Monoclonal antibody inhibitors of factor XIIa

Abstract

To provide an anti-factor XIIa antibody, and use thereof for treating a disease related to contact system activation, plasma prekallikrein signaling (e.g., hereditary angioedema) and a disease related to factor XII such as an eye disease.SOLUTION: Provided is an antibody that specifically binds to factor XIIa (FXIIa) but does not specifically bind to factor XII (FXII). One aspect of the disclosure is a monoclonal antibody that binds to factor XIIa (FXIIa) but does not bind to factor XII (FXII). In some embodiments, this antibody is a full-length antibody or an antigen-binding fragment such as Fab. In some embodiments, this antibody is a human antibody or a humanized antibody.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 194,957, filed July 21, 2015, U.S. Provisional Application No. 62 / 273,657, filed December 31, 2015, and U.S. Provisional Application No. 62 / 316,310, filed March 31, 2016. The entire contents of each of these referenced applications are incorporated herein by reference. [Background technology]

[0002] Factor XII (FXII) is a component of the plasma contact activation system, which is involved in both the initiation of intrinsic coagulation and the conversion of prekallikrein to plasma kallikrein (pKal). Activated pKal cleaves high molecular weight kininogen (HMWK) to release bradykinin, a potent activator of edema and pain. Activation of the intrinsic pathway of blood coagulation leads to fibrin production and clot (thrombus) formation. Summary of the Invention

[0003] The present disclosure is based on the identification of multiple antibodies that specifically bind to factor XIIa (FXIIa) but not to factor XII (FXII). These antibodies have high binding affinity for FXIIa (e.g., K i app <50 pM), successfully reduced plasma kallikrein activation and prolonged the activated partial thromboplastin time (APTT) but had no effect on the prothrombin time (PT).

[0004] Accordingly, one aspect of the present disclosure features a monoclonal antibody that binds to factor XIIa (FXIIa) but not to factor XII (FXII). In some embodiments, the antibody is a full-length antibody or an antigen-binding fragment thereof, such as a Fab. In some embodiments, the antibody is a human antibody or a humanized antibody.

[0005] In some embodiments, the antibody interacts with one or more amino acid residues in the C chain of FXIIa, in some examples, the amino acid residues are selected from L390, Y391, W392, G393, H394, S395, F396, C397, H412, C413, L414, Q415, D416, R432, N433, V456, Y458, H507, F509, E510, G511, A512, E513, Y515, D557, A558, C559, Q560, G561, D562, S563, I584, S585, W586, G587, S588, G589, C590, G591, D592, and G597 in SEQ ID NO: 128. In some embodiments, the antibody binds to an FXIIa fragment comprising residues 390-397, 412-416, 432-433, 456-458, 507-515, 557-563, or 584-592 of SEQ ID NO:128.

[0006] In some examples, the anti-FXIIa antibodies described herein comprise (a) a heavy chain comprising a heavy chain variable region comprising heavy chain complementarity-determining region 1 (CDR1), heavy chain complementarity-determining region 2 (CDR2), and heavy chain complementarity-determining region 3 (CDR3), and optionally (b) a light chain comprising a light chain variable region comprising light chain CDR1, light chain CDR2, and light chain CDR3.

[0007] In some embodiments, the heavy chain variable region comprises a heavy chain CDR3 comprising the amino acid sequence of QRYRGPKYYYYMDV (SEQ ID NO: 111), QRYRGPKYYYYMDA (SEQ ID NO: 112), or QRYRGPRYYYYIDA (SEQ ID NO: 113). Such heavy chain variable regions may further comprise a heavy chain CDR1 comprising the formula X1YX3MX5 (SEQ ID NO: 117), where X1 is R, Q, W, H, F, P, M, or N, X3 is I, V, T, H, S, or N, and X5 is H, G, V, A, R, Q, Y, L, N, or S. In some examples, X1 of CDR1 is W or Q, X3 of CDR1 is S or V, and / or X5 is H. In some examples, the heavy chain CDR1 may comprise the amino acid sequence of any of SEQ ID NOs: 41-73 and 121.

[0008] Alternatively or additionally, the heavy chain variable region of the anti-FXIIa antibodies described herein may be of the formula X1IX3PSGX7X8TX 10 YX 12 X is S, R, V, Y, or G; X is Y, W, V, or S; X is G or S; and X is V, K, M, N, L, F, A, I, S, H, or R; 10 is K, R, T, Q, S, N, H or L, and X 12 In some examples, X1 of CDR2 is V or S, X3 of CDR2 is Y or W, X7 of CDR2 is G, X8 of CDR2 is K or H, and X 10 is R and / or X in CDR2 12 is A. In some examples, the heavy chain CDR2 comprises the amino acid sequence of any of SEQ ID NOs: 74-110, 122-124, and 127.

[0009] In certain embodiments, the heavy chain variable region of the anti-FXIIa antibodies described herein comprises a combination of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 as shown in Table 1.

[0010] In some examples, the heavy chain of an anti-FXIIa antibody described herein comprises a heavy chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 1-39 and SEQ ID NO: 125.

[0011] Any of the heavy chains of the anti-FXIIa antibodies described herein may further comprise a heavy chain constant region. In some embodiments, the heavy chain constant region comprises at least one mutation (e.g., an amino acid substitution) that increases the half-life of the antibody compared to its wild-type counterpart. Such a mutated heavy chain constant region may comprise at least one mutation at a position corresponding to positions 145, 147, or 149 of SEQ ID NO: 119, which represents the amino acid sequence of an exemplary wild-type human heavy chain constant region. The at least one mutation may be an amino acid substitution of M145Y, S147T, and / or T149E. In some examples, the mutated heavy chain constant region comprises amino acid substitutions at positions 145, 147, and 149 of SEQ ID NO: 119 (e.g., M145Y, S147T, and T149E). Such a mutated heavy chain may comprise the amino acid sequence of SEQ ID NO: 120.

[0012] The light chain variable region of an anti-FXIIa antibody described herein may comprise a light chain CDR3 comprising the amino acid sequence of MQALQTPWT (SEQ ID NO: 116). In some examples, the light chain variable region may further comprise a light chain CDR1 comprising the amino acid sequence of RSSQSLLHSNGYNYLD (SEQ ID NO: 114). Alternatively or additionally, the light chain variable region may further comprise a light chain CDR2 comprising the amino acid sequence of LGSNRAS (SEQ ID NO: 115). In some examples, the light chain of an anti-FXIIa antibody described herein may comprise a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 126.

[0013] Any of the anti-FXIIa antibodies described herein may be a full-length antibody or an antigen-binding fragment thereof. In some instances, the antibody may be a human antibody or a humanized antibody.

[0014] Also within the scope of the present disclosure are isolated nucleic acids or sets of nucleic acids that collectively encode any of the anti-FXIIa antibodies described herein (e.g., encoding at least the heavy and light chain variable regions of the antibody), vectors or vector sets (e.g., expression vectors) containing the nucleic acids or sets of nucleic acids, and host cells or sets of host cells containing the vectors or vector sets. Exemplary host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells such as CHO cells.

[0015] Additionally, the present disclosure provides methods for producing any of the anti-FXIIa antibodies described herein, comprising culturing a host cell or set of host cells containing a vector or set of vectors that collectively encode the antibody, and recovering the cultured cells or medium for isolation of the antibody. Such methods may further comprise isolating the antibody from the cultured cells or medium.

[0016] In another aspect, the present disclosure provides methods for treating a factor XII-related disease, e.g., a disease associated with contact system activation, a disease associated with the pKal signaling pathway, such as hereditary angioedema (HAE), or an ocular disease (e.g., macular edema, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, or retinal vein occlusion), in a subject. Such methods may include administering to a subject in need thereof an effective amount of a composition comprising an antibody that binds to FXIIa but cannot bind to FXII (e.g., any of the anti-FXIIa antibodies described herein), one or more nucleic acids collectively encoding the antibody, or an expression vector comprising such nucleic acids. In some embodiments, the antibody specifically binds to the catalytic domain of FXIIa. In other embodiments, the antibody inhibits activation of FXI to FXIa. In still other embodiments, the K domain of the antibody is activated. i app is less than about 110 pM (eg, less than about 50 pM or 10 pM).

[0017] The subject treated by the methods described herein may be a human subject having, suspected of having, or at risk of having a pKal-related disorder (e.g., HAE). In some instances, the subject has, suspected of having, or at risk of having Type I, Type II, or Type III HAE.

[0018] In some embodiments, the subject treated by the methods described herein may be a human subject who has, is suspected of, or is at risk for a factor XII-related disease, such as those disclosed herein (e.g., a disease associated with contact pathway activation). For example, the subject may have, is suspected of, or is at risk for a disease associated with contact pathway activation, such as thrombosis, including thrombosis associated with atrial fibrillation, deep vein thrombosis (DVT), pulmonary embolism, stroke, or arterial or venous thromboembolic events. In another example, the subject may have, is suspected of, or is at risk for a disease associated with the pKal signaling pathway, e.g., HAE, such as type I, type II, or type III HAE. In yet another example, the subject may have, is suspected of, or is at risk for an ophthalmic disease, which may be macular edema, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, or retinal vein occlusion.

[0019] Pharmaceutical compositions comprising any of the anti-FXIIa antibodies described herein and a pharmaceutically acceptable carrier are also within the scope of the present disclosure. Such pharmaceutical compositions may be used to treat diseases associated with the pKal signaling pathway (e.g., HAE, such as type I, type II, or type III HAE), or may be used in the manufacture of a medicament for treating such diseases.

[0020] The following description sets forth the details of one or more embodiments of the invention. Other features or advantages of the invention will become apparent from the following drawings and detailed description of several embodiments, and from the appended claims.

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in conjunction with the detailed description of specific embodiments provided herein. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing the coagulation cascade involving factor XII. [Figure 2] FIG. 1 is a schematic diagram showing antibody selection strategies. [Figure 3] FIG. 1 is a schematic diagram showing the bioassays used to characterize the activity of FXIIa inhibitors, including in vitro activity assays, plasma activity assays, and binding preference assays. [Figure 4] FIG. 1 shows the inhibitory activity of the parent antibody 559C-M71-F06 (human IgG1 antibody) against human FXIIa (left panel) and mouse FXIIa (right panel). [Figure 5] FIG. 1 shows the species cross-reactivity of M71-F06 determined by APTT. [Figure 6] 1 shows the concentrations of exemplary anti-FXIIa antibodies M191-E09 and M192-H11 in the plasma of rats at various time points after injection of anti-FXIIa antibodies. [Figure 7] 1 is a chart showing the time course of APTT and PT prolongation (seconds) in the presence of anti-FXIIa antibody 559C-X211-A01 (A01). A: APTT kinetics after 10 mg / kg A01. B: Time course of APTT relative to baseline in the presence of 10 mg / kg A01. C: Time course of PT (seconds) in the presence of 10 mg / kg A01. [Figure 8]Figure 1 shows APTT activity of antibody DX-4012 at the indicated time points prior to administration of the indicated treatment (pre-drug, gray bars), 5 minutes prior to the start of the study (pre-study, black bars), and 5 minutes prior to the end of the study (post-study, white bars). Values ​​represent the mean APTT ± standard error (seconds). A: APTT time. B: Fold increase in APTT relative to baseline value (average of pre-study and post-study measurements in the same animal relative to control study days). [Figure 9] Figure 1 shows the PT activity of antibody DX-4012 at the indicated time points prior to administration of the indicated treatment (pre-drug, gray bars), 5 minutes prior to the start of the study (pre-study, black bars), and 5 minutes prior to the end of the study (post-study, white bars). Values ​​represent the mean PT ± standard error (seconds). A: PT time. B: Fold increase in PT relative to baseline value (average of pre-study and post-study measurements in the same animal relative to control study days). [Figure 10] Schematic of the external loop of a chronic arteriovenous (AV) shunt during a thrombosis study in non-human primates, where the external loop is extended over a gamma camera for real-time measurement of platelet deposition. [Figure 11] 1 is a chart showing the time course of platelet deposition in a collagen-coated graft. A: Thrombus head. B: Thrombus tail. [Figure 12] 1 is a chart showing the change in platelet deposition over time in collagen-coated grafts. [Figure 13] 1 is a chart showing the change in platelet deposition (total thrombus) over time in collagen-coated grafts. [Figure 14] 1 is a chart showing the rate of thrombus increase in collagen-coated grafts measured as platelet deposition at 5-minute intervals. [Figure 15] 1 is a chart showing the time course of platelet deposition in tissue factor-coated grafts. A: Thrombus head. B: Thrombus tail. [Figure 16] 1 is a chart showing the time course of platelet deposition in tissue factor-coated grafts. [Figure 17]1 is a chart showing the time course of platelet deposition in tissue factor-coated grafts. [Figure 18] 1 is a chart showing the increased rate of thrombosis in tissue factor coated grafts measured as platelet deposition at 5 minute intervals. [Figure 19] Terminal fibrin content and platelet deposition throughout the thrombus (head and tail) in collagen-coated (gray bars) and tissue factor-coated (white bars) grafts. A: Platelet deposition. B: Fibrin content. [Figure 20] 1 is a chart showing the complex formation between the Fab fragment of DX-4012 and FXIIa by SEC analysis. A: FXIIa and Fab. B: FXIIa-Fab complex of FXIIa, Fab, and 50 μM FXIIa and 30 μM Fab. C: FXIIa-Fab complex of FXIIa, Fab, and 50 μM FXIIa and 40 μM Fab. D: FXIIa-Fab complex of FXIIa, Fab, and 50 μM FXIIa and 50 μM Fab. E: FXIIa-Fab complex of FXIIa, Fab, and 50 μM FXIIa and 60 μM Fab. [Figure 21] FIG. 1 shows the inhibitory activity of anti-FXIIa antibody M192-H11 against human FXIIa. [Figure 22] 1 is a chart showing hemostatic measurements associated with DX-4012. A: Bleeding time. B: Bleeding volume. DETAILED DESCRIPTION OF THE INVENTION

[0023] Components of the contact pathway initiate the intrinsic pathway of coagulation and promote inflammation by releasing the proinflammatory peptide bradykinin. Factor XII (FXII), also known as Hageman factor, is a serine protease involved in activating the intrinsic pathway of coagulation and the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid) to produce activated FXIIa. Activated FXIIa has the ability to cleave prekallikrein to produce activated pKal. Activated pKal can then cleave FXII to FXIIa, creating a positive feedback loop in which FXIIa produces more pKal, which in turn activates more FXII to FXIIa. Activated pKal can also cleave high molecular weight kininogen (HMWK) to release bradykinin. The coagulation cascade involving FXII is shown in Figure 1.

