gly Pro Gly Thr Arq Vsl Asp Il. Lys 100 105 <210> 129 < 211› 124 < 212› PRT < 213› Homo Sapiens <400> 129 Giu Val Gln Leu Leu Giu Se: Gly Gly Wear Leu Val Sin ?:0 Wear Wear 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gy Phe Thr Phe Ser Axg Tyr 25 30 40 45 Ser Ser Ile Ser Pro Ser Gly Gly Lou Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Aan Ser Lys Asn Thr Leu Tyr 65 70 75 80 Liu Gln Mit han 5.: Liu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys B5 90 95 Ala Arg Glu Ph: Glu Asn Ala Tyr His Tyr Tyr Ty: Ty: Gly Mct Asp 100 105 110 Val ? tp Gly Gln Wear Thr Thr Val Thr Val Ser Sir 115 120 <210> 130 < 211› 107 < 212> PRT < 213› Homo Sapiens <400> 130 Asp With Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Aia Ser Vai Wear 1 5 10 15 Asp Arg Val Thr Ile rh: Cys Arg Ala Ser Gly Asp Ile Gly Asu Ala 25 30 <210> 131 < 211› 124 < 212> PRT < 213› Homo Sapiens <400> 131 <210> 132 < 211 > 107 < 212> PRT < 213> Homo Sapiens <400> 132 <210>133 < 211› 126 < 212› PRT < 213› Homo Sapiens <400> 133 <210> 134 < 211› 107 < 212› PRT < 213 > Homo Sapiens <400> 134 <210> 135 < 211› 124 < 212› PRT < 213> Homo Sapiens <400> 135 Giu Vai Gin Leu Leu Glu Ser Giy Ser Lou Arg Lou Sar Cys Ala Ala Ser He: Gly Trp Vai Arq Gin Ala Pro Gly Lys Gly Leu Glu <210> 136 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 136 <210> 137 < 211> 120 < 212› PRT < 213› Homo Sapiens <400> 137 Glu Va] Gln Lou Leu Glu Ser Gly Gly GLy Lou Val Gln Pro Gly Gly <210> 138 <211>107 <212>PRT < 213› Homo Sapiens <400> 138 <210> 139 < 211> 120 < 212› PRT < 213› Homo Sapiens <400> 139 <210> 140 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 140 Ser Giy Ser Giy Thr Asp Phe Thr Giu Asp Ile Ala Thr Tyr Tyr Cya Thr Ph. Gly Gly Gly Thr Lys Val <210> 141 < 211› 120 < 212› PRT < 213> Homo Sapiens <400> 141 <210> 142 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 142 With Asp Gln Met Thr Gin Ser Pro Ser Ser Len Ser Ala Ser Vai Wear <210> 143 < 211› 120 < 212› PRT < 213› Homo Sapiens <400> 143 <210> 144 <211> 107 < 212› PRT < 213› Homo Sapiens <400> 144 <210> 145 < 211› 120 < 212› PRT < 213› Homo Sapiens <400> 145 <210> 146 < 211› 107 < 212> PRT < 213> Homo Sapiens <400> 146 <210 > 147 < 211> 120 < 212> PRT < 213› Homo Sapiens <400> 147 Giu Vai Gin Leu Leu Giu Ser Giy Sir Lou Arq Lou Sir Cys Ala Ala Gly Hot Asp Trp Val Arq Gln Ala Giu Asp Thr Aia Vai Tyr Tyr Cya Tyr Gly Mut Asp Val Trp Gly Gln <210> 148 < 211> 107 < 212› PRT < 213› Homo Sapiens <400> 148 <210> 149 < 211› 120 < 212› PRT < 213› Homo Sapiens <400> 149 Mar. Asp <210> 150 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 150 <210> 151 < 211› 120 < 212› PRT < 213> Homo Sapiens <400> 151 <210> 152 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 152 <210> 153 < 211> 120 < 212> PRT < 213› Homo Sapiens <400> 153 <210> 154 < 211> 107 < 212> PRT < 213› Homo Sapiens <400> 154 <210> 155 < 211› 120 < 212› PRT < 213› Homo Sapiens <400> 155 <210> 156 < 211› 107 < 212› PRT < 213> Homo Sapiens <400> 156 <210 > 157 < 211› 120 < 212› PRT < 213> Homo Sapiens <400> 157 65 70 75 80 Leu Gln Met Aan Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Wear Wear Pro Tyr Tyr Ty : Lys GLy Met Asp Val Trp Wear Gin 100 105 110 Gly Thr Thr Val Thr Val Sir Sir 115 120 <210> 158 < 211› 107 < 212> PRT < 213› Homo Sapiens <400> 158 Asp With Gin Met Thr Gin Sex ?:0 Ser Ser Leu Ser Aia Ser Vai Wear 1 5 10 15 Asp Arg vai Thr With Thr Cys Gin Ala Ser Gin Asp With Ser Asn Tyr 25 30 Liu Asn Trp Tyt Gln Gln Lys Pro Gly Lys Ala Pro Ly: Lou Lou Il . 