EA045947B1 - MONOCLONAL ANTIBODY-FACTOR XIIA INHIBITOR - Google Patents

Description

Cross reference to related applications

This application claims priority to the filing dates of US Provisional Application No. 62/194957 filed July 21, 2015, US Provisional Application No. 62/273657 filed December 31, 2015, and US Provisional Application No. 62/316310 filed March 31, 2016. d. The entire contents of each of these specified applications are incorporated herein by reference.

State of the art

Factor XII (FXII) is one of the components of the plasma contact activation system, responsible for both the initiation of intrinsic coagulation and the conversion of prekallikrein to plasma kallikrein (pKal). Active pKal cleaves high molecular weight kininogen (HMWK), releasing bradykinin, which is a potent activator of swelling and pain. Activation of the intrinsic coagulation pathway leads to the formation of fibrin and the formation of blood clots (thrombus).

The essence of the invention

The present invention is based on the identification of a series of antibodies that specifically bind factor XIIa (FXIIa), but not factor XII (FXII). These antibodies have high binding affinity for FXIIa (eg, Ki ^app <50 pM), successfully reduce plasma kallikrein activation and increase activated partial thromboplastin time (aPTT), but have no effect on prothrombin time (PTT).

Accordingly, in one aspect of the present invention, a monoclonal antibody is disclosed that binds to factor XIIa (FXIIa) and does not bind 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.

In some embodiments, the antibody reacts with one or more amino acid residues on the C chain of FXIIa. In some examples, 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 of the invention the antibody binds to the FXIIa fragment containing residues 390-397, 412-416, 432-433, 456-458, 507-515, 557-563, or 584-592 from SEQ ID NO: 128.

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

In some embodiments, the heavy chain variable region comprises a heavy chain CDR3 that contains the amino acid sequence QRYRGPKYYYYMDV (SEQ ID NO: 111), QRYRGPKYYYYMDA (SEQ ID NO: 112), or QRYRGPRYYYYIDA (SEQ ID NO: 113). Such a heavy chain variable region may further comprise a heavy chain CDR1 which contains the sequence of the formula X1YX ₃ MX ₅ (SEQ ID NO: 117) in which: X1 is R, Q, W, H, F, P, M or N ; X ₃ is I, V, T, H, S or N and X ₅ is H, C, V, A, R, Q, Y, L, N or S. In some examples, Xi of CDR1 is W or Q; X ₃ of CDR1 is S or V and/or X ₅ is H. In some examples, the CDR1 heavy chain may comprise the amino acid sequence of any of SEQ ID NOs: 41-73 and 121.

Alternatively or in addition, the heavy chain variable region of the anti-FXIIa antibodies described herein may further comprise a heavy chain CDR2 which contains the sequence of the formula XiIX ₃ PSGX ₇ X ₈ TXi ₀ YXi ₂ DSVKG (SEQ ID NO: 118), in which: Xi represents S, R, V, Y or G; X ₃ represents Y, W, V or S; X ₇ represents G or S; X ₈ represents V, K, M, N, L, F, A, I, S, H or R; X ₁₀ represents K, R, T, Q, S, N, H or L; and X ₁₂ is A or T. In some examples, X 1 of CDR2 is V or S, X ₃ of CDR2 is Y or W, X ₇ of CDR2 is G, X ₈ of CDR2 is K or H, X ₁₀ of CDR2 is R, and/or X ₁₂ of CDR2 is A. In some examples, the heavy chain CDR2 comprises the amino acid sequence of any of SEQ ID NOs: 74110, 122-124, and 127.

In some 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 presented in Table. 1.

In some examples, the heavy chain of the anti-FXIIa antibodies described herein comprises a heavy chain variable region that contains the amino acid sequence of any of SEQ ID NO: 1-39 and SEQ ID NO: 125.

Any of the anti-FXIIa antibody heavy chains described herein may further comprise a heavy chain constant region. In some embodiments, a constant

- 1045947 the heavy chain region contains at least one mutation (eg, amino acid substitution) that increases the half-life of the antibodies compared to the wild type counterpart. Such a mutated heavy chain constant region may contain at least one mutation at a position corresponding to position 145, 147 or 149 in SEQ ID NO: 119, which is the amino acid sequence of a typical naturally occurring heavy chain constant region of a human antibody. The at least one mutation may be an amino acid substitution M145Y, S147T and/or T149E. In some examples, the mutated heavy chain constant region contains amino acid substitutions at positions 145, 147 and 149 in SEQ ID NO: 119 (eg, M145Y, S147T and T149E). Such a mutated heavy chain constant region may comprise the amino acid sequence of SEQ ID NO: 120.

The light chain variable region of the anti-FXIIa antibodies described herein may comprise a light chain CDR3 that contains the amino acid sequence MQALQTPWT (SEQ ID NO: 116). In some examples, the light chain variable region may further comprise a light chain CDR1 that contains the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 114). Alternatively or in addition, the light chain variable region may further comprise a light chain CDR2 that contains the amino acid sequence LGSNRAS (SEQ ID NO: 115). In some examples, the light chain of the anti-FXIIa antibodies described herein may comprise a light chain variable region that contains the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 126.

Any of the anti-FXIIa antibodies described herein can be a full-length antibody or antigen-binding fragments thereof. In some examples, the antibodies may be human antibodies or humanized antibodies.

Also included within the scope of the present invention is an isolated nucleic acid or set of nucleic acids that collectively encodes any of the anti-FXIIa antibodies (e.g., encodes at least the heavy and light chain variable regions of an antibody) described herein, a vector or set of vectors ( for example, expression vectors) that include a nucleic acid or set of nucleic acids, and a host cell or set of host cells that contains the vector or set of vectors. Typical host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells such as CHO cells.

In addition, the present invention provides a method for producing any of the anti-FXIIa antibodies described herein. The method includes culturing a host cell or set of host cells that contains a vector or set of vectors that collectively encode an antibody, and collecting the cultured cells or culture medium to isolate the antibody. Such a method may further include isolating the antibody from cultured cells or culture medium.

In another aspect, the present invention provides a method for treating a factor XII-associated disease, for example, a contact system activation-associated disease, a pKal signaling pathway-associated disease, such as hereditary angioedema or HAE, or an ocular disease (for example, macular edema). , diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, or retinal vein occlusion) in the 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 and cannot bind to FXII (e.g., any of the anti-FXIIa antibodies described herein), one or more nucleic acids, which collectively encode an antibody, or expression vectors containing such nucleic acid(s). In some embodiments, the antibody specifically binds the catalytic domain of FXIIa. In other embodiments, the antibody inhibits the activation of FXI to FXIa. In other embodiments, the Ki ^app of the antibody is below about 110 pM (eg, below about 50 or 10 pM).

The subject to be treated using the method described herein may be a person who has, is suspected of having, or is at risk of a pKal-associated disease (eg, HAE). In some examples, the subject has, is suspected of having, or is at risk for Type I, II, or III HAE.

In some embodiments of the present invention, the subject to be treated by the methods described herein may be a person who has, is suspected of having, or is at risk of a disease associated with factor XII, such as those disclosed herein (for example, a disease associated with activation of contact systems). For example, a subject may have, be expected to have, or be at risk of a disease associated with contact system activation, such as thrombosis, including thrombosis associated with atrial fibrillation, deep vein thrombosis (DVT), pulmonary embolism, stroke, or arterial or venous thrombus formation . In another example, a subject may have, is suspected of having, or is at risk of a disease associated with the pKal signaling pathway, such as HAE, such

- 2 045947 as NAE type I, II or III. In yet another example, the subject may have, is suspected of having, or is at risk of an ocular disease, which may be macular edema, diabetic retinopathy, hypertensive retinopathy, age-related macular degeneration, or retinal vein occlusion.

Also included within the scope of the present invention are pharmaceutical compositions comprising any of the anti-FXIIa antibodies described herein and a pharmaceutically acceptable carrier. Such a pharmaceutical composition may be intended for use in the treatment of a disease associated with the pKal signaling pathway (eg, for example, HAE, such as HAE type I, II or III), or for use in the manufacture of medicaments for use in the treatment of such a disease.

Details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, as well as from the accompanying claims.

Brief description of drawings

The following drawings form part of the present description and are included to further demonstrate certain aspects of the present invention, which may be better understood with reference to the drawings in conjunction with the detailed description of the specific embodiments presented herein.

Fig. 1 is a schematic illustration showing the coagulation cascade in which factor XII is involved.

Fig. 2 is a schematic illustration showing an antibody selection strategy.

Fig. 3 is a schematic illustration showing biological assays used to characterize the activity of FXIIa inhibitors, including an in vitro activity assay, a plasma activity assay, and a preferential binding assay.

Fig. 4 is a graph showing the inhibitory activity of the parent antibody 559C-M71-F06 (human IgG1 antibody) against human (left panel) and mouse (right panel) FXIIa.

Fig. 5 is a chart showing the interspecies cross-reactivity of M71-F06 determined by APTT.

In fig. 6 shows the concentration of exemplary anti-FXIIa antibodies M191-M192 and E09-H11 in rat plasma at various time points after injection of anti-FXIIa antibodies.

Fig. 7 is a graph showing the increase in aPTT and PTW (in seconds) in the presence of anti-FXIIa antibody 559C-X211-A01 (A01). A: APTT kinetics after 10 mg/kg A01. B: APTT kinetics in the presence of 10 mg/kg A01 relative to baseline. C: Kinetics of PTT (sec) in the presence of 10 mg/kg A01.

Fig. 8 is a graph showing the aPTT activity of DX-4012 antibodies at the indicated time points before administration of the indicated treatment (Pre-administration, gray bars), 5 minutes before the start of the study (Pre-test, black bars) and 5 minutes before the end of the study ( After the study, white bars). Values show mean APTT +/- standard error in seconds. A: APTT. B: Fold increase in aPTT compared to baseline value (average of pre- and post-study measurements on one animal on the control day(s) of the study).

Fig. 9 is a graph showing the PTT activity of DX-4012 antibodies at the indicated time points before administration of the indicated treatment (Pre-administration, gray bars), 5 minutes before the start of the study (Pre-test, black bars) and 5 minutes before the end of the study ( After the study, white bars). Values show mean PTT +/- standard error in seconds. A: PTV. B: Fold increase in PTT compared to baseline value (average of pre- and post-study measurements on one animal on the control day(s) of the study).

In fig. Figure 10 shows a diagram of the external loop of a permanent arteriovenous (AV) shunt during thrombosis studies in nonhuman primates. An external loop is routed over the top of the gamma chamber for actual platelet sedimentation time measurement.

Fig. 11 is a graph showing platelet deposition time on a collagen-coated graft. A: Thrombus head. B: Thrombus tail.

Fig. 12 is a graph showing platelet deposition time on a collagen-coated graft.

Fig. 13 is a graph showing the deposition time of platelets (total thrombus) onto a collagen-coated graft.

Fig. 14 is a graph showing the rate of thrombus growth on a collagen-coated graft measured as platelet deposition at 5 minute intervals.

Fig. 15 is a graph showing the deposition time of platelets on the coated

- 3 045947 tissue factor graft. A: Thrombus head. B: Thrombus tail.

Fig. 16 is a graph showing platelet deposition time on a tissue factor-coated graft.

Fig. 17 is a graph showing platelet deposition time on a tissue factor-coated graft.

Fig. 18 is a graph showing the rate of thrombus growth on a tissue factor-coated graft, measured as platelet deposition at 5-minute intervals.

Fig. 19 is a graph showing the final fibrin content and platelet deposition of the entire thrombus (head and tail) on collagen-coated grafts (gray bars) and tissue factor-coated grafts (white bars). A: Platelet sedimentation. Q: Fibrin content.

Fig. 20 is a graph showing the formation of a Fab fragment complex from DX4012 and FXIIa by SEC analysis. A: FXIIa and Fab. B: FXIIa, Fab and Fab-FXIIa complex at 50 µM FXIIa and 30 µM Fab. C: FXIIa, Fab and Fab-FXIIa complex at 50 µM FXIIa and 40 µM Fab. D: FXIIa, Fab and Fab-FXIIa complex at 50 µM FXIIa and 50 µM Fab. E: FXIIa, Fab and Fab-FXIIa complex at 50 µM FXIIa and 60 µM Fab.

Fig. 21 is a graph showing the inhibitory activity of anti-FXIIa antibody M192-H11 on human FXIIa.

Fig. 22 is a chart showing hemostasis measurements associated with DX4012. A: Bleeding time. B: Amount of bleeding.

Detailed Description of the Invention

Components of the contact system initiate the intrinsic coagulation pathway and promote inflammation by releasing the proinflammatory peptide bradykinin. Factor XII (FXII), also known as Hageman factor, is a serine protease that is involved in the activation of the intrinsic coagulation pathway as well as the kallikrein-kinin system. FXII is activated by negatively charged surfaces (eg, polyanionic surfaces, glass, polyphosphate, ellagic acid) to produce the active form, FXIIa. Activated FXIIa has the ability to cleave prekallikrein, generating active pKal. Subsequently, activated pKal is able to cleave FXII to FXIIa, resulting in a positive feedback loop where FXIIa generates even more pKal, which further activates additional FXII to FXIIa. Activated pKal is also able to cleave high molecular weight kininogen (HMWK), releasing bradykinin. The coagulation cascade involving FXII is illustrated in FIG. 1.

In diseases associated with activation of the contact system, such as HAE, elevated levels of bradykinin can cause dilation of blood vessels and inflammation, leading to angioedema. In this regard, it is desirable to create new therapeutic agents for the treatment of a number of diseases that are potentially mediated by activation of the contact system.

Disclosed herein are antibodies that bind and inhibit FXIIa, as well as their use in inhibiting FXIIa and treating diseases associated with contact system activation. In the examples below, a number of exemplary anti-FXIIa antibodies have been prepared and are shown to specifically bind to FXIIa and inhibit its activity. Such antibodies, exemplified herein, are expected to provide superior therapeutic effects in the treatment of diseases associated with activation of the contact system, in particular by reducing bradykinin production, vasodilation and pathological thrombus formation associated with disease symptoms.