[0024] In diseases associated with contact system activation, such as HAE, elevated levels of bradykinin can induce vasodilation and inflammation, leading to edematous HAE attacks. Therefore, it is desirable to develop novel therapeutic agents to treat various diseases potentially mediated by contact system activation.

[0025] Described herein are antibodies that bind to and inhibit FXIIa, and their use in inhibiting FXIIa and treating diseases associated with contact system activation.As shown in the following examples, several exemplary anti-FXIIa antibodies have been produced and found to specifically bind to and inhibit the activity of FXIIa.Such antibodies exemplified herein are expected to show high therapeutic efficacy in treating diseases associated with contact system activation, particularly in reducing bradykinin production, vasodilation, and pathological thrombus formation associated with disease symptoms.

[0026] Antibody binding to FXIIa The present disclosure provides antibodies that bind to FXIIa, for example, the catalytic domain of FXIIa. Such antibodies are unable to bind to FXII.

[0027] Antibodies (the plural forms are used interchangeably) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site with the required specificity, such as glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and the antibody may be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0028] In some embodiments, the anti-FXIIa antibodies described herein are capable of binding to and inhibiting FXIIa activity by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more). The apparent inhibition constant (K) provides a measure of inhibitory potency. i app or K i,app) is related to the inhibitor concentration required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-FXIIa antibodies described herein can be determined by routine methods known in the art.

[0029] The K value of the antibody can be determined by measuring the inhibitory effect of different concentrations of the antibody on the extent of the reaction (e.g., enzyme activity). i app The apparent K value may be determined by fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1). i For competitive inhibitors, the K i app The y-intercept derived from the linear regression analysis of the plot of i app can be obtained.

number

[0030] where A is the initial velocity (v o ) divided by the total enzyme concentration (E) o Equals / E.

[0031] In some embodiments, the anti-FXIIa antibodies described herein have a K of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less for the target antigen or antigen epitope. i app In some embodiments, the anti-FXIIa antibody may have a lower K value for a first target (e.g., FXIIa) compared to a second target (e.g., FXII). i app K i appThe difference in (e.g., for specificity or other comparison) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some examples, the anti-FXIIa antibody inhibits a first antigen (e.g., a first protein or mimetic thereof in a first conformation) better than a second antigen (e.g., the same first protein or mimetic thereof or a second protein in a second conformation). In some embodiments, any of the anti-FXIIa antibodies can be further affinity matured to increase the K of the antibody for the target antigen or antigenic epitope thereof. i app may be reduced.

[0032] The antibodies described herein may be derived from a mouse, rat, human, or any other organism (including chimeric or humanized antibodies). Such antibodies are not naturally occurring, i.e., are not produced in an animal without human action (e.g., immunization of such animal with the desired antigen or a fragment thereof).

[0033] Any of the antibodies described herein may be monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous population of antibodies, while a "polyclonal antibody" refers to a heterogeneous population of antibodies. These two terms do not limit the source of the antibody or the method by which it is made.

[0034] In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. Most often, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in the recipient antibody or in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable regions, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of a human immunoglobulin consensus sequence. The humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more (one, two, three, four, five, six) CDRs that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The humanized antibody may also have undergone affinity maturation.

[0035] In another example, the antibodies described herein are chimeric antibodies that can include heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody that has a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from one mammalian species (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant portions are homologous to the sequences of antibodies from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable and / or constant regions.

[0036] In some embodiments, the anti-FXIIa antibodies described herein specifically bind to a corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts with a particular target antigen more frequently and rapidly, with longer duration, and / or with higher affinity than it reacts with other targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity and avidity, more readily, and / or with longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (FXIIa) or an antigenic epitope therein is an antibody that binds to this target antigen with higher affinity and avidity, more readily, and / or with longer duration than it binds to other antigens or other epitopes of the same antigen. It is understood by this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specifically binds" or "preferentially binds" does not necessarily require (although can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes of the same antigen. In some embodiments, the antibodies described herein specifically bind to FXIIa. In some embodiments, the antibodies described herein specifically bind to FXIIa and do not bind to FXII.

[0037] As used herein, the terms "factor XIIa" or "FXIIa" refer to the activated form of factor XII, and "factor XII" or "FXII" refer to the factor XII proenzyme, i.e., the proenzyme (inactive form). FXII is a single-chain glycoprotein with a molecular weight of approximately 80 kD. Upon activation, FXII is converted to the activated form FXIIa, which contains two chains held together by disulfide bonds: a heavy chain (353 residues of the human FXIIa heavy chain) and a light chain (243 residues of the human FXIIa light chain). Human FXII is encoded by the gene F12. The amino acid sequence of human FXII is well known in the art (e.g., GenBank accession number NP_000496.2).

[0038] In some embodiments, the anti-FXIIa antibodies described herein have suitable binding affinity for a target antigen (e.g., FXIIa) or its antigenic epitope. As used herein, "binding affinity" refers to the apparent binding constant, or K A K A is the dissociation constant (K D ) is the reciprocal of the . The anti-FXIIa antibodies described herein have a binding affinity of at least 10 to the target antigen or antigen epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (K D The increase in binding affinity may be due to the K D The higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a decrease in the K for binding to the second antigen. A (or a number K D ) for binding to the first antigen A (or a smaller number K D) in a specific manner. In such cases, the antibody has specificity for a first antigen (e.g., a first protein or mimetic thereof in a first conformation) compared to a second antigen (e.g., the same first protein or mimetic thereof or a second protein in a second conformation). In some embodiments, the anti-FXIIa antibodies described herein have a higher binding affinity (higher K ) for FXIIa compared to the binding affinity for the FXII zymogen or another protein in the pKal signaling pathway. A or smaller K D The difference in binding affinity (e.g., for specificity or other comparisons) is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 In some embodiments, any of the anti-FXIIa antibodies may be further affinity matured to increase the binding affinity of the antibody to its target antigen or antigenic epitope.

[0039] Binding affinity (or binding specificity) can be determined by various methods, such as equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v / v) surfactant P20). Using these techniques, the concentration of bound binding protein can be measured as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) according to the following equation: [Bound]=[Free] / (Kd+[Free])

[0040] However, K AIt is not necessary to make an accurate determination of K, since it may be sufficient to obtain a quantitative measure of affinity, measured using methods such as, for example, ELISA or FACS analysis, which may be used to determine the K. A , and can be used for comparisons, such as determining whether higher affinity is, for example, 2-fold higher, to obtain a qualitative measure of affinity or to obtain an inference of affinity, for example, by activity in a functional assay, e.g., an in vitro or in vivo assay.

[0041] In some embodiments, an anti-FXIIa antibody comprises a heavy chain comprising a heavy chain variable region comprising heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. In some cases, the heavy chain CDR3 region comprises the amino acid sequence of SEQ ID NO: 111, 112, or 113 (see Table 1). The heavy chain variable region of any of the anti-FXIIa antibodies described herein may comprise a CDR1 region comprising the formula X1YX3MX5 (SEQ ID NO: 117) as described herein and / or the formula X1IX3PSGX7X8TX, also as described herein. 10 YX 12 and may further comprise a CDR2 region comprising DSVKG (SEQ ID NO: 118). In some examples, the anti-FXIIa antibody comprises a heavy chain CDR1 region selected from SEQ ID NOs: 41-73 and 121 (see Table 1) and / or a CDR2 region selected from SEQ ID NOs: 74-110 and 122-124. In some particular examples, the heavy chain variable region of the anti-FXIIa antibodies described herein comprises the amino acid sequence of any of SEQ ID NOs: 1-39.

[0042] Table 1 shows the amino acid sequences of the heavy chain CDRs of exemplary anti-FXIIa antibodies. Antibodies having the same heavy chain CDR1, CDR2 and CDR3 regions as the exemplary anti-FXIIa antibodies are also within the scope of this disclosure. [Table 1] TIFF0007753301000003.tif205158TIFF0007753301000004.tif93158

[0043] The heavy chain variable regions of exemplary anti-FXIIa antibodies listed in Table 1 are shown below (CDR regions are shown in bold): In other embodiments, the HC CDR1 of the anti-FXIIa antibody may be QYSMH ​​(SEQ ID NO: 121), optionally in combination with the HC CDR2 of SEQ ID NO: 74 and / or the HC CDR3 of SEQ ID NO: 111. In yet other embodiments, the HC CDR2 of the anti-FXIIa antibody may be SIYPSGGKTRYADSVKG (SEQ ID NO: 122), VIWPSGGKTRYADSVKG (SEQ ID NO: 123), or VIYPSGGHTRYADSVKG (SEQ ID NO: 124), optionally in combination with the HC CDR1 of SEQ ID NO: 41 and / or the HC CDR3 of SEQ ID NO: 111. In yet another embodiment, the HC CDR2 of the anti-FXIIa antibody may be SIWPSGGHTRYADSVHG (SEQ ID NO: 127), optionally in combination with the HC CDR1 of SEQ ID NO: 58 and / or the HC CDR3 of SEQ ID NO: 111. Heavy chain variable region sequence of M0191-E09 (SEQ ID NO: 1): [ka] Heavy chain variable region sequence of M0192-H11 (SEQ ID NO:2): [ka] Heavy chain variable region sequence of M0184-F12 (SEQ ID NO:3): [ka] Heavy chain variable region sequence of M0292-D07 (SEQ ID NO:4): [ka] Heavy chain variable region sequence of M0182-D04 (SEQ ID NO:5): [ka] Heavy chain variable region sequence of M192-F07 (SEQ ID NO:6): [ka] Heavy chain variable region sequence of M0183-C03 (SEQ ID NO:7): [ka] Heavy chain variable region sequence of M0183-H08 (SEQ ID NO:8): [ka] Heavy chain variable region sequence of M0183-D08 (SEQ ID NO:9): [ka] Heavy chain variable region sequence of M0192-G03 (SEQ ID NO: 10): [ka] Heavy chain variable region sequence of M0310-C06 (SEQ ID NO:11): [ka] Heavy chain variable region sequence of M0192-F01 (SEQ ID NO: 12): [ka] Heavy chain variable region sequence of M0191-A03 (SEQ ID NO: 13): [ka] Heavy chain variable region sequence of M0192-A01 (SEQ ID NO: 14): [ka] Heavy chain variable region sequence of M0191-H10 (SEQ ID NO: 15): [ka] Heavy chain variable region sequence of M0177-C12 (SEQ ID NO: 16): [ka] Heavy chain variable region sequence of M0184-B04 (SEQ ID NO: 17): [ka] Heavy chain variable region sequence of M0308-H03 (SEQ ID NO: 18): [ka] Heavy chain variable region sequence of M0192-D02 (SEQ ID NO: 19): [ka] Heavy chain variable region sequence of M0310-G07 (SEQ ID NO:20): [ka] Heavy chain variable region sequence of M0192-F06 (SEQ ID NO:21): [ka] Heavy chain variable region sequence of M0310-A04 (SEQ ID NO:22): [ka] Heavy chain variable region sequence of M0310-F02 (SEQ ID NO:23): [ka] Heavy chain variable region sequence of M0310-B09 (SEQ ID NO:24): [ka] Heavy chain variable region sequence of M0310-G06 (SEQ ID NO:25): [ka] Heavy chain variable region sequence of M0182-H01 (SEQ ID NO:26): [ka] Heavy chain variable region sequence of M0178-A08 (SEQ ID NO:27): [ka] Heavy chain variable region sequence of M0184-D01 (SEQ ID NO:28): [ka] Heavy chain variable region sequence of M0177-A06 (SEQ ID NO:29): [ka] Heavy chain variable region sequence of M0191-E04 (SEQ ID NO:30): [ka] Heavy chain variable region sequence of M0191-H09 (SEQ ID NO:31): [ka] Heavy chain variable region sequence of M0192-H04 (SEQ ID NO:32): [ka] Heavy chain variable region sequence of M0192-A03 (SEQ ID NO:33): [ka] Heavy chain variable region sequence of M0310-G08 (SEQ ID NO:34): [ka] Heavy chain variable region sequence of M0192-G05 (SEQ ID NO:35): [ka] Heavy chain variable region sequence of M0183-B12 (SEQ ID NO:36): [ka] Heavy chain variable region sequence of M0308-F04 (SEQ ID NO:37): [ka] Heavy chain variable region sequence of M0182-B04 (SEQ ID NO:38): [ka] Heavy chain variable region sequence of M0310-F04 (SEQ ID NO:39): [ka] Heavy chain variable region sequence of X211-A01 (SEQ ID NO: 125) [ka]

[0044] The present disclosure also provides germline variants of any of the exemplary anti-FXIIa antibodies disclosed herein. A germline variant contains one or more mutations in the framework region when compared to its parent antibody with the corresponding germline sequence. To create a germline variant, the heavy or light chain variable region sequence of a parent antibody, or a portion thereof (e.g., framework sequence), can be used to query an antibody germline sequence database (e.g., www.bioinfo.org.uk / abs / , www.vbase2.org, or www.imgt.org) to identify the corresponding germline sequence used by the parent antibody and amino acid residue mutations in one or more framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions based on the germline sequence can then be introduced into the parent antibody to create the germline variant. For example, clone X211-A01 (DX-4012) is a germline variant of clone M192-H11. As described herein, anti-FXIIa antibodies may be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), and single-chain antibodies.