40 45 Tyt Asp Ala Ser Asn Leu Glu Thr Gly Vai Pro Set Axg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr With Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp With Ala Thr Tyr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Val Thr Ph: Gly Gly Wear Thr Lys Val Glu Ili Lys 100 105 <210> 159 < 211> 120 < 212> PRT < 213› Homo Sapiens <400> 159 Glu Vai Gln Leu Leu Glu Ser Gly Wear GLy Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 25 30 <210> 160 < 211› 107 < 212> PRT < 213› Homo Sapiens <400> 160 1.060 <210> 161 < 211> 120 < 212> PRT < 213> Homo Sapiens <400>161 <210>162 <211>107 <212>PRT < 213› Homo Sapiens <400>162 <210> 163 < 211 › 120 < 212› PRT < 213> Homo Sapiens <400> 163 <210> 164 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 164 Gln Met Thr Gin Se with Asp: Pro Ser Ser Leu Ser Ala Ser Vai Giy Asp Arg Val Thr Il. ? hr Cyi Gln Ala Sir Gln Asp Il. Ask Asu Ty: <210> 165 < 211› 122 < 212> PRT < 213› Homo Sapiens <400> 165 <210> 166 < 211> 107 < 212> PRT < 213> Homo Sapiens 501.” <400> 166 <210>167 <211>118 <212> PRT < 213› Homo Sapiens <400> 167 <210> 168 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 168 <210> 169 < 211› 125 < 212› PRT < 213› Homo Sapiens <400> 169 <210> 170 < 211> 107 < 212> PRT < 213› Homo Sapiens <400> 170 <210> 171 < 211› 125 < 212› PRT < 213› Homo Sapiens <400> 171 Glu Val Gln Leu Leu Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly <210> 172 <211> 108 <212> PRT < 213› Homo Sapiens <400> 172 <210> 173 < 211> 121 < 212› PRT < 213› Homo Sapiens <400> 173 <210> 174 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 174 Ser Giy Ser Giy Thr Asp Phe Thr Leu Thr With Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Ph. Ala Thr Tyr Ty: Cys Gln Gln Sir Ty: Sir Thr Pro Pro 85 90 95 Trp Thr Ph. Gly Gln Gly Thr Lys Val Glu II. 100 105 <210> 175 < 211› 118 < 212› PRT < 213› Homo Sapiens <400> 175 Giu Val Gln Leu Leu Giu Ser Gly GLy Gly Leu Val Gin Pro Gly Wear 1 5 10 15 Ser Leu Azg Leu Ser Cys Ala Ala Ser Giy Phe Thr Phe Ser Asp Ty: 25 3D Giu Met Ala Tip Vai Arg Gin Ala Pro Wear Lys Wear Leu Giu sz Vai 40 45 Ser Ser Ile vai Pro Ser Gly Gly Trp Thr Leu Tyt Ala Asp Ser val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Aan Ser Lya Asn Tht Leu Tyr 65 70 75 80 Lou Gln Met Asn Ask Lau Arq Ala Glu Asp Thr Ala Val Tyr Tyt Cys Ala ? hr Trp Gly Asp Ser Trp Gly Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 <210> 176 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 176 Asp with Gln Met Thr Gin Sa: Pro Ser Ser vai Ser kia Ser vai Gly 1 5 10 15 Asp Arg Val Thr With Thr Cys Arg Ala Ser Gln Gly With Ser Ser Trp <210>177 < 211> 118 < 212> PRT < 213› Homo Sapiens <400> 177 <210> 178 < 211> 