Antibodies that bind to FXIIa

The present invention relates to antibodies that bind FXIIa, for example, the catalytic domain of FXIIa. Such antibodies may not bind to FXII.

An antibody (used interchangeably in its plural form) is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the molecule immunoglobulin. As used herein, the term antibody includes not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain fragments (scFv), mutants thereof, antibody site fusion proteins, humanized antibodies, chimeric antibodies, diantibodies, nanoantibodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that contains a recognition site antigen of the required specificity, including antibody glycosylation variants, antibody amino acid sequence variants and covalently modified antibodies. An antibody includes any class of antibody such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), and the antibody does not need to belong to any particular class. Depending on the amino acid sequence

- 4 045947 stant domain of antibody heavy chains, immunoglobulins can be classified into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

In some embodiments, the anti-FXIIa antibodies described herein can bind and inhibit FXIIa activity by at least 50% (eg, 60, 70, 80, 90, 95%, or greater). The apparent inhibition constant (Kiapp or Ki, _app ), which provides a measure of inhibitor activity, is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-FXIIa antibody described herein can be determined using routine methods known in the art.

K value; ^app for an antibody can be determined by measuring the inhibitory effect of different concentrations of the antibody on the magnitude of the response (eg, enzyme activity); and fitting the change in the pseudo-first order rate constant (v) as a function of inhibitor concentration to the modified Morrison equation (Equation 1) gives an estimate of the magnitude of the apparent Ki. For a competitive inhibitor, Ki ^app can be obtained from the y-intercept obtained from a linear regression analysis of a plot of Ki ^W versus substrate concentration.

([£]-[/] -X-0 + 7'I “I-^+ ad - ^k · y = L---—------------ ^; ---- ------η.

*(Equation 1)

Where A is equal to vo/E, the initial rate (vo) of the enzymatic reaction in the absence of inhibitor (I), divided by the total enzyme concentration (E).

In some embodiments, an anti-FXIIa antibody described herein may have a Ki ^app value 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 lower for the target antigen or antigenic epitope. In some embodiments, the anti-FXIIa antibody may have a lower Ki ^app for the first target (eg, FXIIa) relative to the second target (eg, FXII). Differences in Kiapp (for example, regarding specificity or other parameters) can be at least 1, 5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10000 or 10 ⁵ -fold. In some embodiments, an anti-FXIIa antibody better inhibits a first antigen (e.g., a first protein in a first conformation or a mimic thereof) relative to a second antigen (e.g., the same first protein in a second conformation or a mimic thereof; or a second protein). In some embodiments, any of the anti-FXIIa antibodies may be further subjected to affinity maturation to reduce the Kiapp of the antibody to the target antigen or antigenic epitope thereof.

Antibodies described herein may be murine, rat, human, or any other origin (including chimeric or humanized antibodies). Such antibodies will be unnatural, that is, they will not be produced in the animal without human intervention (for example, immunization of such an animal with the desired antigen or fragment thereof).

Any of the antibodies described herein may be monoclonal or polyclonal. A monoclonal antibody refers to a homogeneous population of antibodies, and 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 produced.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies refer to forms of animal (eg, murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain a minimal sequence derived from an animal immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by CDR residues from an animal (donor antibody), such as a mouse, rat, or rabbit, having the desired specificity, affinity, and binding force. In some cases, residues in the Fv framework region (FR) of human immunoglobulin are replaced by corresponding residues from animal immunoglobulin. Moreover, the humanized antibody may contain residues that are not present in either the antibody recipient or the imported CDR or framework sequences, but which are included to further improve and optimize the functions of the antibody. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of an animal immunoglobulin, and all or substantially all of the FR regions are human immunoglobulin consensus sequences. Optimally, the humanized antibody will also contain at least a portion of an immunoglobulin constant region or constant domain (Fc), typically an immunoglobulin

- 5,045,947 people. Antibodies may contain Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are modified from the parent antibody, which are also referred to as one or more CDRs derived from one or more CDRs of the parent antibody. The process of producing humanized antibodies may also involve affinity maturation.

In another example, an antibody described herein is a chimeric antibody that may include a heavy chain constant region and a light chain constant region of a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of a variable region from a first biological species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both the light and heavy chains mimics the variable regions of antibodies derived from the same mammalian species (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant regions are homologous to sequences in the antibodies derived from another mammal, such as a human. In some embodiments, modifications to the amino acids in the variable region and/or constant region may be made.

In some embodiments, 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 known in the art. A molecule is considered to exhibit specific binding if it reacts more frequently, at a faster rate, for a longer duration, and/or with greater affinity with a particular target antigen compared to alternative targets. An antibody binds specifically to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or for a 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 thereon is an antibody that binds that target antigen with greater affinity, avidity, more readily, and/or for a longer duration than it binds with other antigens or other epitopes on the same antigen. In the case of this definition, it should also be understood 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, specific binding or preferential binding does not necessarily require (although may include) exclusive binding. In some examples, an antibody that specifically binds to a target antigen or an epitope thereof cannot bind to other antigens or other epitopes on 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.

As used herein, the term factor XIIa or FXIIa refers to the active form of factor XII, and the term factor XII or FXII refers to the proenzyme or zymogen of factor XII (the inactive form). FXII is a single-chain glycoprotein with a molecular weight of approximately 80 kDa. Once activated, FXII is converted to its active form, FXIIa, which contains two chains, a heavy chain (353 residues of the human FXIIa heavy chain) and a light chain (243 residues of the human FXIIa light chain), held together by a disulfide bond. Human FXII is encoded by the F12 gene. The amino acid sequence of human FXII is well known in the art, for example, under accession number NP_000496.2 in the GenBank database.

In some embodiments, an anti-FXIIa antibody described herein has a suitable binding affinity for a target antigen (eg, FXIIa) or antigenic epitopes thereof. As used herein, the term binding affinity refers to the apparent association constant or K _A . K _A is the reciprocal of the dissociation constant (KD). An anti-FXIIa antibody described herein may have a binding affinity (KD) of at least 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M or lower for the target antigen or antigenic epitope. Increased binding affinity corresponds to a lower KD. The higher binding affinity of an antibody for a first antigen relative to a second antigen may be indicated by a higher K _A (or lower KD score) for binding the first antigen than a K _A (or KD score) for binding the second antigen. In such cases, the antibody has specificity for a first antigen (eg, a first protein in a first conformation or a mimic thereof) relative to a second antigen (eg, the same first protein in a second conformation or a mimic thereof; or a second protein). In some embodiments, the anti-FXIIa antibodies described herein have a higher binding affinity (higher K _A or lower KD) to FXIIa compared to the binding affinity to the FXII zymogen or other protein in the pKal signaling system. Differences in binding affinity (e.g., specificity or other parameters) may be at least 1, 5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100 , 500, 1000, 10000 or 105 times. In some embodiments, any of the anti-FXIIa antibodies may

- 6 045947 affinity maturation may be additionally performed to increase the binding affinity of the antibody to the target antigen or its antigenic epitope.

Binding affinity (or binding specificity) can be determined using various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (eg, using fluorescence analysis). Typical conditions for assessing binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These methods can be used to measure the concentration of bound binding protein as a function of the concentration of the target protein. The concentration of the bound binding protein ([Bound]) is generally related to the concentration of the free target protein ([Free]) by the following equation:

[Bound]=[Free]/(M+[Free])

However, it is not always necessary to make an exact determination of KA, since sometimes it is sufficient to obtain a quantitative measurement of affinity, for example determined using a method such as ELISA or FACS analysis, which is proportional to KA, and can thus be used for comparison, e.g. , to determine whether a higher affinity is, for example, 2-fold higher, it is sufficient to obtain a qualitative measurement of affinity or to infer affinity, for example, from activity in a functional assay, for example, in an in vitro or in vivo assay.

In some embodiments, the anti-FXIIa antibody includes a heavy chain comprising a heavy chain variable region that contains a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3. In some cases, the heavy chain CDR3 region contains the amino acid sequence SEQ ID NO: 111, 112 or 113 (Table 1). The heavy chain variable region of any of the anti-FXIIa antibodies described herein may further comprise a CDR1 containing the sequence of the formula XiYX ₃ MX ₅ (SEQ ID NO: 117) described herein and/or a CDR2 containing the sequence of formula X1IX3PSGX7X8TX10YX12DSVKG (SEQ ID NO: 118), which is also described herein. In some examples, the anti-FXIIa antibody comprises a heavy chain CDR1 selected from SEQ ID NOs: 4173 and 121 (Table 1) and/or a heavy chain CDR2 selected from SEQ ID NOs: 74-110 and 122-124. In certain specific examples, the heavy chain variable region of an anti-FXIIa antibody described herein comprises the amino acid sequence of any of SEQ ID NO: 1-39.

In table 1 shows the heavy chain CDR amino acid sequences for 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 the present invention.

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Table 1. Heavy chain CDR sequences of aHTu-FXIIa antibodies

Exemplary Antibody CDR1 CDR2 CDR3 M0191-E09 WYSMH (SEQ ID NO: 41) VIYPSGGKTRYADSVKG (SEQ ID NO: 74) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0192-Н11 QYVMH (SEQ ID NO: 42) SIWPSGGHTRYADSVKG (SEQ ID NO: 75) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0184-F12 WYVMH (SEQ ID NO: 43) GIWPSGGRTKYADSVKG (SEQ ID NO: 76) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0292-D07 NYVMH (SEQ ID NO: 44) SIWPSGGKTKYADSVKG (SEQ ID NO: 77) QRYRGPKYYYYMDA (SEQ ID NO: 112) M0182-D04 MYTMN (SEQ ID NO: 45) RIYPSGGKTLYADSVKG (SEQ ID NO: 78) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-F07 QYVMS (SEQ ID NO: 46) RIYPSGGVTKYADSVKG (SEQ ID NO: 79) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0183-С03 WYNMH (SEQ ID NO: 47) YISPSGGKTKYTDSVKG (SEQ ID NO: 80) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0183-Н08 RYIMH (SEQ ID NO: 48) SIYPSGGVTRYADSVKG (SEQ ID NO: 81) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0183-D08 RYIMG (SEQ ID NO: 49) SIYPSGGVTRYADSVKG (SEQ ID NO: 82) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-G03 QYNMV (SEQ ID NO: 50) RIWPSGGKTTYADSVKG (SEQ ID NO: 83) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0310-С06 RYVMV (SEQ ID NO: 51) RIYPSGGMTQYADSVKG (SEQ ID NO: 84) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-F01 WYNMA (SEQ ID NO: 52) RIYPSGGMTQYADSVKG (SEQ ID NO: 84) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0191-A03 QYIMH (SEQ ID NO: 53) SIYPSGGNTKYADSVKG (SEQ ID NO: 85) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-A01 HYVMH (SEQ ID NO: 54) SIYPSGGLTKYADSVKG (SEQ ID NO: 86) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0191-Н10 WYTMH (SEQ ID NO: 55) SIYPSGGFTRYADSVKG (SEQ ID NO: 87) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0177-С12 FYHMH (SEQ ID NO: 56) RIVPSGGMTRYADSVKG (SEQ ID NO: 88) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0184-В04 FYSMH (SEQ ID NO: 57) RIYPSGGVTKYADSVKG (SEQ ID NO: 89) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0308-Н03 QYVMH (SEQ ID NO: 58) SIWPSGGKTTYADSVKG (SEQ ID NO: 90) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-D02 QYVMH (SEQ ID NO: 58) SIWPSGGFTKYADSVKG (SEQ ID NO: 91) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-G07 FYNMH (SEQ ID NO: 59) SIYPSGGVTRYADSVKG (SEQ ID NO: 92) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-F06 WYVMH (SEQ ID NO: 43) SIYPSGGKTSYADSVKG (SEQ ID NO: 93) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-A04 PYIMH (SEQ ID NO: 60) VIYPSGSKTNYADSVKG (SEQ ID NO: 94) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-F02 RYTMR (SEQ ID NO: 61) SIWPSGGMTRYADSVKG (SEQ ID NO: 95) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0310-В09 QYVMH (SEQ ID NO: 58) SIYPSGGLTRYADSVKG (SEQ ID NO: 96) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-G06 WYIMG (SEQ ID NO: 62) YIYPSGGNTRYADSVKG (SEQ ID NO: 97) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0182-Н01 RYVMH (SEQ ID NO: 63) SIWPSGGMTKYADSVKG (SEQ ID NO: 98) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0178-A08 FYIMG (SEQ ID NO: 64) RIYPSGGATQYADSVKG (SEQ ID NO: 99) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0184-D01 FYVMG (SEQ ID NO: 65) RIYPSGGLTQYADSVKG (SEQ ID NO: 100) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0177-A06 FYSMH (SEQ ID NO: 66) RIYPSGGITSYADSVKG (SEQ ID NO: 101) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0191-E04 MYIMH (SEQ ID NO: 67) SIYPSGGMTKYADSVKG QRYRGPKYYYYMDV (SEQ ID NO: 102) (SEQ ID NO: 111) М0191-Н09 MYVMH (SEQ ID NO: 68) SIYPSGGLTKYADSVKG (SEQ ID NO: 103) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0192-Н04 QYVMH (SEQ ID NO: 58) RIYPSGGLTNYADSVKG (SEQ ID NO: 104) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0192-A03 WYVMQ (SEQ ID NO: 69) SIYPSGGMTKYADSVKG (SEQ ID NO: 102) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-G08 WYIMG (SEQ ID NO: 62) RIYPSGGSTHYADSVKG (SEQ ID NO: 105) QRYRGPRYYYYIDA (SEQ ID NO: 113) M0192-G05 QYTMV (SEQ ID NO: 70) RIYPSGGVTQYADSVKG (SEQ ID NO: 106) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0183-В12 WYVMY (SEQ ID NO: 71) RIYPSGGITHYADSVKG (SEQ ID NO: 107) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0308-F04 FYVML (SEQ ID NO: 72) SIWPSGGVTKYADSVKG (SEQ ID NO: 108) QRYRGPKYYYYMDV (SEQ ID NO: 111) М0182-В04 WYVMQ (SEQ ID NO: 69) YIYPSGGHTKYADSVKG (SEQ ID NO: 109) QRYRGPKYYYYMDV (SEQ ID NO: 111) M0310-F04 RYSMN (SEQ ID NO: 73) GIYPSGGKTKYADSVKG (SEQ ID NO: 110) QRYRGPKYYYYMDV (SEQ ID NO: 111) Х211-А01 (DX- 4012) QYVMH (SEQ ID NO:58) SIWPSGGHTRYADSVKG (SEQ ID NO:75) QRYRGPKYYYYMDV (SEQ ID NO:111)

The heavy chain variable regions of the exemplary anti-FXIIa antibodies listed in Table 1 are shown below (CDR regions are in bold). In other embodiments, the anti-FXIIa antibody CDR1 HC may be QYSMH (SEQ ID NO: 121), which may be present in combination with the CDR2 HC of SEQ ID NO: 74 and/or with the CDR3 HC of SEQ ID NO: 111. In others In embodiments, the CDR2 HC anti-FXIIa antibody may be SIYPSGGKTRYADSVKG (SEQ ID NO: 122), VIWPSGGKTRYADSVKG (SEQ ID NO: 123), or VIYPSGGHTRYADSVKG (SEQ ID NO: NO: 124), which may be present in combination with the CDR1 HC of SEQ ID NO: 41 and/or HC CDR3 from SEQ

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ID NO: 111. In yet another embodiment, the anti-FXIIa antibody CDR2 HC may be SIWPSGGHTRYADSVHG (SEQ ID NO: 127), which may be present in combination with the CDR1 HC of SEQ ID NO: 58 and/or the CDR3 HC of SEQ ID NO: 111.