[0045] In some embodiments, the heavy chain of any of the anti-FXIIa antibodies described herein may further comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be derived from any suitable organism, for example, human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (γ heavy chain). One exemplary human heavy chain constant region is shown below (SEQ ID NO: 119). [ka]

[0046] Residues shown in bold and underlined represent residues that can be mutated to enhance FcRn binding and thus increase serum half-life.

[0047] In some embodiments, the anti-FXIIa antibody comprises the heavy chain constant region of SEQ ID NO: 119. Alternatively, the heavy chain constant regions of the antibodies described herein may comprise a single domain (e.g., CH1, CH2, or CH3) or any combination of single domains of the constant region (e.g., SEQ ID NO: 119).

[0048] Optionally, the anti-FXIIa antibodies described herein may contain modified constant regions. For example, the antibodies may contain modified constant regions that are immunologically inert, e.g., do not induce complement-mediated lysis or stimulate antibody-dependent cellular cytotoxicity (ADCC). ADCC activity can be assessed using the methods disclosed in U.S. Pat. No. 5,500,362. In other embodiments, the constant regions are modified as described in Eur. J. Immunol. (1999) 29:2613-2624, PCT Application No. PCT / GB99 / 01441, and / or UK Patent Application No. 9809951.8.

[0049] In some embodiments, the heavy chain constant region used in the anti-FXIIa antibodies described herein may contain mutations (e.g., amino acid residue substitutions) to enhance desired properties of the antibody, such as increasing binding activity to the neonatal Fc receptor (FcRn), and thus increasing the serum half-life of the antibody. Binding to FcRn has been known to be important for maintaining antibody homeostasis and regulating the serum half-life of antibodies. One or more (e.g., 1, 2, 3, 4, 5, or more) mutations (e.g., amino acid residue substitutions) may be introduced into the constant region at suitable positions (e.g., within the CH2 region) to enhance FcRn binding and thereby increase the half-life of the antibody. Such mutations may be at positions corresponding to 145, 147, and / or 149 of SEQ ID NO: 119 (positions corresponding to 252, 254, and 256 based on the numbering system reported in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, DC). See also Dall'Acqua et al., JBC, 2006, 281:23514-23524, Robbie et al., Antimicrob. Agents Chemother, 2013, 57(12):6147, and Dall'Acqua et al., J. Immunol. 2002 169:5171-5180. If a constant region other than SEQ ID NO: 119 is used, positions at which mutations can be introduced can be determined by aligning the amino acid sequence of the constant region with SEQ ID NO: 119 and identifying positions corresponding to positions of interest (e.g., positions 145, 147, or 149 in SEQ ID NO: 119), or such positions can be determined by well-known numbering systems, such as the Kabat numbering system described above.

[0050] In some examples, a substitution mutation (e.g., an M to Y substitution) is introduced into the amino acid residue (e.g., a methionine residue) at position 252 (position 145 of SEQ ID NO: 119) of the heavy chain constant region. In some examples, a substitution mutation (e.g., an S to T substitution) is introduced into position 254 (position 147 of SEQ ID NO: 119) of the heavy chain constant region. In some examples, a substitution mutation (e.g., a T to E) is introduced into position 256 (position 149 of SEQ ID NO: 119) of the heavy chain constant region. In some examples, the threonine at position 256 is mutated to a glutamic acid residue. If desired, multiple mutations (e.g., amino acid substitutions) may be introduced into the heavy chain constant region (e.g., any combination of the above substitutions). In a particular example, the modified heavy chain constant region used in any of the anti-FXIIa antibodies can contain amino acid substitutions at positions 252, 254, and 256 (e.g., M252Y, S254T, and T256E, also known as the YTE mutant). See, for example, Dall'Acqua et al., JBC, 2006, 281:23514-23524, Robbie et al., Antimicrob. Agents Chemother, 2013, 57(12):6147, and Dall'Acqua et al., J. Immunol. 2002 169:5171-5180. The following amino acid sequence (SEQ ID NO: 120) represents an exemplary modified heavy chain constant region with improved FcRn binding activity and, therefore, increased serum half-life. Exemplary heavy chain constant region sequence of a YTE variant (SEQ ID NO: 120): [ka]

[0051] The amino acid residues that are mutated in SEQ ID NO:120 compared to SEQ ID NO:119 are shown in bold.

[0052] Any of the anti-FXIIa antibodies described herein may further comprise a light chain comprising a light chain variable region and, optionally, a light chain constant region, which may be any CL known in the art. In some examples, the CL is a κ light chain. In other examples, the CL is a λ light chain. Antibody heavy and light chain constant regions, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php (both of which are incorporated herein by reference), are well known in the art.

[0053] The anti-FXIIa antibodies described herein may comprise a light chain variable region comprising a light chain CDR3 set forth as SEQ ID NO: 116 (see Table 2). In some embodiments, the light chain variable region of the anti-FXIIa antibody may further comprise a light chain CDR1 region set forth as SEQ ID NO: 114 (see Table 2) and / or a light chain CDR2 region set forth as SEQ ID NO: 115 (see Table 2). For example, the anti-FXIIa antibody light chain variable region may comprise a light chain CDR1 of SEQ ID NO: 114, a light chain CDR2 of SEQ ID NO: 115, and a light chain CDR3 of SEQ ID NO: 116. In one example, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40. Alternatively, the light chain variable region is a germline variant of SEQ ID NO: 40, which may contain one or more mutations within one or more frameworks relative to the corresponding germline sequence. Corresponding germline sequences can be identified by methods known in the art and described herein.

[0054] An exemplary light chain variable region of an anti-FXIIa antibody is shown below (CDR regions are shown in bold): Exemplary light chain variable region of an anti-FXIIa antibody (SEQ ID NO: 40): [ka] Exemplary Light Chain Variable Region of an Anti-FXIIa Antibody (SEQ ID NO: 126) [ka] [Table 2]

[0055] In some embodiments, an anti-FXIIa antibody described herein comprises a heavy chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 1-39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40. In other embodiments, an anti-FXIIa antibody described herein (e.g., X211-A01, also known as DX-4012) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 125 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 126.

[0056] Functional variants of any of the exemplary anti-FXIIa antibodies disclosed herein (e.g., the above-mentioned 44 antibody clones) are also within the scope of the present disclosure. In some examples, the anti-FXIIa antibody is a functional variant of an antibody comprising a heavy chain variable region provided by any one of SEQ ID NOS: 1-39 and a light chain variable region provided by SEQ ID NOS: 40. Functional variants may contain up to five (e.g., four, three, two, or one) amino acid residue mutations in one or more of the CDR regions of the antibody, and may have substantially similar affinities (e.g., K of the same order). DThe amino acid mutations bind to the same epitope of FXIIa at the same site (having a specific amino acid residue value). In one example, the amino acid residue mutation is a conservative amino acid substitution. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those described in references that compile such methods, such as "Molecular Cloning: A Laboratory Manual," edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or "Current Protocols in Molecular Biology," edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0057] In some embodiments, an anti-FXIIa antibody comprises a heavy chain CDR that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to any one of the heavy chain CDR sequences provided by SEQ ID NOs: 41-113, 121-124, and 127, and / or a light chain CDR sequence that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to at least one of the corresponding light chain CDRs provided by SEQ ID NOs: 114-116. In some embodiments, an anti-FXIIa antibody comprises a heavy chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of any of SEQ ID NOs: 1-39 and / or a light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region provided by SEQ ID NO: 40.

[0058] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm has been incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0059] Additionally, anti-FXIIa antibodies may contain one or more other residues identified based on the crystal structure discussed in Example 5 herein as being involved in interactions with the C chain of FXIIa. These residues may be located in the VH or VL chain. Examples include T28, S30, Q31, W52, P53, S54, G55, G56, H57, R59, N74, R100, Y101, R102, G103, P104, K105, Y106, Y107, and Y108 in the heavy chain variable region and H31, N33, Y35, Y54, L55, N58, and T99 in the light chain variable region.

[0060] Also provided herein are antibodies that target specific residues in FXIIa. The antibodies can preferentially bind to FXIIa but cannot bind to FXII. In some embodiments, the antibodies that specifically bind to FXIIa include the following residues: L390, Y391, W392, G393, H394, S395, F396, C397, H412, C413, L414, Q415, D416, R432, N433, V456, Y458, H507, F509, E510, G511, A512, E513, Y515, D55 The FXIIa C chain interacts with one or more (e.g., at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, or 45) residues of the FXIIa C chain, such as A558, C559, Q560, G561, D562, S563, I584, S585, W586, G587, S588, G589, C590, G591, D592, and G597. These residues are identified as interacting with one or more residues in the heavy and light chain variable regions of anti-FXIIa antibodies according to the crystal structure described in Example 5 below.

[0061] An exemplary amino acid sequence of human FXIIa (SEQ ID NO: 128) is shown below, with the above residues highlighted in bold. [ka]

[0062] Other exemplary FXIIa sequences can include human, mouse, or rat FXIIa sequences, sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of these sequences, or fragments thereof.

[0063] Interacting means that the distance between two residues in the complex formed by two binding partners is less than a predetermined value, e.g., <6 angstroms, <4 angstroms, or <2 angstroms. For example, an interacting residue in one binding partner can have at least one atom within a given threshold (e.g., <6 angstroms, <4 angstroms, or <2 angstroms) of at least one atom from a residue of the other binding partner in the complex structure. An interaction does not require actual binding. The interacting residues are suggested to be involved in antibody recognition.

[0064] In some embodiments, the antibodies described herein bind to human active FXIIa at an epitope comprising one or more of the residues listed above. "Epitope" refers to the site on a target compound bound by an antibody, such as a Fab or full-length antibody. An epitope may be linear, typically 6-15 aa in length. Alternatively, the epitope may be conformational.

[0065] In some examples, the antibodies described herein that specifically bind to FXIIa bind to an epitope that includes a segment of SEQ ID NO: 128, i.e., residues 390-397, residues 412-416, residues 432-433, residues 456-458, residues 507-515, residues 557-563, and / or residues 584-592.

[0066] Preparation of anti-FXIIa antibody Antibodies capable of binding to FXIIa described herein can be prepared by any method known in the art, see, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0067] In some embodiments, antibodies specific to a target antigen (e.g., FXIIa or its catalytic domain) can be prepared by conventional hybridoma technology. A full-length target antigen or a fragment thereof, optionally conjugated to a carrier protein such as KLH, can be used to immunize a host animal to produce antibodies that bind to the antigen. The route and schedule of immunization of the host animal generally follows established conventional techniques for antibody stimulation and production, as further described herein. General techniques for producing murine, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells therefrom can be engineered to serve as a platform for the production of mammalian (including human) hybridoma cell lines. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of an immunogen, such as those described herein.