107 < 212› PRT < 213› Homo Sapiens <400> 178 <210> 179 < 211› 118 < 212› PRT < 213› Homo Sapiens <400> 179 1.811 Ala rh: Trp Wear Asp Se: Trp Wear Phe Asp Phe Trp Wear Gin Wear Thr 100 105 110 <210> 180 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 180 Asp With Gln Met Thr Gln Ser Pro Ser Vai Ser Ala Ser Val Wear 1 5 10 15 Asp Arg Vai Thr Iii Thr Cys Arg Ala Ask Gln Wear With Sir Trp 25 30 Lou Ala Trp Tyr Gin Gln Arq Pro Gly Lys Ala Pro Lys Lou Leo With 40 45 Ty: Asp Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr With Asn Ser Leu Gln Pro 65 70 75 80 Giu Asp Phe Ala Thr Ty: Ty: Cys Gln Gl n Ala Asp Se: Phe Pro Ile 85 90 95 Ala Phe Gly Gln Gly ?hr Arq Leu Glu Ile Lys 100 105 <210> 181 < 211› 118 < 212› PRT < 213› Homo Sapiens <400> 181 Glu Vai Gin Leu Leu Glu Ser Gly Wear GLy Leu Val Gin Pzo Gly Wear 1 5 10 15 Ser Leu Arg Lou Ser Cys Ala Ala Ser GLy Ph& Thr Phe Ser Asp Tyr 25 30 Giu Hot Ala Trp vii Arq Gin Ala Pro Gly Lys Wear Lou Glu Trp Vai 40 45 <210> 182 < 211› 107 < 212› PRT < 213› Homo Sapiens <400> 182 <210>183 < 211> 118 < 212› PRT < 213› Homo Sapiens <400> 183 <210> 184 < 211 › 107 < 212› PRT < 213› Homo Sapiens <400> 184

Claims (1)

REQUESTS . A human monoclonal antibody capable of binding Factor Xla (FXla) and its antigen-binding fragment, characterized by SEQ ID NO: 19 for the amino acid sequence for the variable light chain domain and SEQ ID NO: 20 for the amino acid sequence for the variable heavy chain. It contains . . A human monoclonal antibody capable of binding Factor Xla and its antigen-binding fragment, characterized by SEQ ID NO: 21 as CDRH1, SEQ ID NO: 22 as CDRH2 and SEQ ID NO: 23 as CDRH3 and SEO as CDRL1. ID NO: 24, SEO ID NO: 25 as CDRL2, and SEQ ID NO: 26 as CDRL3. . A human monoclonal antibody capable of binding FXla and its antigen-binding fragment, characterized by SEO ID NO: 27 for the amino acid sequence for the variable light chain domain and SEQ ID NO: 20 for the amino acid sequence for the variable heavy chain domain. . . A human monoclonal antibody capable of binding FXla and its antigen-binding fragment, characterized by SEQ ID NO: 21 as CDRH1, SEO ID NO: 22 as CDRH2, and SEQ ID NO: 23 as CDRH3, and SEQ ID as CDRL1. NO: 24, SEQ ID NO: 25 as CDRL2, and SEQ ID NO: 28 as CDRL3. . A pharmaceutical composition comprising an antibody according to any one of claims 1 to 4. . A medicament characterized in that it contains an antibody according to any one of claims 1 to 4. . A nucleic acid characterized in that it encodes for an antibody according to any one of claims 1 to 4. . It is a vector characterized in that it contains nucleic acid according to claim 7. 9. A host cell, characterized in that it contains the vector according to claim 8. 10. A method of using the host cell according to claim 8 to produce an antibody according to any one of claims 1 - 4, comprising culturing the host cell under suitable conditions and recovering said antibody. 11. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that it is for use in the treatment and/or prophylaxis of coagulation-related disease in humans or animals.