Sequence of the variable region of the heavy chain M0191E09 (SEQ ID NO: 1):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYSMHWVRQAPGKGLEWVSVIYPSGGKTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0192H11 (SEQ ID NO: 2):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSSIWPSGGHTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence M0184F12 (SEQ ID NO: 3) :

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMHWVRQAPGKGLEWVSGIWPSGGRTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Heavy chain variable region sequence of M0292D07 (SEQ ID NO: 4):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSNYVMHWVRQAPGKGLEWVSSIWPSGGKTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDAWGQGTTVTVSS

Heavy chain variable region sequence M0182D04(SEQ ID NO: 5):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSMYTMNWVRQAPGKGLEWVSRIYPSGGKTL

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

M192F07 heavy chain variable region sequence (SEQ ID NO: 6):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMSWVRQAPGKGLEWVSRIYPSGGVTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Heavy chain variable region sequence M0183003 (SEQ ID NO: 7):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYNMHWVRQAPGKGLEWVSYISPSGGKTK

YTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the heavy chain variable region M0183H08 (SEQ ID NO: 8):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYIMHWVRQAPGKGLEWVSSIYPSGGVTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Heavy chain variable region sequence M0183D08 (SEQ ID NO: 9):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYIMGWVRQAPGKGLEWVSSIYPSGGVTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0192G03 (SEQ ID NO: 10):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYNMWVRQAPGKGLEWVSRIWPSGGKTT

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the heavy chain variable region M0310C06 (SEQ ID NO: 11):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYVMWVRQAPGKGLEWVSRIYPSGGMTQ

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

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Sequence of the variable region of the heavy chain M0192F01 (SEQ ID NO: 12):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYNMAWVRQAPGKGLEWVSRIYPSGGMTQ

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0191A3 (SEQ ID NO: 13):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYIMHWVRQAPGKGLEWVSSIYPSGGNTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0192A01 (SEQ ID NO: 14):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMHWVRQAPGKGLEWVSSIYPSGGLTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

The sequence of the variable region of the heavy chain M0191H10 (SEQ ID NO: 15):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYTMHWVRQAPGKGLEWVSSIYPSGGFTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence M0177012 (SEQ ID NO: 16):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYHMHWVRQAPGKGLEWVSRIVPSGGMTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0184B04 (SEQ ID NO: 17):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYSMHWVRQAPGKGLEWVSRIYPSGGVTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence M0308HOZ (SEQ ID NO: 18):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSSIWPSGGKTT

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Heavy chain variable region sequence of M0192D02 (SEQ ID NO: 19):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSSIWPSGGFTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

The sequence of the heavy chain variable region of M0310G07 (SEQ ID NO: 20):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYNMHWVRQAPGKGLEWVSSIYPSGGVTR YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

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Sequence of the variable region of the heavy chain M0192F06 (SEQ ID NO: 21):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMHWVRQAPGKGLEWVSSIYPSGGKTS

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0310A04 (SEQ ID NO: 22):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYIMHWVRQAPGKGLEWVSVIYPSGSKTN

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

The sequence of the heavy chain variable region of M0310F02 (SEQ ID NO: 23):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYTMRWVRQAPGKGLEWVSSIWPSGGMTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGQGTTVTVSS

The sequence of the variable region of the heavy chain M0310B09 (SEQ ID NO: 24):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSSIYPSGGLTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGQGTTVTVSS

Sequence of the heavy chain variable region of M0310G06 (SEQ ID NO: 25):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYIMGWVRQAPGKGLEWVSYIYPSGGNTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0182H01 (SEQ ID NO: 26):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYVMHWVRQAPGKGLEWVSSIWPSGGMTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M017 8A08 (SEQ ID NO: 27):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYIMGWVRQAPGKGLEWVSRIYPSGGATQ

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Heavy chain variable region sequence M0184D01 (SEQ ID NO: 28):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMGWVRQAPGKGLEWVSRIYPSGGLTQ

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0177A06 (SEQ ID NO: 29):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYSMHWVRQAPGKGLEWVSRIYPSGGITS YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

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Sequence of the heavy chain variable region M0191E04 (SEQ ID NO: 30):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSMYIMHWVRQAPGKGLEWVSSIYPSGGMTK YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0191H09 (SEQ ID NO: 31):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSMYVMHWVRQAPGKGLEWVSSIYPSGGLTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence M0192H04 (SEQ ID NO: 32):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSRIYPSGGLTN YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the heavy chain variable region M0192A3 (SEQ ID NO: 33):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMQWVRQAPGKGLEWVSSIYPSGGMTK YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

The sequence of the heavy chain variable region of M0310G08 (SEQ ID NO: 34):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYIMGWVRQAPGKGLEWVSRIYPSGGSTH

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPRYYYYIDAWGQGTTVTVSS Heavy chain variable region sequence M0192G05 (SEQ ID NO: 35):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYTMVWVRQAPGKGLEWVSRIYPSGGVTQ YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0183B12 (SEQ ID NO: 36):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMYWVRQAPGKGLEWVSRIYPSGGITH YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence M0308F04 (SEQ ID NO: 37):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSFYVMLWVRQAPGKGLEWVSSIWPSGGVTK YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

Sequence of the variable region of the heavy chain M0182B04 (SEQ ID NO: 38):

EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYVMQWVRQAPGKGLEWVSYIYPSGGHTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS Heavy chain variable region sequence of M0310F04 (SEQ ID NO: 39):

EVQLLESGGGLVQPGGSLRLSCAASGTFFSRYSMNWVRQAPGKGLEWVSGIYPSGGKTK

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

X211A01 Heavy Chain Variable Region Sequence (SEQ ID NO:125)

EVQLLESGGGLVQPGGSLRLSCAASGTFFSQYVMHWVRQAPGKGLEWVSSIWPSGGHTR

YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRQRYRGPKYYYYMDVWGKGTTVTVSS

The present invention also provides germline variants for any of the exemplary anti-FXIIa antibodies described herein. The germline variant contains one or more mutations in framework regions relative to its parent antibody in the corresponding germline sequence. To generate a germline variant, the parent antibody heavy or light chain variable region sequence, or part thereof (eg, framework sequence) can be used as a query against a germline antibody sequence database (eg, www.bioinfo.org.uk/abs /,www.vbase2.org or www.imgt.org) to determine the corresponding germline sequence used by the parent antibody and variations in amino acid residues in one or more framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions may be introduced into the parent antibody based on the germline sequence to produce a germline variant. For example, clone X211-A01 (DX-4012) is a germline variant of the clone

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M192-N11. As described herein, the anti-FXIIa antibody can be in any form, including, but not limited to, intact (i.e., full-length) antibodies, antigen binding fragments (such as Fab, Fab', F(ab')2, Fv) and single chain antibodies.

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 region thereof (eg, CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, for example human, mouse, rat or rabbit. In one specific example, the heavy chain constant region is derived from human IgG (gamma heavy chain). One example of a human antibody heavy chain constant region is shown below (SEQ ID NO: 119):

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTDKSRW

QQGNVFSCSV MHEALHNHYT QKSLSLSPG

Bold/underlined residues represent residues that can be mutated to improve FcRn binding and increase serum half-life.

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

If desired, the anti-FXIIa antibody described herein may contain a modified constant region. For example, it may contain a modified constant region that is immunologically inert, eg, does not cause complement-mediated lysis or stimulate antibody-dependent cellular cytotoxicity (ADCC). ADCC activity can be assessed using the methods described in US Pat. No. 5,500,362. In other embodiments, the constant region is 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.

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 a desired characteristic of the antibody, e.g., increasing neonatal Fc receptor (FcRn) binding activity and thus thus increasing the half-life of the antibody in serum. Binding to FcRn is known to be critical for maintaining antibody homeostasis and regulating antibody half-life in serum. One or more (eg, 1, 2, 3, 4, 5 or more) mutations (eg, amino acid residue substitutions) may be introduced into the constant region at suitable locations (eg, in the CH2 region) to enhance binding to FcRn and increasing the half-life of the antibody. Such mutations may be at position(s) corresponding to positions 145, 147 and/or 149 in SEQ ID NO: 119 (corresponding to positions 252, 254 and 256 according to the numbering system published in Rabat et al., 1991, Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, D.C.). See also Dall'Acqua et al., J.B.C., 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, the positions at which mutations may be introduced can be determined by aligning the amino acid sequence of that constant region with SEQ ID NO: 119 to identify positions corresponding to positions of interest (e.g., positions 145, 147, or 149 in SEQ ID NO: 119). Alternatively, such positions may be defined using a well-recognized numbering system such as the Kabat numbering system noted above.

In some examples, a substitution mutation is introduced at an amino acid residue (eg, a methionine residue) at position 252 (position 145 in SEQ ID NO: 119) of the heavy chain constant region, such as an M^Y substitution. In some examples, the replacement mutation is introduced at position 254 (position 147 in SEQ ID NO: 119) of the heavy chain constant region, for example, the Sy-T substitution. In some examples, the replacement mutation is introduced at position 256 (position 149 of SEQ ID NO: 119) of the heavy chain constant region, for example, T^E. In some examples, the threonine at position 256 is mutated to a glutamic acid residue. If desired, multiple mutations (eg, amino acid substitutions) may be introduced into the heavy chain constant region, for example, any combination of the above substitutions. In a specific example, the modified heavy chain constant region used in any of the anti-FXIIa antibodies may contain amino acid substitutions at positions 252, 254 and 256 (eg, M252Y, S254T and T256E, also known as the YTE variant). See, for example, Dall'Acqua et al., J.B.C., 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 amino acid sequence shown below (SEQ ID NO: 120) is an exemplary modified heavy chain constant region capable of better binding to FcRn thereby increasing serum half-life:

Exemplary heavy chain constant region YTE variant sequence (SEQ ID NO: 120):

ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLYITREP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTDKSRW

OOGNVFSCSV MHEALHNHYT OKSLSLSPG

Amino acid residues that are mutated in SEQ ID NO: 120 relative to SEQ ID NO: 119 are shown in bold.

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

The anti-FXIIa antibody described herein may comprise a light chain variable region that contains the light chain CDR3 shown as SEQ ID NO: 116 (see Table 2). In some embodiments, the light chain variable region of an anti-FXIIa antibody may further comprise a light chain CDR1, shown as SEQ ID NO: 114 (see Table 2) and/or a light chain CDR2, shown as SEQ ID NO: 115 ( see table 2). For example, the light chain variable region of an anti-FXIIa antibody may comprise light chain CDR1 of SEQ ID NO: 114, light chain CDR2 of SEQ ID NO: 115, and light chain CDR3 of SEQ ID NO: 116. For example, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40. In an alternative embodiment, the light chain variable region is a germline variant of SEQ ID NO: 40, which may contain one or more mutations in one or more framework regions in the corresponding germline sequence. The corresponding germline sequence can be determined by methods known in the art and those described herein.

An exemplary light chain variable region for anti-FXIIa antibodies is shown below (CDR regions are in bold).

Exemplary light chain variable region for anti-FXIIa antibodies (SEQ ID NO: 40):

DIQMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNR

ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK

Exemplary Light Chain Variable Region for Anti-FXIIa Antibodies (SEQ ID NO: 126) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNR

ASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPWTFGQGTKVEIK

Table 2. CDR sequences of anti-FXIIa antibody light chains

CDR1 CDR2 CD3 Exemplary Antibody RSSQSLLHSNGYNYLD (SEQ ID NO: 114) LGSNRAS (SEQ ID NO: 115) MQALQTPWT (SEQ ID NO: 116)

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 NO: 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; aka 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.

Also included within the scope of the present invention are functional variants of any of the exemplary anti-FXIIa antibodies described herein (eg, the 44 antibody clones described above). In some embodiments, the anti-FXIIa antibody is a functional antibody variant comprising the heavy chain variable region set forth in any of SEQ ID NOs: 1-39 and the light chain variable region set forth in SEQ ID NO: 40. The functional variant

- 14 045947 ant may contain up to 5 (e.g., 4, 3, 2 or 1) amino acid residue variations in one or more CDR regions of the antibody and binds the same FXIIa epitope with substantially similar affinity (e.g., having a KD value of the same order of magnitude ). In one example, variations in amino acid residues are conservative substitutions of amino acid residues. In the context of the present invention, 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 in accordance with methods for altering a polypeptide sequence known to one of ordinary skill in the art, such as can be found in references that combine such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made among 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.