[0068] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or a modified version of Buck, DW et al., In Vitro, 18:377-381 (1982). Available myeloma lines, such as, but not limited to, X63-Ag8.653 and those from the Cell Distribution Center at the Salk Institute, San Diego, California, may be used for hybridization. Generally, the technique involves fusing myeloma cells with lymphoid cells using a fusogen, such as polyethylene glycol, or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Any of the media described herein (with or without serum supplementation) can be used to culture hybridomas secreting monoclonal antibodies. As another alternative to cell fusion techniques, EBV-immortalized B cells may be used to produce the anti-FXIIa monoclonal antibodies described herein. Hybridomas are expanded and subcloned, and if desired, supernatants are assayed for anti-immunogen activity by conventional immunological assays (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0069] Hybridomas that can be used as antibody sources include all derivatives and progeny of the parent hybridoma that produce monoclonal antibodies capable of blocking FXIIa activity. Hybridomas producing such antibodies may be grown in vitro or in vivo using known procedures. If desired, monoclonal antibodies may be isolated from culture media or body fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed by, for example, flowing the preparation over an adsorbent made of immunogen attached to a solid phase, thereby eluting or releasing the desired antibody from the immunogen. Antibody (e.g., monoclonal) populations can be obtained by immunizing a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional agent or derivatizing agent such as maleimidobenzoyl succinimide sulfide ester (attached through a cysteine ​​residue), N-hydroxysuccinimide (through a lysine residue), glutaraldehyde, succinic anhydride, SOCl, or RN=C=NR (where R and R are different alkyl groups).

[0070] If desired, the antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in a vector within a host cell, which may then be propagated and frozen for later use. Alternatively, the polynucleotide sequence may be used for genetic engineering to "humanize" the antibody or improve its affinity (affinity maturation) or other properties. For example, the constant region may be engineered to more closely resemble a human constant region to avoid an immune response when the antibody is used in human clinical trials and treatments. It may be desirable to genetically engineer the antibody sequence to obtain higher affinity for the target antigen and greater efficacy in inhibiting the activity of FXIIa. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody while still maintaining its binding specificity for the target antigen.

[0071] In other embodiments, fully human antibodies can be obtained using commercially available mice engineered to express specific human immunoglobulin proteins. For the production of humanized or human antibodies, transgenic animals designed to generate a more desirable (e.g., fully human) or stronger immune response may also be used. Examples of such technologies are the Xenomouse® from Amgen (Fremont, CA) and the HuMAb-Mouse® and TC Mouse™ from Medarex (Princeton, NJ). In another alternative, antibodies may be prepared recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al. (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) region gene repertoires obtained from unimmunized donors.

[0072] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods, for example, F(ab')2 fragments can be generated by pepsin digestion of the antibody molecule and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment.

[0073] For example, genetically engineered antibodies such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies can be produced using conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be inserted into one or more expression vectors, which can then be transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells (where immunoglobulin proteins would not otherwise be produced), resulting in the synthesis of monoclonal antibodies in the recombinant host cells. See, for example, PCT Application WO 87 / 04462. This DNA can then be modified, for example, by substituting the coding sequences for human heavy and light chain constant regions for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, with the binding specificity of a target antigen can be prepared.

[0074] Techniques developed for the production of "chimeric antibodies" are well known in the art. See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851, Neuberger et al. (1984) Nature 312, 604, and Takeda et al. (1984) Nature 314:452.

[0075] Methods for constructing humanized antibodies are also well known in the art. For example, see Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the VH and VL variable regions of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Then, the framework amino acid residues that are predicted to be important for the formation of accurate CDR structures are identified using the same molecular modeling analysis. In parallel, the parent VH and VL sequences are used as search queries to identify human VH and VL chains with amino acid sequences homologous to the amino acid sequences of the parent non-human antibody from any antibody gene database. Then, human VH and VL acceptor genes are selected.

[0076] The CDR regions in the selected human acceptor gene can be replaced with CDR regions from the parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain predicted to be important in interacting with the CDR regions (see above) can be used to replace the corresponding residues in the human acceptor gene.

[0077] Single-chain antibodies can be prepared by recombinant techniques by linking nucleotide sequences encoding heavy-chain variable regions with nucleotide sequences encoding light-chain variable regions. A flexible linker is preferably incorporated between the two variable regions. Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be modified to construct phage or yeast scFv libraries, from which FXIIa-specific scFv clones can be identified using standard procedures. Positive clones can be further screened to identify those that inhibit FXIIa activity.

[0078] Antibodies obtained according to the methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which an antigen binds, i.e., "epitope mapping." Many methods are known in the art for mapping and characterizing the location of epitopes on proteins, such as analyzing the crystal structure of an antibody-antigen complex, competitive assays, gene fragment expression assays, and assays using synthetic peptides, as described in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope may be a linear epitope (i.e., contained in a single chain of amino acids) or a conformational epitope formed by three-dimensional interactions of amino acids that are not necessarily contained in a single chain (linear sequence of primary structure). Peptides of variable lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, overlapping peptides derived from the target antigen sequence can be used to determine the epitope to which the antibody binds in a systematic screen. In a gene fragment expression assay, the open reading frame encoding the target antigen is fragmented randomly or by specific gene construction, and the reactivity of the antigen fragment expressed by the antibody being tested is determined. Gene fragments can be generated, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries) can also be used to identify specific epitopes.Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding assay. In a further example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify essential, sufficient, and / or necessary residues for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the FXIIa polypeptide are substituted (swapped) with sequences from a closely related but antigenically distinct protein (such as another member of the neurotrophin protein family). By assessing the binding of the antibody to mutant FXIIa, the importance of specific antigen fragments for antibody binding can be assessed.

[0079] Alternatively, competition assays can be performed with other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as other antibodies. Competition assays are well known to those of skill in the art.

[0080] In some instances, anti-FXIIa antibodies are prepared by recombinant techniques, as exemplified below.

[0081] The nucleic acids encoding the heavy and light chains of the anti-FXIIa antibody described herein can be cloned into a single expression vector with each nucleotide sequence operably linked to a suitable promoter. In one example, the nucleotide sequences encoding the heavy and light chains are operably linked to separate promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy chain coding sequence and the light chain coding sequence.

[0082] In some instances, the nucleotide sequences encoding the two chains of the antibody can be cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each of the chains can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow for the formation of the antibody.

[0083] Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector, operably linked to a suitable promoter, using methods known in the art. For example, the nucleotide sequence and vector can be contacted with a restriction enzyme under suitable conditions to form complementary ends on each molecule that can pair and be joined together by a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0084] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) immediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0085] Regulatable promoters can also be used, including those that use the lac repressor from Escherichia coli as a transcriptional regulator to regulate transcription from mammalian cell promoters containing the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] and those that use the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astradiol, RU486, diphenol murislerone or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

[0086] A regulatable promoter containing a repressor can be used in conjunction with an operon. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional regulator to regulate transcription from a mammalian cell promoter with a lac operator [M. Brown et al., Cell, 49:603-612 (1987)], Gossen and Bujard (1992), [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]. The tetracycline repressor (tetR) can be combined with a transcription activator (VP16) to form a tetR mammalian cell transcription activator fusion protein, i.e., tTa(tetR-VP16), which can be combined with a minimal promoter containing tetO from the human cytomegalovirus (hCMV) major immediate-early promoter to form a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, rather than a tetR mammalian cell transcription factor fusion derivative, can function as a powerful transregulator for regulating gene expression in mammalian cells when the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transcription activator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatory effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0087] Additionally, the vector may contain some or all of the following: a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells, an enhancer / promoter sequence from the human CMV immediate early gene for high-level transcription, transcription termination and RNA processing signals from SV40 for mRNA stability, the SV40 polyoma replication origin and ColE1 for proper episomal replication, an internal ribosome entry site (IRES), a versatile multiple cloning site, and T7 RNA and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for generating vectors containing transgenes are well known and available in the art.

[0088] Examples of polyadenylation signals useful in practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0089] To produce the antibodies, one or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies may be introduced into a suitable host cell. The host cell may be cultured under conditions suitable for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains may be recovered from cultured cells (e.g., from the cells or culture supernatant) by conventional methods, such as affinity purification. If necessary, the antibody polypeptide chains may be incubated under suitable conditions and for a suitable period of time to allow production of the antibody.

[0090] In some embodiments, the antibody preparation methods described herein require a recombinant expression vector encoding both the heavy and light chains of an anti-FXIIa antibody, as also described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformant host cells can be selected and cultured under suitable conditions that allow for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow for the formation of the antibody.

[0091] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-FXIIa antibody and the other encoding the light chain of an anti-FXIIa antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under appropriate conditions that allow expression of the antibody polypeptide chains. When the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then incubated under appropriate conditions that allow formation of the antibody. When the two expression vectors are introduced into different host cells, each can be recovered from the corresponding host cell or culture medium. The two polypeptide chains can then be incubated under conditions that allow formation of the antibody.

[0092] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Some antibodies can be isolated, for example, by affinity chromatography using a protein A or protein G-bound matrix.

[0093] Any nucleic acid encoding the heavy chain, light chain, or both, of the anti-FXIIa antibodies described herein, a vector (e.g., an expression vector) containing the same, and a host cell containing the vector are within the scope of the present disclosure.

[0094] Pharmaceutical Composition The antibodies described herein, as well as nucleic acids or nucleic acid sets encoding them, vectors containing them, or host cells containing those vectors, can be mixed with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions used to treat target diseases. By "acceptable," it is meant that the carrier must be compatible with the active ingredient of the composition (preferably capable of stabilizing the active ingredient) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20th Ed. (2000) Lippincott Williams and Wilkins, K. E. Hoover (eds.).

[0095] The pharmaceutical compositions used in the methods can include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, eds. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used and include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; serum albumin. , proteins such as gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, counterions that form salts such as sodium, metal complexes (e.g., Zn-protein complexes) and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0096] In some examples, the pharmaceutical compositions described herein include liposomes containing the antibody (or a nucleic acid encoding it), which can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0097] The antibody or nucleic acid encoding it may also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or interfacial polymerization, or in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. Such techniques are known in the art; see, for example, Remington, The Science and Practice of Pharmacy 20th Ed., Mack Publishing (2000).

[0098] In other examples, the pharmaceutical compositions described herein can be formulated for sustained release. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0099] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0100] The pharmaceutical compositions described herein may be in unit dosage form such as tablets, pills, capsules, powders, granules, solutions, suspensions or suppositories for oral, parenteral or rectal administration or for administration by inhalation or insufflation.

[0101] To prepare solid compositions such as tablets, the primary active ingredient can be mixed with a pharmaceutical carrier, e.g., corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, and other pharmaceutical diluents (e.g., water), to form a solid preformulation composition containing a homogeneous mixture of the compounds of the present invention or their non-toxic pharmaceutically acceptable salts. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredients are dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of this novel composition can be coated or otherwise compounded to provide a dosage form offering the advantage of prolonged action. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of an envelope surrounding the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including many polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0102] Suitable surfactants include non-ionic agents, such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85), among others. Compositions containing surfactants advantageously contain 0.05 to 5% of the surfactant, and may contain 0.1 to 2.5%. Of course, other ingredients, such as mannitol or other pharmaceutically acceptable vehicles, may be added if necessary.

[0103] Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient may be dissolved in a premixed emulsion composition or in an emulsion formed by mixing an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and a phospholipid (e.g., egg phospholipid, soybean phospholipid, or soybean lecithin) with water. Of course, other ingredients, such as glycerin or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, e.g., 5-20%. The fat emulsion may contain 0.1-1.0 μm, particularly 0.1-0.5 μm, of fat droplets and have a pH in the range of 5.5-8.0.

[0104] The emulsion composition may be a composition prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerin, and water).

[0105] Pharmaceutical compositions for inhalation or insufflation include pharmaceutically acceptable aqueous or organic solvents or mixtures thereof in solutions and suspensions, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0106] Compositions, preferably dissolved in sterile, pharmaceutically acceptable solvents, may be nebulized using a gas. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizer may be attached to a face mask tent or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[0107] Treatment method Any of the antibodies described herein, as well as nucleic acids or nucleic acid sets encoding same, vectors containing same, or host cells containing the vectors, are useful for treating diseases or disorders associated with abnormal contact system activation, such as diseases associated with contact system activation, diseases associated with abnormal contact system activation (e.g., HAE), or eye diseases.

[0108] To practice the methods disclosed herein, an effective amount of the pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in need of such treatment by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), or by a suitable route, such as intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical. Commercially available nebulizers for liquid formulations, such as jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered-dose inhalers, or inhaled as a lyophilized and milled powder.

[0109] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, livestock, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment may be a human patient who has, is at risk for, or is suspected of having a target disease / disorder, such as hereditary angioedema (HAE), thrombosis, or eye disease. A subject with a target disease or disorder can be identified by routine medical examination, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any of these target diseases / disorders may exhibit one or more symptoms of the disease / disorder. A subject at risk for the disease / disorder may also have one or more risk factors for the disease / disorder.

[0110] The methods and compositions described herein can be used to treat any disease or disorder associated with contact system activation and / or the pKal signaling pathway. In some embodiments, the target disease is thrombosis, such as thrombosis associated with atrial fibrillation, DVT, pulmonary embolism, stroke, or other arterial or venous thromboembolic events. Thrombosis (e.g., venous thrombosis or arterial thrombosis) refers to the formation of a blood clot inside a blood vessel that can obstruct the flow of blood through the circulatory system. Subjects with or at risk of thrombosis can be identified by routine medical procedures.

[0111] In other embodiments, diseases that can be treated with the anti-FXIIa antibodies described herein can be diseases associated with the kallikrein system (e.g., the pKal system), such as, but not limited to, macular edema, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, and retinal vein occlusion. Examples of diseases or disorders associated with the contact activation system include, but are not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, systemic lupus erythematosus, systemic lupus erythematosus nephritis, systemic mastocytosis, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, vasculitis, edema, acquired angioedema, idiopathic angioedema, anaphylaxis, and especially inflammatory bowel disease. These include acute anaphylaxis, cerebral edema, pulmonary embolism, stroke, clotting on a ventricular assist device or stent, clotting associated with the use of an indwelling catheter or peripherally inserted central catheter, clotting associated with the use of a membrane oxygenator, clotting associated with the use of a graft or shunt for dialysis, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) injury, ischemic event (stroke), restenosis (e.g., after angioplasty), or burns.

[0112] In some embodiments, diseases that can be treated with the anti-FXIIa antibodies described herein are ocular diseases associated with the contact activation system, such as, but not limited to, macular edema, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, and retinal vein occlusion.

[0113] In one example, a disease or condition associated with contact system activation is hereditary angioedema (HAE). Hereditary angioedema (HAE) is also known as "Quincke's edema," C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent episodes of severe swelling (angioedema), which can affect, for example, the hands, feet, face, genitals, gastrointestinal tract, and respiratory tract. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction, which may be accompanied by throat swelling and sudden hoarseness; recurrent episodes of abdominal cramps without apparent cause and / or intestinal swelling, which can become severe and cause abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with HAE develop a non-itchy rash called erythema marginatum during attacks.

[0114] Airway swelling can be life-threatening and can even be fatal in some patients. Mortality rates are estimated at 15-33%. HAE causes approximately 15,000-30,000 emergency room visits per year.

[0115] Trauma or stress, such as dental procedures, illness (e.g., viral illnesses such as colds and influenza), menstruation, and surgery can trigger attacks of angioedema. To prevent acute attacks of HAE, patients can try to avoid specific stimuli that have previously triggered attacks. However, attacks often occur without known triggers. Typically, HAE symptoms first appear in early childhood and worsen during adolescence. On average, untreated individuals have attacks every 1–2 weeks, with most episodes lasting approximately 3–4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, even among members of the same family.

[0116] There are three types of HAE, known as types I, II, and III. HAE is estimated to affect 1 in 50,000 people, with type I accounting for approximately 85% of cases, type II accounting for approximately 15% of cases, and type III being extremely rare. Type III is the newest form and was initially thought to occur only in women, but families with affected males have been identified.

[0117] HAE is inherited in an autosomal dominant manner, such that affected individuals may inherit a mutation from one affected parent. New mutations in genes can also occur, and thus, HAE can occur in people with no history of the disease in their family. It is estimated that 20-25% of cases are due to new, spontaneous mutations.

[0118] Mutations in the SERPING1 gene cause type I and type II hereditary angioedema. The SERPING1 gene provides instructions for the production of the C1 inhibitor protein, which is important for controlling inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema result in reduced levels of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of abnormally functioning C1 inhibitor. In the absence of adequate levels of functional C1 inhibitor, excessive amounts of bradykinin are produced. Bradykinin promotes inflammation by increasing fluid leakage from blood vessel walls into body tissues. Excessive accumulation of fluid in body tissues causes the swelling manifestations seen in individuals with type I and type II hereditary angioedema.

[0119] Mutations in the F12 gene have been associated with some cases of type III hereditary angioedema, also known as HAE with normal C1 inhibitor. The F12 gene provides the instructions for producing coagulation factor XII. Factor XII not only plays a key role in blood clotting (clotting) but is also an important stimulator of inflammation and is involved in the production of bradykinin. Specific mutations in the F12 gene result in the production of highly active factor XII. As a result, more bradykinin is produced, making blood vessel walls leakier, leading to the swelling episodes. The cause of other cases of type III hereditary angioedema remains unknown. Mutations in one or more as-yet-unidentified genes may be involved in the disorder in these cases.

[0120] Although HAE may present similarly to other forms of angioedema resulting from allergies or other medical conditions, it differs significantly in cause and treatment. When hereditary angioedema is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are generally ineffective in HAE, although epinephrine can be used for life-threatening reactions. Misdiagnosis has also led to unnecessary exploratory surgery for patients with abdominal swelling, and some patients with HAE have had their abdominal pain misdiagnosed as psychogenic.

[0121] Symptoms of HAE can be assessed, for example, using a questionnaire (e.g., a questionnaire completed by the patient, a clinician, or a family member). Such questionnaires are known in the art, and include, for example, visual analog scales. See, for example, McMillan, CV et al. Patient. 2012;5(2):113-26.

[0122] As used herein, "effective amount" refers to the amount of each active agent, alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is a decrease in FXIIa activity, a decrease in the amount of pKal or bradykinin, or a decrease in vasodilation. An antibody inhibitor that specifically binds to FXIIa but not to FXII may require a lower effective dose than an antibody inhibitor that also binds to FXII. Methods for determining whether the amount of antibody achieves a therapeutic effect will be clear to those of skill in the art. As will be recognized by those skilled in the art, effective amounts will vary depending on the specific condition being treated, the severity of the condition, individual patient parameters such as age, health, size, sex, and weight, the duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those of skill in the art and can be addressed with no more than routine experimentation. It is generally preferable to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment.

[0123] Empirical considerations such as half-life generally contribute to determining the dosage. For example, antibodies compatible with the human immune system, such as humanized or fully human antibodies, may be used to prolong the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration may be determined and adjusted over the course of treatment, and is generally (but not necessarily) based on the treatment and / or suppression and / or remission and / or delay of the target disease / disorder. Alternatively, sustained continuous release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0124] In one example, dosages of the antibodies described herein may be determined empirically in individuals who have received one or more doses of the antibody. The individual is given increasing doses of the antagonist. Indicators of the disease / disorder can be tracked to assess the efficacy of the antagonist.

[0125] Generally, for administration of any of the antibodies described herein, an initial candidate dosage may be about 2 mg / kg. For purposes of the present disclosure, typical daily dosages may range from anywhere from 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, up to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is continued until a desired suppression of symptoms occurs or sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms. An exemplary dosing regimen includes an initial dose of about 2 mg / kg, followed by a maintenance dose of about 1 mg / kg of the antibody once a week or once every two weeks at about 1 mg / kg. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the practitioner desires to achieve. For example, dosing one to four times a week is contemplated. In some embodiments, dosages ranging from about 3 μg / mg to about 2 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 2 mg / kg) may be used. In some embodiments, the dosing frequency is once per week, once per 2 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, or once per 10 weeks, or once per month, once per 2 months, or once per 3 months or more. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) may be varied over time.

[0126] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.3 to 5.00 mg / kg may be administered. In some examples, the dose of an anti-FXII antibody described herein (e.g., DX-4012) may be 10 mg / kg. The specific dosing regimen, i.e., dose, timing, and repetition, will depend on the particular individual and their medical history, as well as the characteristics of the individual drug (such as the drug's half-life and other considerations known in the art).

[0127] For purposes of this disclosure, appropriate dosages of the antibodies described herein will depend on the particular antibody, antibody and / or non-antibody peptide (or composition thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to antagonists, and the discretion of the attending physician. Typically, a clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in thrombosis. Methods for determining whether administration has produced the desired result will be apparent to those of skill in the art. Administration of one or more antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the administration is for therapeutic or prophylactic purposes, and other factors known to those of skill in the art. Administration of the antibody may be essentially continuous over a preselected period of time, or may be in a series of spaced doses, e.g., either before, during, or after the onset of the target disease or disorder.

[0128] The term "treating" as used herein refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, with the intent to cure, cure, alleviate, relieve, alter, repair, ameliorate, improve or affect the disorder, symptom of the disease, or predisposition to the disease or disorder.

[0129] Alleviating the target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Alleviating the disease does not necessarily require a curative result. As used herein, "delaying" the onset of the target disease or disorder means prolonging, preventing, slowing down, delaying, stabilizing, and / or postponing the progression of the disease. This delay may be of varying duration depending on the medical history and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the probability of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame compared to not using the method. Such comparisons are typically based on clinical trials using a large enough number of subjects to obtain statistically significant results.

[0130] "Onset" or "progression" of a disease refers to the first signs of disease and / or confirmed progression. Disease onset can be detected and assessed using standard clinical techniques as are well known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and / or recurrence.

[0131] In some embodiments, the antibodies described herein are administered to a subject in need of such treatment in an amount sufficient to inhibit the activity of one or both target antigens in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). In other embodiments, the antibodies are administered in an amount effective to reduce the activity level of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0132] The pharmaceutical composition can be administered to a subject using conventional methods known to those skilled in the art, depending on the type or location of the disease being treated. The composition can also be administered by other conventional routes, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Furthermore, the composition can be administered to a subject via a depot injection route, such as using a 1-, 3-, or 6-month depot injection material or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0133] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethyllactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerin, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by infusion, resulting in the infusion of a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient. Physiologically acceptable excipients include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular formulations, for example, sterile formulations of the antibody in the form of a suitable soluble salt, can be administered by dissolving them in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0134] In one embodiment, the antibody is administered by site-specific or targeted local delivery method.Examples of site-specific or targeted local delivery method include various implantable depot sources or local delivery catheters such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents, other implantable devices, site-specific carriers, direct injection or direct application.See, for example, PCT Application WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0135] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery methods are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (ed. JA Wolff) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0136] Therapeutic compositions containing polynucleotides (e.g., polynucleotides encoding antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for local administration in gene therapy protocols. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or greater can also be used during gene therapy protocols.

[0137] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be viral or non-viral in origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be either constitutive or regulated.

[0138] Viral vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Applications WO 90 / 07936, WO 94 / 03622, WO 93 / 25698, WO 93 / 25234, WO 93 / 11230, WO 93 / 10218, WO 91 / 02805, U.S. Patent Nos. 5,219,740 and 4,777,127, British Patent No. 2,200,651, and European Patent No. 0345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-1246)). Examples of suitable vectors include ATCC VR-923, ATCC VR-1250, ATCC VR1249, and ATCC VR-532) and adeno-associated virus (AAV) vectors (see, e.g., PCT Publications WO 94 / 12649, WO 93 / 03769, WO 93 / 19191, WO 94 / 28938, WO 95 / 11984, and WO 95 / 00655). Administration of DNA bound to killed adenovirus, as described in Curiel, Hum. Gene Ther. (1992) 3:147, can also be used.

[0139] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycationically condensed DNA alone, with or without binding to killed adenovirus (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147), DNA bound to a ligand (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985), eukaryotic cell delivery vehicles (see, e.g., U.S. Patent No. 5,814,482, PCT Publications WO 95 / 07994, WO 96 / 17072, WO 95 / 30763, and WO 97 / 42338), and nucleic acid charge neutralization or fusion with the cell membrane. Naked DNA can also be used. Exemplary methods for introducing naked DNA are described in PCT Publication WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can function as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT applications WO 95 / 13796, WO 94 / 23697, WO 91 / 14445, and EP 0 524 968. Further techniques are described in Philip, Mol. Cell. Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0140] The particular dosing regimen, i.e., dosage, timing, and repetition, employed in the methods described herein will depend on the particular subject and the subject's medical history.

[0141] In some embodiments, a combination of two or more antibodies, or an antibody and another suitable therapeutic agent, may be administered to a subject in need of such treatment. The antibodies may also be used in conjunction with other agents that function to enhance and / or complement the effectiveness of the agent in question.

[0142] The efficacy of a treatment for a target disease / disorder can be assessed by methods well known in the art.

[0143] Kits for use in alleviating diseases associated with contact system activation The present disclosure also provides kits for use in alleviating diseases / disorders associated with contact system activation, such as HAE. Such kits can include one or more containers containing an anti-FXIIa antibody, such as any of the antibodies described herein.

[0144] In some embodiments, the kit can include instructions for use in accordance with any of the methods described herein. The included instructions can include instructions for administering an anti-FXIIa antibody to treat, delay the onset of, or alleviate a target disease described herein. The kit can further include instructions for selecting an individual suitable for treatment based on identifying whether or not the individual has a target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for a target disease.

[0145] The instructions for use of the anti-FXIIa antibody typically include information regarding the dosage, administration schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable.

[0146] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or alleviating a disease or disorder associated with the pKal signaling pathway, such as HAE. Instructions may be provided for practicing any of the methods described herein.

[0147] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packaging for use in combination with a specific device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device, such as a minipump, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-FXIIa antibody as described herein.

[0148] The kit may optionally provide additional components, such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit.

[0149] General Technology The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of those in the art. Such techniques are described in such publications as "Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press," "Oligonucleotide Synthesis (MJ Gait, ed., 1984)," "Methods in Molecular Biology (Humana Press)," "Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press," "Animal Cell Culture (RI Freshney, ed., 1987)," "Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press," "Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-98) J. Wiley and Sons," and "Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (eds. D.M. Weir and C.C. Blackwell), Gene Transfer Vectors for Mammalian Cells (eds. J.M. Miller and M.P. Calos, 1987), Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Current Protocols in Immunology (JE Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory Antibodies are well explained in such references as the "Antibody Use, Laboratory Manual" by E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999) and "The Antibodies" by M. Zanetti and JD Capra (eds.), Harwood Academic Publishers, 1995.