In some embodiments, the anti-FXIIa antibody comprises a heavy chain CDR that is at least 80% (e.g., 85, 90, 95, or 98%) identical to any one heavy chain CDR sequence set forth in SEQ ID NO: 41-113 , SEQ ID NO: 121-124 and SEQ ID NO: 127, and/or light chain CDR sequences that are at least 80% (e.g., 85, 90, 95, or 98%) identical to at least one of the corresponding Light chain CDRs given in SEQ ID NO: 114-116. In some embodiments, the anti-FXIIa antibody contains 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 NO: 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 set forth in SEQ ID NO: 40.

The percentage identity of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is included in the programs NBLAST and XBLAST (version 2.0) from Altschul, et al. J. Mol. Biol. 215:403-10, 1990. A protein BLAST search can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to protein molecules of interest. If there are gaps between two sequences, Gapped BLAST, described in Altschul et al., Nucleic Acids Res., can be used. 25 (17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (for example, XBLAST and NBLAST) may be used.

In addition, the anti-FXIIa antibody may include one or more other residues that are identified based on the crystal structures discussed in Example 5 herein as being involved in interaction with the FXIIa C chain. These residues can 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.

The present invention also relates to antibodies that target certain residues in FXIIa. The antibody may preferentially bind to FXIIa but not to FXII. In some embodiments, an antibody that specifically binds FXIIa reacts with one or more residues (e.g., at least 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, or 45) of C- chains in FXIIa, including 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. These residues are identified as interacting with one or more residues in the heavy chain variable region and light chain variable region of the anti-FXIIa antibody according to the crystal structures described in Example 5 below.

The amino acid sequence (SEQ ID NO: 128) of an exemplary human FXIIa is given below and the above residues are shown in bold:

- 15 045947 mrallllgfl Ivslestlsi ppweapkehk ykaeehtvvl tvtgepchfp fqyhrqlyhk cthkgrpgpq pwcattpnfd qdqrwgycle pkkvkdhcsk hspcqkggtc vnmpsgphcl

121 cpqhltgnhc qkekcfepql Irffhkneiw yrteqaavar cqckgpdahc qrlasqacrt

181 npclhggrcl eveghrlchc pvgytgafcd vdtkascydg rglsyrglar ttlsgapcqp

241 waseatyrnv taeqarnwgl gghafcrnpd ndirpwcfvl nrdrlsweyc dlaqcqtptq

301 aapptpvspr Ihvplmpaqp appkpqpttr tppqsqtpga Ipakreqpps Itrngplscg

361 qrlrkslssm trvvgglval rgahpyiaal ywqhsfcaqs liapcwvlta ahclqdrpap

421 edltvvlgqe rrnhscepcq tlavrsyrlh eafspvsyqh dlallrlqed adgscallsp

481 yvqpvclpsg aarpsettlc qvaqwghqfe qaeeyasfIq eaqvpflsle rcsapdvhgs

541 silpgmlcag fleqqtdacq qdsqqplvce dqaaerrltl qgiiswqsqc qdrnkpqvyt

601 dvayylawir ehtvs

Other exemplary FXIIa sequences may include human, mouse, or rat FXIIa sequences, a sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to one of these sequences or fragment.

An interaction means that the distance between two residues in a complex formed by two binding partners is less than a specified value, for example, <6 A, <4 A, or <2 A. For example, the interacting residue in one binding partner may have at least 1 atom in within a given threshold (eg, <6 A, <4 A, or <2 A) from at least 1 atom from the residue of the other binding partner per complex structure. Interaction does not require actual binding. The interacting residues have been proposed to be involved in antibody recognition.

In some embodiments, an antibody described herein binds active human FXIIa at an epitope containing one or more of the residues listed above. An epitope refers to a region on a target compound that is bound by an antibody, such as a Fab fragment or a full-length antibody. The epitope can be linear, typically 6-15 aa in length. Alternatively, the epitope may be conformational.

In some embodiments, an antibody that specifically binds to FXIIa described herein binds an epitope that contains the following segments from SEQ ID NO: 128: residues 390-397, residues 412-416, residues 432-433, residues 456- 458, residues 507-515, residues 557-563 and/or residues 584-592.

Preparation of anti-FXHa antibodies

Antibodies capable of binding 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.

In some embodiments, an antibody specific for a target antigen (eg, FXIIa or its catalytic domain) can be produced using conventional hybridoma technology. The full-length target antigen or fragment thereof, optionally in combination with a carrier protein such as KLH, can be used to immunize a host animal to produce antibodies that bind to the antigen. The method and schedules for immunizing the host animal generally follow well-known standard methods for generating and producing antibodies, as further described herein. General methods for producing murine, humanized and human antibodies are known in the art and are described herein. It is contemplated that any mammal, including humans, or their antibody-producing cells may be manipulated to serve as a basis for the production of mammalian, including human, hybridoma cell lines. Typically, an amount of the immunogen is administered to the host animal intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally, including the methods described herein.

Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, S. (1975) Nature 256:495-497 or a modified technique of Buck, D. W., et al., In Vitro , 18:377-381 (1982).

- 16 045947

Available myeloma lines, including but not limited to X63-Ag8.653 and lines from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used in hybridization. In general, these techniques involve fusing myeloma cells and lymphoid cells using a fusion promoting agent such as polyethylene glycol or using electricity, as is 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 can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV immortalized B cells can be used to produce the anti-FXIIa monoclonal antibodies described herein. Hybridomas are propagated and subcloned as needed, and supernatants are assayed for anti-immunogenic activity using conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that can be used as a source of antibodies include all derivatives and progeny cells of the parent hybrid that produce monoclonal antibodies capable of interfering with FXIIa activity. Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids using conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration, as desired. Impurity (unwanted) activity (if present) can be removed, for example, by passing the drug through adsorbents from the immunogen attached to the solid phase and eluting or releasing the desired antibodies from the immunogen. A population of antibodies (eg, monoclonal antibodies) can be obtained by immunizing a host animal with a target antigen or fragment thereof containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, for example, hemocyanin, serum albumin, bovine thyroglobulin or soy trypsin inhibitor, using a bifunctional or derivatizing agent such as maleimidobenzoylsulfosuccinimide ester (for conjugation via cysteine residues), Nhydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOC1 or R1N=C=NR, where R and R1 are various alkyl groups.

If desired, the nucleotide sequence of an antibody of interest (monoclonal or polyclonal, eg, produced by a hybridoma) can be determined by sequencing, and the polynucleotide sequence can then be cloned into an expression or propagation vector. The sequence encoding the antibody of interest can be stored in a vector in a host cell, and the host cells can then be expanded and frozen for later use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to humanize the antibody or to increase affinity (affinity maturation), or to change other characteristics of the antibody. For example, the constant region may be modified to be more similar to the constant region of a human antibody in order to avoid an immune response if the antibody is used in human clinical trials and treatment. It may be desirable to genetically modify antibody sequences to obtain higher affinity for the target antigen and greater effectiveness in inhibiting FXIIa activity. One skilled in the art will appreciate that one or more changes can be made to a polynucleotide encoding an antibody while still retaining the binding specificity of the antibody to its target antigen.

In other embodiments, fully human antibodies can be produced using commercially available mice that have been engineered to express certain human immunoglobulins. Transgenic animals that have been created to produce more desirable antibodies (such as fully human antibodies) or a stronger immune response can also be used to produce humanized or human antibodies. Examples of such technology include Xenomouse™ from Amgen, Inc. (Fremont, Calif.), as well as HuMAb-Mouse™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies can be produced recombinantly using phage or yeast display technology. See, for example, US patents. Nos. 5565332, 5580717, 5733743 and 6265150; 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 the immunoglobulin variable (V) domain gene repertoire of non-immunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody) can be prepared by conventional methods. For example, F(ab)2 fragments can be obtained by pepsin digestion of the antibody molecule, and Fab fragments can be obtained by reduction of disulfide

- 17 045947 bridges in E(ab)2-(|) fragments.

Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single chain antibodies and bispecific antibodies, can be produced using, for example, conventional recombinant technology. In one example, DNA encoding monoclonal antibodies specific for a target antigen can be readily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as the preferred source of such DNA. Once isolated, the DNA can be transferred into one or more expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins, to produce synthesis monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. The DNA can then be modified, for example, by replacing the human antibody constant domain heavy and light chain coding sequences with homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851) or by covalent attachment to the immunoglobulin coding sequence of all or part of the coding sequence of a non-immunoglobulin polypeptide. Thus, antibodies, such as chimeric or hybrid antibodies, that specifically bind a target antigen can be produced through genetic engineering.

Techniques developed for the production of chimeric antibodies are well known in the art. See, for example, 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.

Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the VH and VL variable regions of a parent animal antibody are subjected to three-dimensional molecular modeling analysis following methods known in the art. Scaffold amino acid residues believed to be important for the formation of correct CDR structures are then identified using the same molecular modeling. In parallel, human antibody VH and VL chains having amino acid sequences that are homologous to those of the parent animal antibody are identified from any antibody gene database using the parent VH and VL sequences as a search query. The human VH and VL acceptor genes are then selected.

CDR regions within selected human acceptor genes may be replaced by CDR regions from the parent animal antibody or functional variants thereof. If necessary, residues in framework regions of the parent chain that are predicted to be important for interaction with the CDRs (see description above) can be used to replace the corresponding residues in human acceptor genes.

A single chain antibody can be produced by recombinant technology by linking a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region. Preferably, a flexible linker is included between the two variable regions. Alternatively, the techniques described for the production of single chain antibodies (US Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce a phage or yeast scF library, and FXIIa-specific scFv clones can be identified from the library using routine procedures. . Positive clones can be further screened to identify those that inhibit FXIIa activity.

Antibodies produced by a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to determine the epitope to which the antigen binds, or epitope mapping. There are a variety of methods known in the art for mapping and characterizing the location of epitopes on proteins, including solution of the crystal structure of the antigen-antibody complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 books Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional 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 stretch of amino acids, or may be a conformational epitope formed by the three-dimensional interaction of amino acids that are not necessarily contained in a single stretch (linear sequence of primary structure). Peptides of various lengths (eg, at least 4-6 amino acids in length) can be isolated or synthesized (eg, recombinantly) and used for antibody binding assays. In another example, the epitope to which an antibody binds can be determined in a systematic screen using overlapping peptides derived from the target antigen sequence and determining binding to an

- 18 045947 titel. In gene fragment expression assays, the open reading frame encoding the target antigen is fragmented randomly or using specific genetic constructs, and the reactivity of the expressed antigen fragments with the antibody of interest is determined. Gene fragments can, for example, be obtained by PCR and then transcribed and translated into protein in vitro in the presence of radiolabeled amino acids. The binding of the antibody to radiolabeled antigen fragments is then determined using immunoprecipitation and gel electrophoresis. Some epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a limited library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. As a further example, antigen binding domain mutagenesis, domain swap experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or essential for epitope binding. For example, domain swap experiments can be performed using a mutant of a target antigen in which various fragments of the FXIIa polypeptide have been replaced with sequences from a closely related but antigenically distinct protein (eg, another member of the neurotrophin family). By assessing antibody binding to mutant FXIIa, the importance of a particular antigen fragment for antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies that are known to bind to the same antigen to determine whether the antibody binds to the same epitope as other antibodies. Competitive assays are well known to those skilled in the art.

In some examples, the anti-FXIIa antibody is produced using recombinant technology, as illustrated below.

Nucleic acids encoding the heavy and light chain of an anti-FXIIa antibody described herein can be cloned into a single expression vector, each nucleotide sequence being operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy chain and light chain variable regions is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy chain and the light chain may be operably linked to a single promoter such that the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome binding site (IRES) can be inserted between the heavy chain and light chain coding sequences.

In some examples, nucleotide sequences encoding two chains of an antibody are cloned into two vectors that can be introduced into the same or different cells. When the two chains are expressed in different cells, each can be isolated from the host cells expressing them, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to promote antibody formation.

In general, nucleotide sequences encoding one or all chains of an antibody can be cloned into a suitable expression vector operably linked to an appropriate promoter using methods known in the art. For example, the nucleotide sequence and the vector can be treated under suitable conditions with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined to each other by a ligase. Alternatively, synthetic nucleotide linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleotide sequences that match a specific restriction site in the vector. The choice of expression vectors/promoter will depend on the type of host cells used for antibody production.

Various promoters can be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, early promoter simian virus 40 (SV40), the LacUV5 promoter from E. coli, and the herpes simplex virus tyrosine kinase (tk) promoter.

Regulated promoters can also be used. Such regulated promoters include promoters that use the lac repressor from E. coH as a transcriptional modulator to regulate transcription from 1ac operator-carrying promoters from mammalian cells [Brown, M. et al., Cell, 49:603-612 (1987 )], promoters 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, VP 16 or p65 dimer using astradiol, RU486, diphenolmurislerone or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulated promoters that include a repressor with an operon can be used. In one

- 19 045947 embodiment, the 1ac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-carrying promoters from mammalian cells [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (in 1992) [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined a tetracycline repressor (tetR) with a transcription activator (VP-16) to create a tetR-mammalian transcription activator fusion protein, tTa (tetR-VP 16), with a tetO-carrying minimal promoter derived from the core immediate early promoter of human cytomegalovirus (HCMV) to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment of the invention, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, rather than fusion derivatives of tetR and a mammalian cell transcription factor, can function as a potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is correctly positioned downstream of the TATA element in 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 a tetracycline repressor-transactivator or mammalian repressor fusion protein, which in some cases can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89: 5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)) to provide a transcriptional regulatory effect.

In addition, the vector may contain, for example, some or all of the following elements: a selectable marker gene, such as a neomycin gene for selection of stable transfectants or transient transfectants in mammalian cells;

enhancer/promoter sequence from human cytomegalovirus immediate early gene for high level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; origin of replication from SV40 and ColE1 for proper episomal replication; internal ribosome binding sites (IRES), universal multiple cloning sites; and T7- and SP6-nmotors for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

Examples of polyadenylation signals useful in the practice of 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.