[0150] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter discussed herein. [Example]

[0151] Example 1: Production of anti-FXIIa antibodies

[0152] Phage display selections using Dyax's FAB310 phagemid library were performed using biotinylated β-fragments of FXIIa immobilized on streptavidin-coated beads. Fab molecules that bound to the immobilized FXIIa were isolated. This selection resulted in a K of 800 pM. i app We generated a selective Fab inhibitor that specifically binds to the catalytic domain of FXIIa. The isolated Fab showed no cross-reactivity with the following proteases tested at a concentration of 1 μM: urokinase plasminogen activator, hepatocyte growth factor activator, activated protein C, cathepsin G, C1s, elastase, factor VIIa, factor Xa, factor XIa, plasmin, thrombin α, trypsin, urokinase, plasma kallikrein, hepatocyte growth factor activator, and urokinase-type plasminogen activator.

[0153] The isolated Fab, M71-F06, was affinity matured by multiple strategies, including heavy chain CDR1 / 2 shuffling, light chain shuffling, heavy chain CDR3 wobble, and a combination of heavy chain CDR3 wobble and heavy chain CDR1 / 2 shuffling. The CDR1 / 2 region of M71-F06 was removed and replaced with sufficient library diversity to yield approximately 10 8 A heavy chain CDR1 / 2 shuffling library was constructed by generating an affinity maturation library with a diversity of 1000 kDa. The heavy chain CDR3 was varied at each amino acid position from the parent CDR3 sequence of M71-F06 to generate a new library, which was then also combined with the HC CDR1 / 2 shuffling diversity. A second selection was performed using these libraries. In contrast to selections performed with immobilized FXIIa as used for direct library selection, the affinity maturation library was selected using the parent K i appThe FXIIa antibody was incubated in solution with the biotinylated target at a concentration lower than 1000 kJ / mL. Subsequently, the biotinylated FXIIa and any bound Fabs from the library were removed from the streptavidin-coated beads. Figure 2 shows a schematic diagram of the selection strategy for the identification and production of anti-FXIIa antibodies.

[0154] The inhibitory activity of clone M71-F06 against human and mouse FXIIa, as determined by the "in vitro" activity assay described in Example 2 below, is shown in Figure 4. The APTT values ​​of clone M71-F06 compared to DX-2930 (see Example 2 below) are shown in Figure 5. The results also showed that clone M71-F06 had no inhibitory activity against activated protein C, CCC 1s, cathepsin G, elastase, factor VIIa, factor Xa, factor XIa, activated plasma kallikrein, plasmin, thrombin α, trypsin, urokinase, HGFA, and uPA, indicating that its inhibitory activity is specific to FXIIa.

[0155] All isolated Fabs from the selection were screened by ELISA, and 39 unique isolates were obtained. The amino acid sequences of the heavy and light chain variable regions are shown above. These clones are expected to have the same antigen-binding activity and specificity as the parent clones, which have higher binding affinity.

[0156] Example 2: Characterization of anti-FXIIa antibodies

[0157] method: Activated partial thromboplastin time (APTT) assay Inhibitor (or control dilution buffer = 25 mM HEPES (pH 7.5), 125 mM NaCl) was added to pure plasma to a 1:1 mixture and pre-equilibrated at 37°C for 5 minutes. 2 x 50 μl of this mixture was dispensed into two separate KC4Delta assay cuvettes (with metal spheres). After 60 seconds, 50 μl of APTT reagent (activator, Pacific Hemostasis APTT-XL) was added to the rotating cuvette, and 180 seconds after APTT addition (t = 0 seconds), 50 μl of CaCl2 was added. Clotting times (seconds) were recorded on the KC4Delta instrument.

[0158] Prothrombin time (PT) assay Inhibitor (or control dilution buffer = 25 mM HEPES (pH 7.5), 125 mM NaCl) was added to pure plasma in a 1:1 mixture and pre-equilibrated at 37°C for 5 minutes. 2 x 50 μl of this mixture was dispensed into two separate KC4Delta assay cuvettes (with metal spheres). After 4 minutes, PT activator (Pacific Hemostasis Thromboplastin D) was added (t = 0 seconds). Clotting times were automatically recorded by the KC4Delta instrument.

[0159] Inhibition assay of purified components The inhibition assay of purified components is shown in Figure 3 (Panel 1). 20 pM FXIIa was incubated with various concentrations of inhibitors in a 96-well microplate at 30°C for 1 h. 10 nM prekallikrein was added for 20 min at 30°C, followed by a 5-min incubation with 100 nM corn trypsin inhibitor (CTI). Proteolysis was then assessed over time by the addition of 10 μM of the final fluorogenic peptide substrate (PFR-AMC). The initial rate of substrate proteolysis (y-axis) was plotted against the inhibitor concentration (x-axis), and the resulting data were fitted to the Morrison equation (Equation 1), modified for tight-binding inhibitors. All reagents were diluted in assay buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% PEG-8000, and 0.1% Triton X-100).

[0160] Plasma inhibition assay The plasma inhibition assay is shown in Figure 3 (panel 2). Pooled normal human plasma was diluted 1:40 in assay buffer (described above) and inhibitors were added at various concentrations to a 96-well microplate at room temperature. Contact activation was then initiated by the addition of 25% (2.5% final) ellagic acid, and the microplate was gently shaken to mix and allowed to proceed for 2 minutes at room temperature before the addition of 100 nM CTI. 10 μl of this mixture was then removed and placed in a replicate microplate containing 80 μl of assay buffer pre-equilibrated at 30°C. The dilution plate was then incubated for an additional 5 minutes at 30°C and a standard IC 50 Proteolysis of PFR-AMC was assessed as above, except that the back-calculated concentration of inhibitor was used on the x-axis for curve fitting to the equation (plasma was diluted 1:400 for the final assay reading).

[0161] One or more anti-FXIIa antibodies disclosed herein were also tested in a non-human primate model at two different doses. The study was conducted for 30 days, after which blood samples were drawn to assess pharmacokinetics, effects on APTT, and effects on plasma kallikrein activation by Western blot analysis.

[0162] Binding preference assay To determine the binding preference of anti-FXIIa antibodies, each antibody was incubated with FXII zymogen (another relevant protease of the contact / coagulation system). Binding affinity was then assessed by surface plasmon resonance (SPR) analysis using a Biacore biosensor. See Figure 3, panel 3.

[0163] Flow model / capillary blockage assay The antibodies were tested in an in vitro flow model, in which human blood spiked with the antibodies was passed through collagen-coated capillaries at different flow rates. Platelet and fibrin deposition was assessed by fluorescence. In this system, platelet and fibrin deposition was inhibited by antibodies that bind to the enzyme precursors factor XI or factor XII. In a typical experiment, the antibodies were bound to their targets before blood flow was initiated.

[0164] Mouse thrombosis model The antibody is tested in a mouse model of ferric chloride-induced carotid artery thrombosis. The model incorporates different FeCl concentrations, starting with the lowest concentration (3.5%) that consistently induces thrombus formation in C57B1 / 6 mice. If the antibody exhibits an antithrombotic effect at low FeCl concentrations, progressively higher concentrations are tested until the antibody's antithrombotic effect surpasses other concentrations.

[0165] result: Thirty-nine anti-FXIIa antibody isolates were tested in various in vitro activity assays and demonstrated the following properties: apparent K i ,I C 50 The binding preferences, including binding to the FXII zymogen, cross-reactivity with other related proteases in the contact / coagulation system, cross-reactivity with closely related sequence homologues, effect on plasma kallikrein (pKal) production, activity in human plasma, partial thromboplastin time (PT), activated partial thromboplastin time (APTT), and lag time for thrombin generation were determined.

[0166] The results of the assay are shown in Table 3. All 39 isolates were found to delay APTT without affecting PT. All 39 affinity-matured isolates each exhibited a 10- to 100-fold improvement in K over the parent isolate. i app The antibody reduced pKal production as measured in two independent inhibition assays: a purified component inhibition assay and a plasma inhibition assay.

[0167] SPR analysis (Biacore) showed that the antibody did not bind to the FXII zymogen and showed specificity for the catalytic domain when tested against full-length FXIIa. The antibody also prevented the activation of FXI to FXIa. [Table 3]

[0168] The inhibitory activity of clone M0192-H11 against human FXIIa is shown in Figure 21 and was found to be approximately 4.7±0.6 pM.

[0169] The anti-FXIIa antibodies M192-H11 and M192-E09 were also tested in an in vivo pharmacokinetic study in rats. Groups of rats were injected with 20 mg / kg of either the anti-FXIIa antibody M191-E09 or M192-H11. After various days of injection, samples were collected from the rats to assess the concentrations and pharmacokinetic parameters of the anti-FXIIa antibodies (Figure 6 and Table 4). [Table 4]

[0170] Example 3: Expression and characterization of anti-FXIIa antibody 559C-X211-A01

[0171] 559C-X211-A01 is an affinity matured, partially germlined version of the previously described parent antibody 559C-M71-F06. The amino acid sequences of the heavy and light chain variable regions of this antibody are shown below. 559C-X211-A01_HV (CDRs are underlined and in bold) [ka] 559C-X211-A01_LV (CDRs are underlined and in bold) [ka]

[0172] 559C-X211-A01 was tested in various in vitro activity assays and demonstrated the following properties: apparent K i ,I C 50 The binding to FXII zymogen, cross-reactivity with other related proteases in the contact / coagulation system, cross-reactivity with closely related sequence homologues, effect on plasma kallikrein (pKal) production, and activity in human plasma were determined. The partial thromboplastin time (PT) and activated partial thromboplastin time (APTT) in mouse plasma were also tested in vitro.

[0173] Cross-reactivity assay 559C-X211-A01 showed no cross-reactivity with the following proteases tested at 1 μM: urokinase plasminogen activator, human growth factor activator, activated protein C, cathepsin G, C1s, elastase, factor VIIa, factor Xa, factor XIa, plasmin, thrombin α, trypsin, urokinase, and plasma kallikrein.

[0174] Activated partial thromboplastin time (APTT) and prothrombin time assays Inhibitor (or control dilution buffer = 25 mM HEPES (pH 7.5), 125 mM NaCl) was added to both pure mouse and human plasma in a 1:1 mixture and pre-equilibrated at 37°C for 5 minutes. 2 x 50 μl of this mixture was dispensed into two separate KC4Delta assay cuvettes (with metal spheres). After 60 seconds, 50 μl of APTT reagent (activator, Pacific Hemostasis APTT-XL) was added to the rotating cuvette, and 180 seconds after this (t = 0 seconds), 50 μl of CaCl2 was added. Clotting times (seconds) were recorded on the KC4Delta instrument.

[0175] As above, 2 x 50 μl of the inhibitor / plasma mixture was dispensed into two separate KC4Delta assay cuvettes, but not after 4 min, and PT activator (Pacific Hemostasis Thromboplastin D) was added at t = 0 s. Clotting times were recorded automatically on the KC4Delta instrument.

[0176] 559C-X211-A01 delayed APTT in mouse plasma but had no effect on PT (Figure 7), supporting species cross-reactivity with mouse factor XII.

[0177] Inhibition assay of purified components: In a 96-well microplate, 20 pM FXIIa was incubated with various concentrations of inhibitors at 30°C for 1 hour. Then, 10 nM prekallikrein was added for 20 minutes at 30°C, followed by incubation with 100 nM corn trypsin inhibitor (CTI) for 5 minutes. Proteolysis was then assessed over time by adding 10 μM of the final fluorogenic peptide substrate (PFR-AMC). The initial rate of substrate proteolysis (y-axis) was plotted against the inhibitor concentration (x-axis), and the resulting data were fitted to the Morrison equation, modified for tight-binding inhibitors. All reagents were diluted in assay buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% PEG-8000, and 0.1% Triton X-100).

[0178] Plasma Inhibition Assay: Pooled normal human plasma was diluted 1:40 in assay buffer (described above), and various concentrations of inhibitor were added to a 96-well microplate at room temperature. Contact activation was then initiated by the addition of 25% (2.5% final) APTT-XL reagent (diluted ellagic acid, Pacific Hemostasis). The microplate was gently shaken to mix and the reaction was allowed to proceed at room temperature for 2 minutes, after which 100 nM CTI was added. 10 μl of this mixture was then removed and placed in a replicate microplate containing 80 μl of assay buffer pre-equilibrated at 30°C. The dilution plate was then incubated for an additional 5 minutes at 30°C, and proteolysis of PFR-AMC was assessed as described above, except that the back-calculated inhibitor concentration was used on the x-axis for curve fitting to the standard IC50 equation and / or modified Morrison equation (plasma was diluted 1:400 for the final assay reading).

[0179] The activity of 559C-X211-A01 observed in the above assays is summarized in Table 5 below. [Table 5]

[0180] SPR analysis showed that this antibody binds to FXIIa but not to the FXII zymogen. Enzyme inhibition experiments with FXIIa-β demonstrate specific targeting to the catalytic domain.