One or more vectors (eg, expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into suitable host cells to produce antibodies. Host cells can be cultured under suitable conditions to express the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof can be recovered from cultured cells (eg, cells or culture supernatant) using a conventional method, such as affinity purification. If necessary, the antibody polypeptide chains can be incubated under suitable conditions for a suitable period of time to allow antibody production.

In some embodiments, the methods for producing an antibody described herein include a recombinant expression vector that encodes the heavy chain and light chain of an anti-FXIIa antibody also described herein. The recombinant expression vector can be introduced into a suitable host cell (eg, a dhfr-CHO- cell) by a conventional method, such as calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under suitable conditions allowing expression of two polypeptide chains that form antibodies, which can be isolated from the cells or from the culture medium. If necessary, these two chains, isolated from host cells, can be incubated under suitable conditions to allow the formation of antibodies.

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 a suitable host cell (eg, a dhfr-CHO cell) by conventional means, such as calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into suitable host cells. Positively transformed host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains. When two expression vectors are introduced into the same host cells, the antibodies produced therein can be isolated from the host cells or from the culture medium. If necessary, polypeptide chains can be isolated from host cells or culture medium and then incubated under suitable conditions to promote antibody formation. When two expression vectors are introduced into different host cells, each of the chains can be isolated from the corresponding host cells or from the corresponding culture medium. These two polypeptide chains can then be incubated under conditions suitable for antibody formation.

Standard molecular biology techniques are used to produce a recombinant expression vector, transfect host cells, select transformants, culture host cells, and

- 20 045947 isolation of antibodies from the culture medium. For example, some antibodies can be isolated using affinity chromatography on a protein A or protein G support.

Any of the nucleic acids encoding the heavy chain, light chain, or both chains of the anti-FXIIa antibody described herein, vectors (e.g., expression vectors) containing these nucleic acids, and host cells containing the vectors are within the scope of the present invention .

Pharmaceutical compositions

Antibodies, as well as nucleic acids or sets of nucleic acids encoding them, vectors containing them, or host cells containing vectors described herein, can be mixed with a pharmaceutically acceptable carrier (excipient) to obtain a pharmaceutical composition for use in treatment target disease. Acceptable means that the carrier must be compatible with the active ingredient of the composition (and, preferably, capable of stabilizing the active ingredient) and not be harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers), including buffers, are well known to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

Pharmaceutical compositions for use in the methods of the present invention may 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, Ed. K. E. Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used and may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechin, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight polypeptides (shorter than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, zinc protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some embodiments, the pharmaceutical composition described herein includes liposomes containing antibodies (or nucleic acids encoding them), which can be prepared by methods known in the art, for example, as 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 US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased blood residence time are described in US Pat. No. 5,013,556. Particularly suitable liposomes can be prepared by a reverse phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with a certain pore size, obtaining liposomes of the required diameter.

Antibodies or nucleic acids encoding them can also be enclosed in microcapsules obtained, for example, by coacervation methods or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively; in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein may be formulated in a sustained release format. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are molded articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl 7-c-glutamate. non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid-glycolic acid and leuprolide acetate), acetate-isobutyrate sucrose and poly^(-)-3-hydroxybutyric acid.

Pharmaceutical compositions to be used for in vivo administration must be sterile. This is easily achieved by, for example, filtration through sterile filter media.

- 21 045947 membranes. Therapeutic antibody compositions are typically placed in a container having a sterile access port, such as an intravenous bag or vial having a stopper pierced by a hypodermic needle.

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

To prepare solid compositions such as tablets, the main active ingredient may be mixed with a pharmaceutical carrier, for example standard tabletting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or various gums, and other pharmaceutical diluents, for example, water, to form a solid pre-composition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. The indication that these preformulations are homogeneous means that the active ingredient is uniformly distributed throughout the composition so that the composition can be easily divided into equally effective units of dosage forms such as tablets, pills and capsules. This solid preformulation is then divided 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 the novel composition may be coated or otherwise formulated to provide a dosage form that provides the benefit of a sustained release. For example, a tablet or pill may contain an internal dose and an external dose component, the latter being a coating for the former. The two components may be separated by an enteric layer, which prevents degradation in the stomach and allows the internal component to pass intact into the duodenum or delay its release. Various materials can be used for such enteric layers or shells, including a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include, but are not limited to, nonionic agents such as polyoxyethylene sorbitans (eg, Tween™ 20, 40, 60, 80 or 85) and other sorbitans (eg, Span™ 20, 40, 60, 80 or 85). Surfactant compositions will typically contain from 0.05 to 5% surfactant, and the content may range from 0.1 to 2.5%. It should be understood that other ingredients, such as mannitol or other pharmaceutically acceptable carriers, may be added as necessary.

Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient can either be dissolved in a premixed emulsion composition or, alternatively, it can be dissolved in an oil (eg, soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion is formed when mixed with a phospholipid (eg egg phospholipids, soy phospholipids or soy lecithin) and water. It should be borne in mind that other ingredients, such as glycerin or glucose, may be added to regulate the tonicity of the emulsion. Suitable emulsions typically contain up to 20% oil, for example 5 to 20%. The fat emulsion may contain fat droplets from 0.1 to 1.0.im, in particular from 0.1 to 0.5.im, and has a pH in the range from 5.5 to 8.0.

Emulsion compositions can be compositions that are prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerin and water).

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

The compositions in preferably sterile pharmaceutically acceptable solvents can be nebulized using gases. Nebulized solutions can be inhaled directly from a nebulizing device (nebulizer), or the nebulizer can be connected to a face mask, chamber, or positive pressure breathing machine. The solution, suspension or powder composition can be administered, preferably orally or nasally, from devices that deliver the drug in an appropriate manner.

Treatment options

Any of the antibodies, as well as the nucleic acids or sets of nucleic acids encoding them, vectors containing them or containing host cell vectors described herein are useful for treating a disease or disorder associated with aberrant

- 22 045947 contact system activation, including diseases associated with contact system activation, diseases associated with aberrant contact system activation (for example, HAE), or eye diseases.

To practice the method described herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject in need of treatment (e.g., a human) by a suitable route, such as intravenous administration, for example, as a bolus or continuous infusion, in over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, inhalation or topical. Commercially available nebulizers for liquid formulations, including jet and ultrasonic nebulizers, are suitable for administration. Liquid drugs can be sprayed directly, and lyophilized powder can be sprayed after reconstitution. Alternatively, the antibody described herein may be administered as an aerosol using a fluorocarbon formulation and a metered dose inhaler, or the antibody may be inhaled as a lyophilized and micronized powder.

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

The methods and compositions described herein can be used to treat any disease or disorder associated with activation of the contact system and/or pKal signaling pathways. In some embodiments, the target disease is thrombosis, including thrombosis associated with atrial fibrillation, deep vein thrombosis, pulmonary embolism, stroke, or other arterial or venous thrombosis. Thrombosis (such as venous thrombosis or arterial thrombosis) refers to the formation of blood clots within a blood vessel, which can impede the flow of blood in the circulatory system. Subjects with thrombosis or at risk of thrombosis can be identified using routine medical procedures.

In other embodiments of the invention, a disease that can be treated with anti-FXIIa antibodies described herein may be a disease associated with the kallikrein system (eg, pKal system), including, 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 activation of the contact system include, but are not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, systemic lupus erythematosus, systemic lupus erythematous nephritis, systemic mastocytosis, gout, intestinal diseases, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis, degenerative diseases of the spine, arterial or venous thrombosis, postoperative ileus, aortic aneurysm, vasculitis, edema, acquired angioedema, idiopathic angioedema, anaphylactic shock, idiopathic anaphylactic shock, cerebral edema, pulmonary embolism, stroke , clotting on ventricular assist devices or stents, clotting associated with the use of catheters or peripherally inserted central catheters, blood clotting associated with the use of an extracorporeal membrane oxygenation device, clotting associated with the use of a graft or dialysis fistula, head trauma or edema brain tissue around the tumor, sepsis, acute middle cerebral artery (MCA) infarction, ischemic events (stroke), restenosis (eg, after angioplasty), or burns.

In some embodiments, the disease that can be treated with the anti-FXIIa antibodies described herein is an eye disease associated with contact system activation, including, but not limited to, macular edema, diabetic retinopathy, hypertensive retinopathy, age-related degeneration macula and retinal vein occlusion.

In one example, a disease or condition that involves activation of the contact system is hereditary angioedema (HAE). Hereditary angioedema (HAE) is also known as angioedema, C1-esterase inhibitor deficiency, C1 inhibitor deficiency and hereditary angioedema.

- 23 045947 edema (NANO). HAE is characterized by repeated attacks of severe swelling (angioedema), which can affect, for example, the limbs, face, genitals, gastrointestinal tract and respiratory tract. Symptoms of HAE include, for example, increased volume of the arms, legs, lips, eyes, tongue and/or throat; airway blockage, which may include swelling of the throat and sudden hoarseness; repeated episodes of cramping abdominal pain for no apparent reason; and/or swelling of the intestines, which can be severe and may lead to cramping abdominal pain, vomiting, dehydration, diarrhea, pain and/or shock. About one third of individuals with such HAE develop a non-itching rash called erythema marginatum during an attack.

Swelling in the airways can be life-threatening and can lead to death in some patients. Mortality rates are estimated at 15-33%. HAE is responsible for approximately 15,000 to 30,000 emergency department visits per year.

Trauma or stress, such as dental procedures, illness (such as viral illnesses such as colds and flu), menstruation, and surgery, can trigger angioedema. To prevent acute attacks of HAE, patients may try to avoid certain stimuli that previously triggered attacks. However, in many cases the attack occurs without a known trigger. Typically, HAE symptoms first appear in childhood and worsen during puberty. On average, untreated people have attacks every 1 to 2 weeks, and most episodes last approximately 3 to 4 days (ghr.nlm.nih.gov/condition/hereditary-angioedema).

The frequency and duration of attacks vary greatly among people with hereditary angioedema, even among people in the same family.

There are three types of HAE, known as types I, II and III. HAE is estimated to occur in 1:50,000 people, with type I accounting for about 85% of cases, type II accounting for about 15% of cases, and type III being very rare. Type III is the most recently described type and was originally thought to occur only in women, but families with affected men have been identified.

HAE is inherited in an autosomal dominant manner, for example, an affected person may inherit the mutation from one affected parent. Also, new mutations can occur in the gene, and thus HAE can also occur in people with no history of the disease in their family. It is estimated that 20-25% of HAE cases are the result of a new spontaneous mutation.

Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene is required for the production of C1 inhibitory protein, which is very important for controlling inflammation. A C1 inhibitor blocks the activity of certain proteins that increase inflammation. Mutations that cause type I hereditary angioedema lead to a decrease in the level of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of a C1 inhibitor that does not function properly. Without adequate levels of functional C1 inhibitor, excess bradykinin is generated. Bradykinin promotes inflammation by increasing the leakage of fluid through the walls of blood vessels into body tissue. Excessive accumulation of fluid in body tissue causes the swelling seen in individuals with hereditary angioedema types I and II.

Mutations in the F12 gene are associated with some cases of hereditary angioedema type III, also known as HAE with normal C1 inhibitor. The F12 gene is required for FXII coagulation. In addition to its important role in the process of blood clotting (coagulation), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Some mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is produced and the walls of the blood vessels become more leaky, leading to swelling. The cause of other cases of type III hereditary angioedema remains unknown. Mutations in one or more as yet unknown genes may be responsible for the pathology in these cases.

HAE may be similar to other forms of angioedema resulting from allergies or other pathological causes, but it differs significantly in cause and treatment. When hereditary angioedema is diagnosed as an allergy, it is most often treated with antihistamines, steroids, and/or epinephrine, which are generally ineffective for HAE, although epinephrine may be used for a life-threatening reaction. Misdiagnosis also leads to unnecessary exploratory surgery in patients with bloating, and in some patients with HAE, abdominal pain is misdiagnosed as psychosomatic.

Symptoms of HAE can be assessed, for example, using questionnaires, such as those completed by patients, doctors, or family members. Such questionnaires are known in the art and include, for example, a visual analogue scale. See, for example, McMillan, C.V. et al. Patient. 2012; 5 (2):113-26.

As used herein, the term effective amount refers to the amount of each active agent required to produce a therapeutic effect in a subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect reduces FXIIa activity, reduces the amount of pKal or bradykinin, or reduces vasodilation. Inhibitory antibodies that specifically bind

- 24 045947

FXIIa but do not bind FXII may have a lower effective dose than inhibitory antibodies that also bind FXII. Determining whether an amount of antibody produces a therapeutic effect will be apparent to one skilled in the art. Effective amounts will vary, as will be known to those skilled in the art, depending on the specific condition being treated, the severity of the condition, individual patient characteristics including age, physical condition, size, sex and weight, duration of treatment, nature of concomitant therapy (if any) ), the specific route of administration, and similar factors known to the practitioner. These factors are well known to those skilled in the art and can be determined through routine experimentation. In general, it is preferable to use the highest dose of the individual components or their combination, that is, the highest dose that is medically safe.

Empirical parameters such as half-life are usually taken into account when determining the dose. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to increase the half-life of the antibody and prevent the antibody from being attacked by the host immune system. The frequency of administration may be determined and adjusted over the course of treatment and generally, but not necessarily, based on the success of treatment and/or suppression and/or attenuation and/or delay in progression of the target disease or disorder. Alternatively, continuous sustained release antibody formulations may be suitable. Various sustained release formulations and devices are well known in the art.

In one example, dosages for an antibody described herein can be determined empirically in individuals who have been administered the antibodies one or more times. Individuals are given additional doses of the antagonist. In order to assess the effectiveness of an antagonist, it is necessary to monitor indicators of the disease or disorder.

In general, for administration of any of the antibodies described herein, the initial dose may be about 2 mg/kg. For purposes of the present invention, a typical daily dose may vary from about any of 0.1 μg/kg to 3 μg/kg, 30 μg/kg, 300 μg/kg, 3 mg/kg, 30 mg/kg, 100 mg/kg, or more depending on the above factors. When administered multiple times over several days or longer, depending on the condition, treatment is continued until desired symptom suppression or until therapeutic levels sufficient to relieve the target disease or disorder or symptom are achieved. An exemplary dosing regimen includes administering an initial dose of about 2 mg/kg followed by a weekly maintenance dose of about 1 mg/kg of antibody, or followed by a maintenance dose of about 1 mg/kg every two weeks. However, other treatment regimens may also be appropriate, depending on the nature of the pharmacokinetic profile desired by the treating physician. For example, administration is provided 1-4 times a week. In some embodiments, administration in the range of 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 mcg/mg, approximately 1 mg/kg and approximately 2 mg/kg). In some embodiments, the frequency of administration is once per week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once a month, once every 2 months or every 3 months or at greater intervals. The success of this treatment is easily monitored by conventional methods and tests. The administration regimen (including the antibody used) may change over time.

In some embodiments, dosages in the range of about 0.3 to 5.00 mg/kg may be administered to an adult patient of normal weight. In some examples, the dose of an anti-FXII antibody described herein (eg, DX-4012) may be 10 mg/kg. The specific treatment regimen, namely dose, timing and frequency, will depend on the individual and his medical history, as well as the properties of the particular agents (such as the half-life of the agent and other parameters well known in the art).

For purposes of the present invention, the appropriate dose of an antibody described herein will depend on the specific antibody, antibodies and/or other non-antibody peptide (or composition thereof) used, the type and severity of the disease/disorder, whether the antibody is administered to prophylactic or therapeutic purposes, on previous therapy, the patient's medical history and response to the antagonist, and the judgment of the treating physician. Typically, the doctor will administer the antibody until the dose is reached to achieve the desired result. In some embodiments, the desired result is a reduction in thrombus formation. Methods for determining whether a dose has produced the desired result will be apparent to one skilled in the art. Administration of one or more antibodies may be continuous or at intervals, depending, for example, on the physical condition of the patient, whether the administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the antibody may be substantially continuous over a preselected period of time, or may be

- 25 045947 is a series of discrete doses spaced apart in time, for example, before, during or after the development of the disease or disorder being treated.

In the context of the present invention, the term treatment refers to the use or administration of a composition comprising one or more active agents to a subject who has the target disease or disorder, a symptom of a disease/disorder, or a predisposition to a disease/disorder, with the purpose of treating, healing, alleviating, reducing, change, assist, cure, improve, or influence a disorder, a symptom of a disease, or a predisposition to a disease or disorder.

Alleviation of the target disease/disorder includes delaying the development or progression of the disease, or reducing the severity of the disease. Alleviation of the disease does not necessarily require a cure. In the context of the present invention, delaying the development of a target disease or disorder means delaying, preventing, retarding, retarding, stabilizing and/or delaying the progress of the disease. This delay may vary in time depending on the medical history and/or individuals being treated. A method that delays or attenuates the development of a disease, or delays the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of a disease in a certain period of time, and/or reduces the severity of symptoms in a certain period of time, compared to when method is not used. Such comparisons are typically based on clinical studies using a sufficient number of subjects to produce statistically significant results.

Development or progression of a disease means the initial manifestations and/or subsequent development of a disease. Disease progression can be identified and assessed using standard clinical techniques well known in the art. However, the term development also refers to progress that may not be detectable. In the present invention, development or progress refers to the biological aspect of the symptoms. Development includes emergence, relapse and onset. In the context of the present invention, the term onset or occurrence of the target disease or disorder includes initial onset and/or relapse.

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

Standard methods known to those skilled in the art of medicine may be used to administer the pharmaceutical composition to a subject, depending on the type of disease being treated or the location of the disease. The composition may also be administered by other standard routes, for example, orally, parenterally, inhalation, topically, rectally, nasally, buccally, 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 methods. Additionally, the composition may be administered to a subject via depot injections, such as 1-, 3-, or 6-month depot injections, or via biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous administration, water-soluble antibodies can be administered by drip, in which a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is administered by infusion. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline or 5% glucose solution.

In one embodiment, the antibody is administered using site-specific or targeted local delivery methods. Examples of site-specific or targeted local techniques include various implantable antibody depots or local delivery catheters, such as infusion catheters, cavity or needle catheters, synthetic grafts, adventitial wraps, shunts and stents, or other implantable devices that are site-specific. specific carriers, direct injection or direct application. See, for example, PCT Publication No. WO 00/53211 and US Patent No. 5981568.

Targeted delivery of therapeutic compositions containing

- 26 045947 antisense polynucleotide, expression vector or subgenomic polynucleotides. 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 (J. A. Wolff, ed.) (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.

Therapeutic compositions containing a polynucleotide (eg, encoding the antibodies described herein) are administered in the range of about 100 ng to about 200 mg of DNA for topical administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 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 more may also be used in the protocol gene therapy.

The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery may be viral or non-viral (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 promoters or heterologous promoters and/or enhancers. Expression of a coding sequence may be constitutive or regulated.

Viral vectors for delivery of the desired polynucleotide and expression in the desired cell are well known in the art. Examples of viral carriers include, but are not limited to, recombinant retroviruses (see, for example, PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/ 10218; WO 91/02805; US Patent Nos. 5219740 and 4777127; UK Patent No. 2200651; and European Patent No. 0345242), alphavirus vectors (for example, vectors based on Sindbis virus, 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-923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532)) and adeno-associated virus vectors (AAV) (see, for example, PCT publications Nos. WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984 and WO 95/00655). Injection of DNA associated with a killed adenovirus may also be used, as described in Curiel, Hum. Gene Ther. (1992) 3:147.

Non-viral delivery vehicles and methods can also be used, including, but not limited to, condensed DNA on a polycationic carrier, bound or not bound to a killed adenovirus (see, for example, Curiel, Hum. Gene Ther. (1992) 3:147), ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985), vehicles for delivery to eukaryotic cells (see, e.g., US Pat. No. 5,814,482; PCT Publication No. WO 95/ 07994, WO 96/17072, WO 95/30763 and WO 97/42338) and neutralizing the charge of nucleic acids or fusion with cell membranes. DNA without carriers (naked) can also be used. Examples of methods for introducing naked DNA are described in PCT Publication No. WO 90/11092 and US Patent No. 5,580,859. Liposomes that can act as carriers for gene delivery are described in US Pat. No. 5,422,120; in PCT Publication Nos. WO 95/13796, WO 94/23697, WO 91/14445 and in European Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14: 2411, and in Woffendin, Proc. Natl. Academician Sci. (1994) 91:1581.

The specific schedule of administration, ie, dose, timing and frequency, used in the method described herein will depend on the individual subject and the medical history of that subject.

In some embodiments, multiple antibodies or a combination of an antibody and another suitable therapeutic agent may be administered to a subject in need of treatment. Antibodies may also be used in combination with other agents that serve to enhance and/or complement the effectiveness of the agents.

The effectiveness of treatment of the target disease/disorder can be assessed using methods well known in the art.

Kits for use in the treatment of diseases associated with activation of the contact system

The present invention also relates to kits for use in the treatment of diseases/disorders associated with activation of the contact system, such as HAE. Such kits may include one or more containers containing an anti-FXIIa antibody, such as any of those described herein.

In some embodiments, the kit may include instructions for use in accordance with any of the methods described herein. The included instructions may describe the administration of an anti-FXIIa antibody to treat, delay the onset of, or ameliorate the target disease described herein. The kit may further include a description of selecting an individual suitable for treatment based on whether the individual has the target disease. In other embodiments, the instructions include a description of administering the antibody to a person at risk for the target disease.

- 27 045947

Instructions related to the use of anti-FXIIa antibodies generally include information regarding dosage, dosage regimen, and route of administration for the intended treatment. Containers may be single doses, larger packages (eg, multi-dose packages), or multiple doses. The instructions provided in the kits of the invention are typically written instructions on a label or insert (eg, a sheet of paper included with the kit), but machine-readable instructions (eg, instructions provided on a magnetic or optical disk) are also acceptable.

The label or package insert indicates that the composition is used to treat, delay the onset of, and/or alleviate a disease or disorder associated with the pKal signaling pathway, such as HAE. Instructions may be provided for performing any of the methods described herein.

The kits of the present invention are provided in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar bags or plastic bags), and the like. Packages are also provided for use in combination with a specific device, such as an inhaler, a nasal delivery device (eg, a nebulizer), or an infusion device, such as a mini-pump. The kit may have a sterile entry port (eg, the container may be an intravenous solution bag or a vial having a stopper pierced by a hypodermic needle).

The container may also have a sterile entry port (eg, the container may be an intravenous solution bag or a vial having a stopper pierced by a hypodermic needle). At least one active agent in the composition is an anti-FXIIa antibody, such as those described herein.

The kits may optionally contain additional components, such as buffers, and information for interpreting the results. Typically, the kit includes a container and a label or insert on or associated with the container. In some embodiments, the present invention relates to articles including the contents of the kits described above.

General techniques

In the practice of the present invention, unless otherwise indicated, conventional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are known to those skilled in the art, will be used. Such methods are described in detail in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and S. S. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 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 (J. E. 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 manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, use the present invention to its fullest extent. The following specific embodiments are, therefore, to be considered illustrative only and not limiting of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter of the invention stated herein.

Examples

Example 1: Preparation of FXIIa Anti-Antibodies

Phage display selection using the FAB310 phagemid library from Dyax was performed using biotinylated FXIIa beta fragment immobilized on streptavidin-coated beads. Fab molecules that bind to immobilized FXIIa have been isolated. The selection yielded a selective Fab inhibitor that specifically bound to the catalytic domain of FXIIa with a Ki ^app of 800 pM. Isolated Fabs were not cross-reactive to the following proteases tested at 1 µM concentration: urokinase plasminogen activator, hepatocyte growth factor activator, activated protein C, cathepsin G, protein C1s, elastase, factor VIIa, factor Xa, factor XL·, plasmin, alpha-thrombin, trypsin, urokinase,

- 28 045947 plasma kallikrein, hepatocyte growth factor activator and urokinase-type plasminogen activator.

For the isolated Fab, M71-F06, affinity maturation was performed using several strategies, including heavy chain CDR1/2 shuffling, light chain shuffling, heavy chain CDR3 wobbling, and a combination of heavy chain CDR3 wobbling and heavy chain CDR1/2 shuffling. A heavy chain CDR1/2 shuffled library was constructed by removing the CDR1/2 region from M71-F06 and replacing it with a random sequence library to produce an antibody affinity maturation library with ~10 ⁸ diversity. The heavy chain CDR3 was swapped at each amino acid position in the parent CDR3 sequence from M71-F06 to generate a new library, which was also further combined with the heavy chain CDR1/2 shuffle. The second selection was performed using these libraries. In contrast to the selection performed with immobilized FXIIa on the parent library, affinity maturation libraries were incubated with the biotinylated target in solution at concentrations below the parent Ki ^app .

Biotinylated FXIIa and any bound Fab from the libraries were subsequently deposited onto streptavidin-coated beads. In fig. Figure 2 provides a diagram of the selection strategy for identifying and generating anti-FXIIa antibodies.

The inhibitory activity of clone M71-F06 against human and mouse FXIIa, determined by the in vitro activity assay described in Example 2 below, is shown in FIG. 4. The APTT value for clone M71-F06 (see Example 2 below) compared to DX-2930 is shown in FIG. 5. The results also show that clone M71-F06 has no inhibitory activity against activated protein C, protein C1s, cathepsin G, elastase, factor VIIa, factor Xa, factor XL·, activated plasma kallikrein, plasmin, alpha-thrombin, trypsin , urokinase, HGFA and uPA, indicating that its inhibitory activity is specific to FXIIa.

Any isolated Fabs after selection were screened by ELISA, resulting in 39 unique isolates. The amino acid sequences of the heavy chain variable regions and light chain variable regions are given above. These clones should have the same antigen-binding activity and specificity as the parent clone, with higher binding affinity.

Example 2: Characterization of Anti-FXIIa Antibodies

Methods:

Activated partial thromboplastin time (APTT) analysis

Inhibitors (or control dilution buffer: 25 mM HEPES, pH 7.5, 125 mM NaCl) were added to pure blood plasma (without additives) in a 1:1 mixture and pre-equilibrated at 37°C for 5 min. 2 x 50 µl of this mixture was dispensed into 2 separate KC4 Delta analytical cuvettes (with metal ball). After 60 s, 50 μl of the APTT reagent (activator, Pacific Hemostasis APTT-XL) was added to the rotating cuvettes and 180 s after the addition of the APTT reagent (at t = 0 sec), 50 μl of CaCl ₂ was added. The KS4 Delta device recorded the coagulation time in seconds.

Prothrombin time (PTT) analysis

Inhibitors (or control dilution buffer: 25 mM HEPES, pH 7.5, 125 mM NaCl) were added to pure blood plasma (without additives) in a 1:1 mixture and pre-equilibrated at 37°C for 5 min. 2 x 50 µl of this mixture was dispensed into 2 separate KC4 Delta analytical cuvettes (with metal ball). After 4 min, PTB activator (Pacific Hemostasis Thromboplastin D) was added (at t=0 s). Coagulation time was automatically recorded by the KS4 Delta device.

Inhibition assay of purified components

The inhibition assay of the purified components is depicted in FIG. 3, panel 1. 20 pM FXIIa was incubated with inhibitors at various concentrations for 1 hour at 30°C in a 96-well microplate. 10 nM prekallikrein was added for 20 min at 30°C, followed by incubation for 5 min with 100 nM corn trypsin inhibitor (CTI). Proteolysis was then assessed over time by adding a final concentration of 10 μM fluorogenic peptide substrate (PFR-AMC), with the initial rate of substrate proteolysis plotted (y-axis) versus inhibitor concentration (x-axis) and fitting the resulting data to a modified equation Morrison (equation 1) for highly 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.