[0181] 559C-X211-A01 is expected to reduce fibrin generation and deposition, prolong the time it takes blood to occlude collagen-coated capillaries in a flow model / capillary occlusion assay, and / or reduce the incidence of platelet aggregation and arterial occlusion. 559C-211-A01 is expected to reduce the incidence of thrombus formation in both mouse models of thrombosis and non-human primate models of thrombosis.

[0182] Example 4: In vivo thrombosis study with DX-4012

[0183] Pharmacokinetic studies The anticoagulant effect of a single dose of DX-4012 was evaluated in healthy baboons as a pharmacodynamic marker of the antibody's presence in the circulation. The animals were gently sedated, and blood was collected from the antecubital vein for baseline coagulation values. The antibody was then administered at a saturating dose. A single dose of the antibody was delivered intravenously, and blood samples were collected at intervals sufficient to confirm anticoagulation by APTT assay against baseline. Plasma samples were also frozen for further testing.

[0184] Following completion of the pharmacokinetic studies in baboons and determination of the effective half-life of the antibody, pharmacodynamic thrombosis and hemostasis studies were carried out.

[0185] Pharmacodynamic thrombosis research Acute thrombosis and hemostasis experiments were performed before and during anticoagulation to determine the efficacy and safety of the antibody at a single saturating dose compared with positive and negative controls. Thrombosis experiments were performed in trained baboons by deploying vascular graft segments into chronic superficialized femoral AV shunts. On each day of the study, 1 mL of citrated blood was collected from the shunt loop at each time point for serial bleeding time, volumetric measurements, and anticoagulation assays. Sample collection times included pre-drug (on the day the animals received enoxaparin or DX-4012, before administration of enoxaparin or DX-4012), pre-study (5 minutes before the start of the study), and post-study (5 minutes before the end of the study, when the shunt loop was removed). Prior to the thrombosis and hemostasis experiments, the baboons were 111 In-radiolabeled autologous platelets and 125 1 I radiolabeled fibrinogen was administered. The antithrombotic activity of DX-4012 was compared with that of enoxaparin (low molecular weight heparin).

[0186] After baseline blood samples were collected, the thrombosis experiment was initiated. A chronic AV shunt was temporally stretched (60 min) using a silicon tube, and a short collagen ePTFE graft segment or a tissue factor-coated ePTFE vascular graft segment (20 mm) was deployed within the external AV loop (Figure 10). A thrombus formed within the graft (thrombus head) and extended distally (thrombus tail). The accumulation of radiolabeled platelets within the graft was monitored in real time for 60 min using gamma camera imaging and calculated as the number of platelets present within the graft at 5-min intervals. After 60 min of graft perfusion, the graft was removed, the shunt was reconnected, and 111 After decay of In (30+ days - approximately 10 half-lives), the amount of fibrin deposited within the graft was measured at 60 minutes (endpoint). Fibrin and platelet counts represent the size of the thrombus.

[0187] result APTT and PT assays were performed as described in Examples 2 and 3. APTT was measured using citrated plasma, SynthA Sil® (Instrumentation Laboratories) reagent, and a KC-4 coagulometer. PT was measured using citrated plasma, Dade® Innovin® reagent (Siemens), and a KC-4 coagulometer. Both APTT and PT were measured immediately after blood collection and centrifugation of the blood samples to generate platelet-poor plasma. The fold change in APTT measured 1 hour after administration of DX-4012 was 2.2-fold (expected >2.2-fold). The fold change in APTT measured 24-48 hours later was 1.8-fold (expected 1.8-2.0-fold), and the fold change in APTT measured 1 week later was 1.3-fold (expected 1.3-1.5-fold). No change in PT was observed.

[0188] Collagen graft For the thrombosis study, animals were administered 10 mg / kg of the antibody DX-4012 and the study was performed at 1 hour, 24 hours, 48 ​​hours, 168 hours, and 192 hours after antibody administration. For analysis, data from the 24-hour and 48-hour time points were combined, and data from the 168-hour and 192-hour time points were combined. As expected, control animals formed baseline collagen-induced thrombus heads. The thrombus heads were significantly reduced in animals receiving enoxaparin. Up to 48 hours after antibody administration, the size of the thrombus heads was reduced relative to control animals and comparable to that of animals receiving enoxaparin. One week after antibody administration (168-192 hours), the size of the thrombus heads was comparable to that of control animals (Figure 11A). Up to 48 hours after antibody administration, the thrombus tails had almost disappeared, comparable to that of animals receiving enoxaparin. One week (168-192 hours) after antibody administration, thrombus tails remained reduced, but to a lesser extent than after enoxaparin treatment (FIG. 11B).

[0189] In another experiment, up to 48 hours after antibody administration, the size of the thrombus heads was reduced compared to control animals and was comparable to that of animals receiving enoxaparin. One week after antibody administration (168-192 hours), the size of the thrombus heads remained reduced, but to a lesser extent than after enoxaparin treatment (Figure 12). Figure 13 shows thrombus formation (total thrombi) and demonstrates that thrombus formation was comparable at 1 hour, 24 hours, and 48 hours after antibody administration. The thrombus growth rate (platelet deposition at 5-minute intervals) is shown in Figure 14. Control thrombi showed an increased growth rate in the first 30 minutes before reaching a plateau. After enoxaparin treatment, the growth rate reached a lower plateau at 20 minutes, began to decrease after 40 minutes, and dissolved after 60 minutes (negative growth rate). Within the first 48 hours after DX-4012 administration, the growth rate plateaued at 15-20 minutes and showed a low growth rate. One week after antibody administration, the amount of thrombus was between that at earlier time points (1 hour and 24-48 hours) and that of the control.

[0190] Overall, administration of 10 mg / kg DX-4012 reduced the size of collagen-induced thrombi, with the effect observed in both the size of the thrombus head and the thrombus tail. The reduction in thrombus size was observed up to 48 hours after antibody administration and was comparable to that observed with enoxaparin treatment (1 mg / kg). After one week, thrombus heads returned to baseline in animals receiving the antibody, while thrombus tail size remained low.

[0191] Tissue factor graft In general, thrombus size was significantly more variable in tissue factor-coated shunts compared with collagen-coated shunts. A large variability was observed in the described experiment (7.4, 1.40, and 230 million platelets). As expected, control animals formed baseline tissue factor-induced thrombus heads. Thrombus heads were significantly reduced in animals receiving enoxaparin. Up to 48 hours after antibody administration, thrombus head size was comparable to that of control animals (Figure 15A). One week after antibody administration (168–192 hours), thrombus head size significantly changed. Enoxaparin treatment nearly abolished tissue factor-induced thrombus tail formation. Although no significant reduction in thrombus tail size was observed in antibody-treated animals, the data suggest a slight trend toward a reduction in thrombus tail size (Figure 15B).

[0192] In another experiment, enoxaparin treatment resulted in a greater than 50% reduction in thrombus size, primarily due to the near disappearance of thrombus tails. Although no significant reduction in thrombus tails was observed in antibody-treated animals, the data suggested a slight trend toward a smaller thrombus tail (Figure 16). Figure 17 shows thrombus formation (total thrombus). RM ANOVA analysis of the data revealed a reduction in total thrombus size 1 hour after antibody treatment compared to controls. The thrombus growth rate (platelet deposition at 5-minute intervals) is shown in Figure 18. Control thrombi showed an increased growth rate in the first 30 minutes before reaching a plateau. The growth rate after enoxaparin treatment plateaued at 20 minutes, began to decrease after 40 minutes, and dissolved after 60 minutes (negative growth rate). Within the first 48 hours after DX-4012 administration, the thrombus growth rate was similar to that observed in control animals (Figure 18).

[0193] Overall, administration of 10 mg / kg of DX-4012 had no effect on the size of the thrombus head in tissue factor-induced thrombus formation, and had only a slight effect on the size of the thrombus tail.

[0194] Terminal fibrin content and platelet deposition At the end of the experiment, the shunt was flushed with saline and images were acquired to assess the final platelet count. Loops were also saved for fibrin content analysis of the thrombus head and tail.

[0195] Distal thrombus size was reduced in animals receiving enoxaparin. Animals receiving DX-4012 also had a significant reduction in thrombus size as measured by both platelet deposition and fibrin content in collagen-coated, but not tissue factor-coated, shunts (Figures 19A and 19B).

[0196] Hemostasis research Hemostasis was measured using the FDA-approved Surgicutt™ bleeding time device and protocol. Blood volume was also measured. Bleeding time and bleeding volume measurements were performed once per study day. As shown in Figures 22A and 22B, DX-4012 was effective in reducing bleeding time and bleeding volume compared to enoxaparin.

[0197] Example 5: Identification of essential residues in the catalytic domain of FXIIa based on the crystal structure of the DX-4012-FXIIa complex

[0198] The recombinant Fab fragment of DX-4012 was prepared and purified by conventional recombinant techniques in E. coli. FXIIa was produced and purified by conventional methods.

[0199] The Fab fragment of DX-4012 and FXIIa were mixed at various concentrations under suitable conditions to form Fab-FXIIa complexes, which were purified using SEC and visualized by the trace shown in Figure 20.

[0200] The Fab-FXIIa complex was crystallized under various conditions. Diffraction analysis was performed on the crystallized complex. Based on the diffraction statistics, the crystal structures (2.6 Å and 2.25 Å) were determined.

[0201] According to the crystal structure, the residues in the C chain of FXIIa involved in the interaction with the Fab fragment of DX-4012 were identified: L390, Y391, W392, G393, H394, S395, F396, C397, H412, C413, L414, Q415, D416, R432, N433, V456, Y458, H507, F509, E510, G511, A512, E513, Y515, D557, A558, C559, Q560, G561, D562, S563, I584, S585, W586, G587, S588, G589, C590, G591, D592, and G597.

[0202] Furthermore, residues in the Fab of DX-4012 that interact with FXIIa were also identified based on the crystal structure, including T28, S30, Q31, W52, P53, S54, G55, G56, H57, R59, N74, R100, Y101, R102, G103, P104, K105, Y106, Y107, and Y108 in the heavy chain variable region and H31, N33, Y35, Y54, L55, N58, and T99 in the light chain variable region.

[0203] These results indicated that the heavy chain of DX-4012 is the main region that interacts with FXIIa, and that 2-3 residues in the LC CDR1 contribute to the interaction.

[0204] The crystal structure also revealed that residue R102 of the anti-FXIIa Fab fragment binds to the S1 pocket of FXIIa and interacts with residue D557 through a water-mediated interaction. This positions the backbone of the Fab residues R102-G103-P104 in a catalytically inactive orientation close to the catalytic triad of FXIIa. P104 contributes to rigidifying this orientation, thereby inhibiting FXIIa activity.

[0205] To determine further crystal structures, complexes of other anti-FXIIa antibodies and their Fab fragments with FXIIa will also be formed and co-purified.

[0206] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by another feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0207] Those skilled in the art can easily ascertain the essential characteristics of the present invention from the above description, and can make various changes and modifications to the present invention to adapt it to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0208] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision numerous other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, it should be understood that the above-described embodiments are provided by way of example only and that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Any combination of two or more such features, systems, articles, materials, kits and / or methods is within the inventive scope of this disclosure, unless such features, systems, articles, materials, kits and / or methods are mutually inconsistent.

[0209] All definitions defined and used herein should be understood to supersede dictionary definitions, definitions within documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0210] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may in some cases include the entire document.

[0211] As used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless there is a clear contradiction.

[0212] The term "and / or," as used herein in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are sometimes conjunctively present and otherwise disjunctively present. Multiple elements listed with "and / or" should be interpreted similarly, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether or not related to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used with open-ended language such as "comprising," can, for example, in one embodiment refer to A only (optionally including elements other than B); in another embodiment, refer to B only (optionally including elements other than A); and in yet another embodiment, refer to both A and B (optionally including other elements).

[0213] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, "or" or "and / or" when separating listed items should be construed as inclusive, i.e., the inclusion of at least one of the multiple or listed elements, but also two or more, and optionally additional unlisted items. Only clearly contradictory terms, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of the multiple or listed elements. In general, the term "or" as used herein should be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") only when accompanied by terms of exclusivity, such as "either one of," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0214] The phrase "at least one," as used herein in the specification and claims, when referring to one or more elements in a list, should be understood to mean at least one element selected from any one or more elements in the list, but not necessarily including at least one of each and every specifically listed element in the list, and not excluding any combinations of elements in the list. This definition also recognizes that elements other than the specifically identified elements in the list to which the phrase "at least one" refers may optionally be present, whether or not related to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer, for example, in one embodiment, to at least one (optionally including more than one) A (and optionally including elements other than B) in the absence of B; in another embodiment, to at least one (optionally including more than one) B (and optionally including elements other than A) in the absence of A; and in yet another embodiment, to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements).