Plasma Inhibition Assay

The plasma inhibition assay is depicted in FIG. 3, panel 2. Pooled normal human plasma was diluted 1:40 in assay buffer (see above) and inhibitors were added at various concentrations to a 96-well microplate at room temperature. Contact activation was then initiated by adding 25% (2.5% final concentration) ellagic acid, the contents of the microplate were mixed by gentle shaking, and the reaction was allowed to proceed for 2 min at room temperature, after which 100 nM CTI was added. 10 µl of this mixture was then transferred to a replica microplate containing 80 µl of assay buffer, pre-equilibrated

- 29 045947 ny at 30°C. This dilution plate was incubated for an additional 5 min at 30°C, and PFR-AMC proteolysis was assessed as described above, but with the inhibitor concentration calculated back as the x-axis when fitting the curve to the standard IC ₅₀ equation (plasma dilution was 1: 400 at the final spectrophotometric measurement in the analysis).

One or more of the anti-FXIIa antibodies disclosed herein were also tested in a non-human primate model at two different dosages. The study was conducted for 30 days, after which blood samples were collected to evaluate pharmacokinetics, effects on aPTT, and effects on plasma kallikrein activation using Western blot analysis.

Preferential binding assay

To determine the preferential binding of anti-FXIIa antibodies, each antibody was incubated with FXII zymogen or other related contact/coagulation system proteases. Binding affinity was then assessed by surface plasmon resonance (SPR) analysis using the Biacore biosensor. See fig. 3, panel 3.

Occlusion Analysis in a Flow/Capillary Model

The antibodies are tested in an ex vivo flow model in which human blood spiked with antibodies is passed at varying flow rates through collagen-coated capillaries. Platelet and fibrin sedimentation is assessed by fluorescence. Platelets and fibrin deposition are inhibited in this system by antibodies that bind to factor XI or factor XII zymogen. In a typical experiment, antibodies are allowed to bind to their targets before blood flow is initiated.

Mouse model of thrombosis

The antibodies are tested in a mouse model of ferric chloride-induced carotid artery thrombosis. The model includes different concentrations of FeC'l·, starting with the lowest concentration, which successively induce thrombus formation in C57B1/6 mice (3.5%). If the antibody exhibits an antithrombotic effect at a low concentration of FeC'l·, the concentration is increased until the antithrombotic activity of the antibody is depleted.

Results:

anti-FXIIa antibody isolates were tested in various in vitro activity assays to determine the following properties: apparent Ki, IC ₅₀ , preferential binding, e.g. binding to FXII zymogen, cross-reactivity with other related proteases in the contact/coagulation system, cross-reactivity with closely related sequence homologs, effect on plasma kallikrein (pKal) formation, activity in human plasma, prothrombin time (PTT), activated partial thromboplastin time (APTT) and thrombin latency time.

The results of the analyzes are presented in table. 3. All 39 isolates were found to increase aPTT without having any effect on PT. Each of the 39 affinity mature isolates had a 10- to 100-fold increase in Ki ^app compared to the parent isolate. The antibodies reduced pKal production, which was confirmed by two independent inhibition assays: a purified component inhibition assay and a plasma inhibition assay.

By SPR assay (Biacore), the antibodies do not bind FXII zymogen and are specific for the catalytic domain when tested with full-length FXIIa. The antibodies also prevent the activation of FXI to FXIa.

- 30 045947

Table 3

ISOLATE Plasmatic Ki ^a ₽₽ (nM) APTT (fold increase) Ki ^app (pm) M0191-E09 31 3.70 5 М0183-С03 33 3, 17 6 M0184-F12 38 3.74 4 М0183-Н08 76 3.36 5 M0182-D04 96 3, 13 eleven М0192-Н11 101 3.33 6 M0292-D07 115 3, 11 14 M0183-D08 118 2.08 eleven M0192-F07 125 3.00 7 M0192-G03 125 3, 14 18 М0310-С06 135 2.46 57 M0192-F01 174 3, 18 8 M0191-A03 176 2.88 13 M0192-A01 198 3, 11 17 М0191-Н10 216 3, 81 13 М0177-С12 217 3.86 18 М0184-В04 269 3.76 8 М0308-Н03 275 3.38 18 M0192-D02 279 3.30 5 M0310-G07 295 4.48 22 M0192-F06 304 3.49 9 M0310-A04 316 3.48 15 M0310-F02 319 1.86 106 М0310-В09 331 3.73 12 M0310-G06 353 3, 18 7 М0182-Н01 359 No data No data M0178-A08 381 3.26 7 M0184-D01 435 3.32 4 M0177-A06 458 4.08 17 M0191-E04 460 3.59 7 М0191-Н09 463 3, 61 16 М0192-Н04 485 3.51 9 M0192-A03 514 3.34 13 M0310-G08 796 2.76 24 M0192-G05 852 3.38 15 М0183-В12 1004 3, 06 eleven M0308-F04 1570 3.29 17 М0182-В04 No data No data No data M0310-F04 No data No data No data

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

Anti-FXna antibodies M192-M192 and H11-E09 were also tested in in vivo pharmacokinetic experiments in rats. Groups of rats were injected with 20 mg/kg of anti-FXIIa antibodies M191-E09 or M192-H11. On various days after injections, samples were collected from rats and assessed for anti-FXIIa antibody concentrations and pharmacokinetic parameters (Fig. 6 and Table 4).

Table 4. Pharmacokinetic characteristics of antibodies M192-H11 and M191-E09

Antibody of interest Smack (µg/ml) AUCo-last (h*mcg/ml) CI (ml/h/kg) Vss (ml/kg) tl/2 (h) M192-N11 360 18375 0.92 242 220 M191-E09 269 7612 2.28 489 218

Example 3. Preparation and characterization of anti-FXIIa antibody 559C-X211-A01

559C-X211-A01 is an affinity mature, partially germline version of the parent antibody 559C-M71-F06 described above.

The amino acid sequences of the variable heavy chain and variable light chain of this antibody are shown below: 559C-X211-A01_HV (CDRs are underlined and in bold) EVQLLESGGGLVQPGGSLRLSCAASGTFFS QYVMH WVRQAPGKGLEWVS

SIWPSGGHTRYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR

QRYRGPKYYYYMDV WGKGTTVTVSS (SEQ ID NO:125)

559C-X211-A01_LV (CDRs are underlined and in bold) DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPGQSPQLLIY LGSNRAS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQALQTPWT FGQGTKVEIK (SEQ ID NO:126)

- 31 045947

559C-X211-A01 was tested in various in vitro activity assays to determine the following properties:

apparent K _i , IC ₅₀ , binding to zymogen FXII, cross-reactivity with other related proteases in the contact/coagulation system, cross-reactivity with closely related sequence homologues, effect on plasma kallikrein (pKal) formation and activity in human plasma. Prothrombin time (PTT) and activated partial thromboplastin time (APTT) in mouse plasma were also determined in vitro.

Cross-reactivity assay

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

Activated partial thromboplastin time (aPTT) and prothrombin time (PTT) tests

Inhibitors (or control dilution buffer: 25 mM HEPES, pH 7.5, 125 mM NaCl) were added to pure (no additives) mouse and human plasma in a 1:1 mixture and pre-equilibrated at 37°C for 5 min. 2 x 50 µl of this mixture was dispensed into 2 separate KC4 Delta analytical cuvettes (with metal ball). After 60 s, 50 μl of APTT reagent (activator, Pacific Hemostasis APTT-XL) was added to the rotating cuvettes and 180 s after this (at t = 0 s) 50 μl of CaCl ₂ was added. The KS4 Delta device recorded the coagulation time in seconds.

As above, except that after a 4-minute incubation, after dividing 2x50 μl of the inhibitor mixture with plasma into 2 separate analytical cuvettes KC4 Delta, the PTB activator (Pacific Hemostasis Thromboplastin D) was added at t = 0 s. Coagulation time was automatically recorded by the KS4 Delta device.

559C-X211-A01 increases aPTT in mouse plasma, while simultaneously having no effect on PTT. See fig. 7. These data support species cross-reactivity with murine factor XII.

Inhibition assay of purified FXIIa PM components are incubated with inhibitors at various concentrations for 1 hour at 30°C in a 96-well microplate. 10 nM prekallikrein was then added for 20 min at 30°C, followed by incubation with 100 nM corn trypsin inhibitor (CTI) for 5 min. Proteolysis is then assessed over time by adding a final concentration of 10 μM fluorogenic peptide substrate (PFR-AMC), with the initial rate of substrate proteolysis plotted (y-axis) versus inhibitor concentration (x-axis) and fitting the resulting data to a modified equation Morrison for highly 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.

Plasma Inhibition Assay

Pooled normal human plasma is diluted 1:40 in assay buffer (see above) and inhibitors are added at various concentrations to a 96-well microplate at room temperature. Contact activation is then initiated by adding 25% (2.5% final concentration) APTT-XL reagent (dilute ellagic acid; Pacific HAOmostasis), the contents of the microplate are mixed by gentle shaking, and the reaction is allowed to proceed for 2 min at room temperature. after which 100 nM CTI is added. 10 μl of this mixture is then transferred to a replica microplate containing 80 μl of assay buffer, pre-equilibrated at 30°C. This dilution plate is incubated for an additional 5 min at 30°C, and PFR-AMC proteolysis is assessed as described above, but with the inhibitor concentration calculated back as the x-axis when the curve is fitted to the standard FE equation and/or the modified Morrison equation ( plasma dilution is 1:400 at the final spectrophotometric measurement in the assay).

Data on the activity of 559C-X211-A01 observed in the above analyzes are given in table below. 5.

Table 5. Activity of 559С-Х211-А01

Isolate Plasma I IC50 (nM) APTT (fold increase) using 1 µM X211AO1 in human plasma APTT (fold increase) using 1 µM X211-A01 in mouse plasma Ki ^a pp (pm) 559С-Х211-А01 289 3.4 2.8 5

According to SPR analysis, this antibody binds to FXIIa and does not bind the FXII zymogen. Enzyme inhibition experiments with FXIIa-beta showed specificity for the catalytic domain.

It is expected that 559C-X211-A01 reduces the formation and deposition of fibrin, increasing the time

- 32 045947 which requires blood to occlude a collagen-coated capillary in a flow/capillary model occlusion assay, and/or to reduce platelet aggregation and the incidence of arterial occlusion. 559C-211-A01 is expected to reduce the incidence of blood clots in both a mouse model of thrombosis and a non-human primate model of thrombosis.

Example 4: In Vivo Thrombosis Studies Using DX-4012

Pharmacokinetic studies

The anticoagulant effect of a single dose of DX-4012 was assessed in healthy baboons as a pharmacodynamic marker for the presence of antibody in circulating blood. Animals were lightly sedated and blood was collected from the cubital vein to obtain baseline coagulation values. Then the antibody was administered in a saturating dose. A single dose of antibody was administered intravenously and blood samples were collected at various time points until an anticoagulant effect was observed in the aPTT assay relative to baseline. Plasma samples were frozen for additional testing.

After completion of pharmacokinetic studies and determination of the effective half-life of the antibody in baboons, pharmacodynamic studies of thrombosis and hemostasis were continued.

Pharmacodynamic study of thrombosis and hemostasis

Acute thrombosis and hemostasis experiments were performed before and during anticoagulation therapy to determine the efficacy and safety of the antibody as a single loading dose compared to positive and negative controls. Thrombosis experiments were performed in trained baboons by inserting a segment of vascular graft into a permanent externalized femoral AV shunt. On each study day, 1 mL of blood (with citrate) was withdrawn from the shunt loop at various time points for serial bleeding time tests, volume measurements, and anticoagulant effect assays. Sampling times include pre-test drug administration (on the day the animal received enoxaparin or DX-4012 before enoxaparin or DX-4012 administration), pre-study (5 min before the start of the study), and post-study (5 min before the end of the study). studies when the shunt loop was removed). Before experiments studying thrombosis and hemostasis, baboons received In-radiolabeled autologous platelets and ²⁵ 1-radiolabeled fibrinogen. The antithrombotic activity of DX-4012 was compared with enoxaparin (low molecular weight heparin).

After baseline blood samples were collected, thrombosis experiments began. The permanent AV shunt was temporarily (60 min) extended with silicone tubing, and a short segment of collagen-coated ePTFE graft or growth factor-coated ePTFE vascular graft (20 mm) was inserted into the external AV loop (Fig. 10). A thrombus forms on the graft (thrombus head) and elongates on the distal side (thrombus tail). The accumulation of radiolabeled platelets in the graft was monitored in real time using a gamma imaging camera for 60 minutes and calculated as the number of platelets deposited onto the graft every 5 minutes. After 60 min of perfusion of the graft, it was removed, the shunt was reconnected, and the amount of fibrin deposited on the graft at 60 min (end point) was determined after decay '''In ( ³⁰⁺ days after approximately '0 half-lives). Fibrin and platelet count represent the size of the clot. Results APTT and PTT assays were performed as described in Examples 2 and 3. APTT was measured using citrated plasma, Syntha Sil® reagent (Instrumentation Laboratories) and a KS-4 coagulometer. PTT was measured using citrated plasma, Dade® Innovin® reagent (Siemens) and a KS-4 coagulometer. Both aPTT and PTW were measured immediately after blood collection and centrifugation of blood samples to obtain platelet-poor plasma. One hour after administration of DX-40'2, the measured change in aPTT was 2.2-fold (expected >2.2-fold). After 24-48 hours, the measured change in APTT was '.8 times (with the expected '.8-2.0 times), and after ' a week the measured change in APTT was '.3 times (with the expected '.3-'.5 times) . No changes in PTT were observed.

Collagen-coated graft

For thrombosis studies, animals received '0 mg/kg DX-40'2 antibody and studies were performed at ', 24, 48, '68 and '92 hours after antibody administration. For analysis, data from the 24 and 48 hour time points were grouped together and data from the '68 and '92 hour time points were also grouped together. As expected, control animals developed a collagen-initiated thrombus head (baseline). The thrombus head was significantly smaller in animals treated with enoxaparin. At time points up to 48 hours after antibody administration, thrombus head size was smaller compared to control animals and was comparable to that of animals that received enoxaparin. One week after antibody administration ('68-'92 hours), the size of the thrombus head was comparable to that of control animals (Fig. 'A'). At time points up to 48 hours after antibody administration, the thrombus tail was virtually absent and was comparable to that in animals treated with enoxaparin. One week after antibody administration ('68-'92 hours), the thrombus tail remained reduced, but to a lesser extent

- 33045947 degrees than after the administration of enoxaparin (Fig. 11B).