[0215] It is also to be understood that, unless otherwise clearly contradicted, in any method claimed herein including two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. 1. Use of a monoclonal antibody that binds to factor XIIa (FXIIa) but not to factor XII (FXII) in the manufacture of a medicament for the treatment of a disease associated with factor XII (FXII), comprising: The medicament further comprises a pharmaceutically acceptable carrier; the antibody comprises a heavy chain comprising a heavy chain variable region comprising heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3); and a light chain comprising a light chain variable region comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3); (i) the HC CDR1 comprises WYSMH (SEQ ID NO:41), the HC CDR2 comprises VIYPSGGKTRYADSVKG (SEQ ID NO:74), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (ii) the HC CDR1 comprises WYVMH (SEQ ID NO:43), the HC CDR2 comprises GIWPSGGRTKYADSVKG (SEQ ID NO:76), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (iii) the HC CDR1 comprises NYVMH (SEQ ID NO:44), the HC CDR2 comprises SIWPSGGKTKYADSVKG (SEQ ID NO:77), and the HC CDR3 comprises QRYRGPKYYYYMDA (SEQ ID NO:112); or (iv) the HC CDR1 comprises MYTMN (SEQ ID NO:45), the HC CDR2 comprises RIYPSGGKTLYADSVKG (SEQ ID NO:78), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (v) the HC CDR1 comprises QYVMS (SEQ ID NO:46), the HC CDR2 comprises RIYPSGGVTKYADSVKG (SEQ ID NO:79), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (vi) the HC CDR1 comprises WYNMH (SEQ ID NO:47), the HC CDR2 comprises YISPSGGKTKYTDSVKG (SEQ ID NO:80), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (vii) the HC CDR1 comprises RYIMH (SEQ ID NO:48), the HC CDR2 comprises SIYPSGGVTKYADSVKG (SEQ ID NO:81), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (viii) the HC CDR1 comprises RYIMG (SEQ ID NO:49), the HC CDR2 comprises SIYPSGGVTRYADSVKG (SEQ ID NO:82), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (ix) the HC CDR1 comprises QYNMV (SEQ ID NO:50), the HC CDR2 comprises RIWPSGGKTTYADSVKG (SEQ ID NO:83), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (x) the HC CDR1 comprises RYVMV (SEQ ID NO:51), the HC CDR2 comprises RIYPSGGMTQYADSVKG (SEQ ID NO:84), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xi) the HC CDR1 comprises WYNMA (SEQ ID NO:52), the HC CDR2 comprises RIYPSGGMTQYADSVKG (SEQ ID NO:84), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xii) the HC CDR1 comprises QYIMH (SEQ ID NO:53), the HC CDR2 comprises SIYPSGGNTKYADSVKG (SEQ ID NO:85), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xiii) the HC CDR1 comprises HYVMH (SEQ ID NO:54), the HC CDR2 comprises SIYPSGGLTKYADSVKG (SEQ ID NO:86), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xiv) the HC CDR1 comprises WYTMH (SEQ ID NO:55), the HC CDR2 comprises SIYPSGGFTRYADSVKG (SEQ ID NO:87), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xv) the HC CDR1 comprises FYHMH (SEQ ID NO:56), the HC CDR2 comprises RIVPSGGMTRYADSVKG (SEQ ID NO:88), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xvi) the HC CDR1 comprises FYSMH (SEQ ID NO: 57), the HC CDR2 comprises RIYPSGGVTKYADSVKG (SEQ ID NO: 89), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xvii) the HC CDR1 comprises QYVMH (SEQ ID NO:58), the HC CDR2 comprises SIWPSGGKTTYADSVKG (SEQ ID NO:90), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xviii) the HC CDR1 comprises QYVMH (SEQ ID NO:58), the HC CDR2 comprises SIWPSGGFTKYADSVKG (SEQ ID NO:91), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xix) the HC CDR1 comprises FYNMH (SEQ ID NO:59), the HC CDR2 comprises SIYPSGGVTRYADSVKG (SEQ ID NO:92), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xx) the HC CDR1 comprises WYVMH (SEQ ID NO:43), the HC CDR2 comprises SIYPSGGKTSYADSVKG (SEQ ID NO:93), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxi) the HC CDR1 comprises PYIMH (SEQ ID NO: 60), the HC CDR2 comprises VIYPSGSKTNYADSVKG (SEQ ID NO: 94), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxii) the HC CDR1 comprises RYTMR (SEQ ID NO: 61), the HC CDR2 comprises SIWPSGGMTRYADSVKG (SEQ ID NO: 95), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxiii) the HC CDR1 comprises QYVMH (SEQ ID NO:58), the HC CDR2 comprises SIYPSGGLTRYADSVKG (SEQ ID NO:96), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxiv) the HC CDR1 comprises WYIMG (SEQ ID NO: 62), the HC CDR2 comprises YIYPSGGNTRYADSVKG (SEQ ID NO: 97), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxv) the HC CDR1 comprises RYVMH (SEQ ID NO: 63), the HC CDR2 comprises SIWPSGGMTKYADSVKG (SEQ ID NO: 98), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxvi) the HC CDR1 comprises FYIMG (SEQ ID NO: 64), the HC CDR2 comprises RIYPSGGATQYADSVKG (SEQ ID NO: 99), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxvii) the HC CDR1 comprises FYVMG (SEQ ID NO: 65), the HC CDR2 comprises RIYPSGGLTQYADSVKG (SEQ ID NO: 100), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxviii) the HC CDR1 comprises FYSMH (SEQ ID NO: 66), the HC CDR2 comprises RIYPSGGITSYADSVKG (SEQ ID NO: 101), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxix) the HC CDR1 comprises MYIMH (SEQ ID NO: 67), the HC CDR2 comprises SIYPSGGMTKYADSVKG (SEQ ID NO: 102), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxx) the HC CDR1 comprises MYVMH (SEQ ID NO: 68), the HC CDR2 comprises SIYPSGGLTKYADSVKG (SEQ ID NO: 103), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO: 111); or (xxxi) the HC CDR1 comprises QYVMH (SEQ ID NO:58), the HC CDR2 comprises RIYPSGGLTNYADSVKG (SEQ ID NO:104), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxxii) the HC CDR1 comprises WYVMQ (SEQ ID NO:69), the HC CDR2 comprises SIYPSGGMTKYADSVKG (SEQ ID NO:102), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxxiii) the HC CDR1 comprises WYIMG (SEQ ID NO: 62), the HC CDR2 comprises RIYPSGGSTHYADSVKG (SEQ ID NO: 105), and the HC CDR3 comprises QRYRGPRYYYYIDA (SEQ ID NO: 113); or (xxxiv) the HC CDR1 comprises QYTMV (SEQ ID NO:70), the HC CDR2 comprises RIYPSGGVTQYADSVKG (SEQ ID NO:106), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxxv) the HC CDR1 comprises WYVMY (SEQ ID NO:71), the HC CDR2 comprises RIYPSGGITHYADSVKG (SEQ ID NO:107), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxxvi) the HC CDR1 comprises FYVML (SEQ ID NO:72), the HC CDR2 comprises SIWPSGGVTKYADSVKG (SEQ ID NO:108), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); (xxxvii) the HC CDR1 comprises WYVMQ (SEQ ID NO:69), the HC CDR2 comprises YIYPSGGHTKYADSVKG (SEQ ID NO:109), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); or (xxxviii) the HC CDR1 comprises RYSMN (SEQ ID NO:73), the HC CDR2 comprises GIYPSGGKTKYADSVKG (SEQ ID NO:110), and the HC CDR3 comprises QRYRGPKYYYYMDV (SEQ ID NO:111); the LC CDR1 comprises RSSQSLLHSNGYNYLD (SEQ ID NO: 114), the LC CDR2 comprises LGSNRAS (SEQ ID NO: 115), and the LC CDR3 comprises MQALQTPWT (SEQ ID NO: 116); use.

2. The use according to claim 1 , wherein the antibody is a full-length antibody or an antigen-binding fragment thereof, and / or the antibody is a human antibody or a humanized antibody.

3. The use according to claim 2 , wherein the antibody is a Fab.

4. (i) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYSMHWVRQAPGKGLEWVSVIYPSGGKTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 1); or (ii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMHWVRQAPGKGLEWVSGIWPSGGRTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 3); or (iii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFFSNYVMHWVRQAPGKGLEWVSSIWPSGGKTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDAWGQGTTVTVSS (SEQ ID NO: 4); or (iv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYTMNWVRQAPGKGLEWVSRIYPSGGKTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 5); or (v) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMSWVRQAPGKGLEWVSRIYPSGGVTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 6); or (vi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYNMHWVRQAPGKGLEWVSYISPSGGKTKYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 7); or (vii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYIMHWVRQAPGKGLEWVSSIYPSGGVTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 8); or (viii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYIMGWVRQAPGKGLEWVSSIYPSGGVTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 9); or (ix) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYNMVWVRQAPGKGLEWVSRIWPSGGKTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 10); or (x) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMVWVRQAPGKGLEWVSRIYPSGGMTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 11); or (xi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYNMAWVRQAPGKGLEWVSRIYPSGGMTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 12); or (xii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYIMHWVRQAPGKGLEWVSSIYPSGGNTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 13); or (xiii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMHWVRQAPGKGLEWVSSIYPSGGLTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 14); or (xiv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMHWVRQAPGKGLEWVSSIYPSGGFTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 15); or (xv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYHMHWVRQAPGKGLEWVSRIVPSGGMTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 16); or (xvi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYSMHWVRQAPGKGLEWVSRIYPSGGVTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 17); or (xvii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMHWVRQAPGKGLEWVSSIWPSGGKTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 18); or (xviii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMHWVRQAPGKGLEWVSSIWPSGGFTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 19); or (xix) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYNMHWVRQAPGKGLEWVSSIYPSGGVTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 20); or (xx) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMHWVRQAPGKGLEWVSSIYPSGGKTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 21); or (xxi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYIMHWVRQAPGKGLEWVSVIYPSGSKTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 22); or (xxii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYTMRWVRQAPGKGLEWVSSIWPSGGMTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGQGTTVTVSS (SEQ ID NO: 23); or (xxiii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMHWVRQAPGKGLEWVSSIYPSGGLTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGQGTTVTVSS (SEQ ID NO: 24); or (xxiv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYIMGWVRQAPGKGLEWVSYIYPSGGNTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 25); or (xxv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMHWVRQAPGKGLEWVSSIWPSGGMTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 26); or (xxvi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYIMGWVRQAPGKGLEWVSRIYPSGGATQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 27); or (xxvii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMGWVRQAPGKGLEWVSRIYPSGGLTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 28); or (xxviii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYSMHWVRQAPGKGLEWVSRIYPSGGITSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 29); or (xxix) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYIMHWVRQAPGKGLEWVSSIYPSGGMTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 30); or (xxx) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYVMHWVRQAPGKGLEWVSSIYPSGGLTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 31); or (xxxi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYVMHWVRQAPGKGLEWVSRIYPSGGLTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 32); or (xxxii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMQWVRQAPGKGLEWVSSIYPSGGMTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 33); or (xxxiii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYIMGWVRQAPGKGLEWVSRIYPSGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPRYYYYIDAWGQGTTVTVSS (SEQ ID NO: 34); or (xxxiv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSQYTMVWVRQAPGKGLEWVSRIYPSGGVTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 35); or (xxxv) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMYWVRQAPGKGLEWVSRIYPSGGITHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 36); or (xxxvi) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMLWVRQAPGKGLEWVSSIWPSGGVTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 37); or (xxxvii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMQWVRQAPGKGLEWVSYIYPSGGHTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 38); or (xxxviii) the heavy chain variable region comprises the amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYSMNWVRQAPGKGLEWVSGIYPSGGKTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS (SEQ ID NO: 39); (xxxix) the light chain variable region comprises the amino acid sequence DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK (SEQ ID NO: 126); or (xxxx) the light chain variable region comprises the amino acid sequence DIQMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK (SEQ ID NO: 40); Use according to any one of claims 1 to 3.

5. The use according to any one of claims 1 to 4, wherein the heavy chain further comprises a heavy chain constant region.

6. The use of claim 5, wherein the heavy chain constant region comprises at least one mutation that increases the half-life of the antibody compared to its wild-type counterpart.

7. The use of claim 6, wherein the heavy chain constant region comprises amino acid substitutions at positions corresponding to positions 145, 147 and 149 of SEQ ID NO:

119.

8. The use of claim 7, wherein the heavy chain constant region comprises the amino acid substitutions M145Y, S147T and T149E.

9. The use of claim 6, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

120.

10. The use according to any one of claims 1 to 9, wherein the medicament is for the treatment of a subject having, suspected of having or at risk of having a disease associated with FXII.

11. The use of claim 10, wherein the subject has one or more symptoms of a disease associated with FXII.

12. 12. The use of claim 11, wherein the one or more symptoms are selected from the group consisting of swelling of the arms, legs, lips, eyes, tongue and / or throat; airway obstruction; sudden hoarseness; recurring episodes of abdominal cramps with no apparent cause; intestinal swelling; abdominal cramps; vomiting; dehydration; diarrhea; pain; shock; and / or marginal erythema.

13. The use of claim 10, wherein the subject has no symptoms of a disease associated with FXII, no history of symptoms of a disease associated with FXII, or no history of a disease associated with FXII.

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