In another experiment, at time points up to 48 hours after antibody administration, the size of the thrombus head was smaller compared to control animals and was comparable to that in animals that received enoxaparin. One week after antibody administration (168-192 hours), the size of the thrombus head was still reduced, but to a lesser extent than after enoxaparin administration (Fig. 12). Fig. 13 represents thrombus formation (total thrombus), indicating that thrombus formation was comparable at 1, 24 and 48 hours after antibody administration. The rate of thrombus growth (platelet deposition every 5 minutes) is shown in Fig. 14. In the control thrombus, the growth rate increased during the first 30 minutes until a plateau was reached. Growth rate after enoxaparin administration reached a lower plateau at 20 min, began to decline at 40 min, and thrombosed at 60 min (negative growth rate). During the first 48 hours after administration of DX-4012, the growth rate reached a plateau after 15-20 minutes and was reduced. One week after antibody administration, blood clots were between the early time points (1 hour and 24-48 hours) and control.

Overall, administration of 10 mg/kg DX-4012 reduced thrombus size after initiation of thrombus formation by collagen, an effect that was observed for both thrombus head and thrombus tail size. A reduction in thrombus size was observed up to 48 hours after antibody administration and was comparable to enoxaparin (1 mg/kg). After one week, the head of the clot returned to baseline levels in animals that received the antibody, but the size of the tail clot remained reduced.

Tissue Factor Coated Graft

In general, thrombus size tends to vary more in tissue factor-coated shunts compared to collagen-coated shunts. In the experiments described, large variations were observed (7.4, 14.0 and 2.3 billion platelets). As expected, control animals developed a tissue factor-initiated thrombus head (baseline). The thrombus head was significantly reduced in animals treated with enoxaparin. At time points up to 48 hours after antibody administration, the size of the thrombus head was comparable to that of control animals (Fig. 15A). One week after antibody administration (168-192 hours), thrombus head sizes varied greatly. Administration of enoxaparin virtually eliminated tissue factor-initiated tail thrombus formation. There was no significant reduction in thrombus tail in antibody-treated animals, although the data suggested that there was a slight trend towards a reduction in thrombus tail (FIG. 15B).

In another experiment, administration of enoxaparin resulted in a reduction in thrombus size by more than 50%, largely due to the virtual disappearance of the thrombus tail. There was no significant reduction in thrombus tail in antibody-treated animals, although the data suggested that there was a slight trend towards a reduction in thrombus tail (FIG. 16). In fig. 17 shows the formation of a thrombus (total thrombus). Analysis of the data using RM ANOVA showed that there was a decrease in total thrombus size 1 hour after antibody administration compared to control. The rate of thrombus growth (platelet deposition every 5 is shown in Fig. 18. For the control thrombus, the growth rate increased during the first 30 minutes until a plateau was reached. The growth rate after enoxaparin administration reached a lower plateau after 20 minutes, began to decrease after 40 minutes, and the thrombus was lysed after 60 minutes (negative growth rate).During the first 48 hours after administration of DX-4012, the rate of thrombus growth was similar to that observed in control animals (Fig. 18).

In general, administration of 10 mg/kg DX-4012 has no effect on thrombus head size and has little effect on thrombus tail size in tissue factor-initiated thrombus formation.

Final fibrin content and platelet sedimentation

At the end of the experiment, the shunts were flushed with saline and images were obtained to assess the final platelet count. The loop was also saved for analysis of fibrin content in the head and tail of the thrombus.

The final thrombus size was reduced in animals treated with enoxaparin. Thrombus size was also significantly reduced, as measured by platelet sedimentation and fibrin content, in collagen-coated shunts in animals that received DX-4012, but was not reduced in tissue factor-coated shunts (FIGS. 19A and 19B).

Hemostasis studies

Hemostasis was measured using the FDA-approved Surgicutt™ bleeding time device and protocol. Blood volume was also measured. One measurement of bleeding time and bleeding volume was performed on one study day. As shown in FIG. 22A and 22B, DX-4012 was effective in reducing bleeding time and bleeding volume compared with enoxaparin.

Example 5: Identification of Critical Residues in the FXIIa Catalytic Domain Based on the Crystal Structure of the DX-4012-FXIIa Complex

The recombinant Fab fragment of DX-4012 was produced using conventional recombinant technology in E. coli and purified. FXIIa was prepared and purified using conventional methods.

- 34 045947

The DX-4012 Fab fragment and FXIIa were mixed at various concentrations under suitable conditions to allow the formation of the Fab-FXIIa complex. The complexes were purified using SEC and visualized using the traces shown in Fig. 20.

The Fab-FXIIa complex was maintained under various conditions to promote crystallization. Diffraction analysis was carried out on the crystallized complex. Crystal structures (2.6 A and 2.25 A) were determined based on statistical processing of diffraction data.

In accordance with the crystal structures, residues in the C-chain of FXIIa were identified that are involved in the interaction with the Fab fragment from DX-4012: 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, G5 89 , C590, G591, D592, G597.

In addition, residues in Fab from 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.

These results indicate that the heavy chain of DX-4012 is the main region that interacts with FXIIa, and several residues in the LC CDR1 were found to contribute to the interaction.

Crystal structures also showed that residue R102 of the anti-FXIIa antibody Fab fragment binds the S1 pocket of FXIIa and interacts with residue D557 via water-mediated interactions. This positions the framework of Fab residues R102-G103-P104 close to the FXIIa catalytic triad, but in a catalytically incompetent orientation. P104 may contribute to the rigid retention of this orientation, thereby inhibiting FXIIa activity.

Complexes of other anti-FXIIa antibodies and their Fab fragments with FXIIa were also prepared and co-purified to determine additional crystal structures.

Other embodiments

All features disclosed in this description can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

From the above description, one skilled in the art can readily determine the essential characteristics of the present invention and, without departing from the spirit and scope of the invention, can make various changes and modifications to the invention to adapt it to various applications and conditions. Thus, other embodiments are also included within the scope of the claims.

Equivalents

Although some innovative embodiments have been described and illustrated herein, those of ordinary skill in the art will readily imagine many other means and/or structures for performing the function and/or obtaining the results and/or one or more benefits described herein , and each such variation and/or modification is considered to be within the scope of the innovative embodiments described herein. More generally, those skilled in the art will readily understand that all parameters, dimensions, materials and configurations described herein are provided by way of example, and that actual parameters, dimensions, materials and/or configurations will depend on the particular embodiment. or applications that use innovative ideas. Those skilled in the art will recognize, or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is therefore to be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced other than as specifically described and claimed. Embodiments of the invention herein are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, products, materials, kits and/or methods, unless such features, systems, products, materials, kits and/or methods are mutually incompatible, is included within the scope of the present invention.

All definitions described and used herein are to be understood to supersede the standard definitions given in the documents incorporated by reference and/or the ordinary meanings of the terms defined.

All references, patents and patent applications disclosed in this specification are incorporated by reference to the subject matter to which each reference is provided, which in some cases may include the entire document.

-

Claims (23)

The singular number used herein in the description and claims, unless expressly stated to the contrary, is to be understood as at least one. The phrase and/or, as used herein in the specification and claims, is to be understood to mean one or both of the elements thus combined, that is, elements that are present together in some cases and are present separately in other cases. The plurality of elements listed with and/or should be interpreted in the same way, that is, one or more elements combined in this way. Optionally, other elements may be present other than those specifically identified by the clause and/or whether or not related to those specifically identified elements. Thus, by way of non-limiting example, a reference to A and/or B, when used in conjunction with an open expression such as containing, may refer to: in one embodiment, only A (optionally including elements other than B); in another version - only to B (optional, including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on. In the context of the present specification and claims, the conjunction or should be considered to have the same meaning as the conjunction of conjunctions and/or, as defined above. For example, when separating items in a list, the conjunctions either or and/or should be interpreted as inclusive, that is, including at least one, but also including more than one of a series or list of items and, optionally, additional unspecified items. Only terms clearly indicating the contrary, such as only one of or exactly one of, or (when used in a claim) consisting of, will refer to the inclusion of one element from a series or list of elements. In general, the term or, as used herein, should be interpreted only as indicating exclusive alternatives (i.e., one or the other, but not both) when preceded by an indication of exclusivity, such as either, one of, only one of or exactly one of. The phrase consisting essentially of when used in a claim will have its ordinary meaning as used in the field of patent law. As used herein and in the claims, the phrase at least one, with respect to a list of one or more elements, is to be understood to mean at least one element selected from one or more elements in the list of elements, but not necessarily including at least one of each individual element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified in the list of elements to which at least one phrase applies, whether or not they are related to those specifically identified elements. Thus, by way of non-limiting example, at least one of A and B (or likewise at least one of A or B, or likewise at least one of A and/or B) may be, in one embodiment, at least to one, optionally including more than one A in the absence of B (and, optionally, including elements other than B); in another embodiment, to at least one, optionally including more than one B, in the absence of A (and optionally including elements other than B); in yet another embodiment, to at least one, optionally including more than one A, and to at least one, optionally including more than one B (and, optionally, including other elements); and so on. It should also be understood that, unless expressly stated to the contrary, in any methods disclosed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the stated order of steps or acts of the method. CLAIM 1. A monoclonal antibody that binds to factor XIIa (FXIIa) and does not bind to factor XII (FXII), comprising a heavy chain that contains a heavy chain variable region, and a light chain that contains a light chain variable region, wherein the heavy chain variable region contains complementarity determining region 1 (CDR1) of the heavy chain (HC), HC CDR2 and HC CDR3), where: (i) HC CDR1 contains QYVMH (SEQ ID NO: 58), HC CDR2 contains SIWPSGGHTRYADSVKG (SEQ ID NO: 75), HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (ii) HC CDR1 contains WYSMH (SEQ ID NO: 41), HC CDR2 contains VIYPSGGKTRYADSVKG (SEQ ID NO: 74) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (iii) HC CDR1 contains QYVMH (SEQ ID NO: 42), HC CDR2 contains SIWPSGGHTRYADSVKG (SEQ ID NO: 75) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (iv) HC CDR1 contains WYVMH (SEQ ID NO: 43), HC CDR2 contains GIWPSGGRTKYADSVKG (SEQ ID NO: 76) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (v) HC CDR1 contains NYVMH (SEQ ID NO: 44), HC CDR2 contains SIWPSGGKTKYADSVKG - 36 045947 (SEQ ID NO: 77) and HC CDR3 contains QRYRGPKYYYYMDA (SEQ ID NO: 112); (vi) HC CDR1 contains MYTMN (SEQ ID NO: 45), HC CDR2 contains RIYPSGGKTLYADSVKG (SEQ ID NO: 78) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (vii) HC CDR1 contains QYVMS (SEQ ID NO: 46), HC CDR2 contains RIYPSGGVTKYADSVKG (SEQ ID NO: 79) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (viii) HC CDR1 contains WYNMH (SEQ ID NO: 47), HC CDR2 contains YISPSGGKTKYTDSVKG (SEQ ID NO: 80) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (ix) HC CDR1 contains RYIMH (SEQ ID NO: 48), HC CDR2 contains SIYPSGGVTKYADSVKG (SEQ ID NO: 81) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (x) HC CDR1 contains RYIMG (SEQ ID NO: 49), HC CDR2 contains SIYPSGGVTRYADSVKG (SEQ ID NO: 82) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xi) HC CDR1 contains QYNMV (SEQ ID NO: 50), HC CDR2 contains RIWPSGGKTTYADSVKG (SEQ ID NO: 83) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xii) HC CDR1 contains RYVMV (SEQ ID NO: 51), HC CDR2 contains RIYPSGGMTQYADSVKG (SEQ ID NO: 84) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xiii) HC CDR1 contains WYNMA (SEQ ID NO: 52), HC CDR2 contains RIYPSGGMTQYADSVKG (SEQ ID NO: 84) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xiv) HC CDR1 contains QYIMH (SEQ ID NO: 53), HC CDR2 contains SIYPSGGNTKYADSVKG (SEQ ID NO: 85) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xv) HC CDR1 contains HYVMH (SEQ ID NO: 54), HC CDR2 contains SIYPSGGLTKYADSVKG (SEQ ID NO: 86) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xvi) HC CDR1 contains WYTMH (SEQ ID NO: 55), HC CDR2 contains SIYPSGGFTRYADSVKG (SEQ ID NO: 87) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xvii) HC CDR1 contains FYHMH (SEQ ID NO: 56), HC CDR2 contains RIVPSGGMTRYADSVKG (SEQ ID NO: 88) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xviii) HC CDR1 contains FYSMH (SEQ ID NO: 57), HC CDR2 contains RIYPSGGVTKYADSVKG (SEQ ID NO: 89) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xix) HC CDR1 contains QYVMH (SEQ ID NO: 58), HC CDR2 contains SIWPSGGKTTYADSVKG (SEQ ID NO: 90) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xx) HC CDR1 contains QYVMH (SEQ ID NO: 58), HC CDR2 contains SIWPSGGFTKYADSVKG (SEQ ID NO: 91) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxi) HC CDR1 contains FYNMH (SEQ ID NO: 59), HC CDR2 contains SIYPSGGVTRYADSVKG (SEQ ID NO: 92) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxii) HC CDR1 contains WYVMH (SEQ ID NO: 43), HC CDR2 contains SIYPSGGKTSYADSVKG (SEQ ID NO: 93) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxiii) HC CDR1 contains PYIMH (SEQ ID NO: 60), HC CDR2 contains VIYPSGSKTNYADSVKG (SEQ ID NO: 94) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxiv) HC CDR1 contains RYTMR (SEQ ID NO: 61), HC CDR2 contains SIWPSGGMTRYADSVKG (SEQ ID NO: 95) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxv) HC CDR1 contains QYVMH (SEQ ID NO: 58), HC CDR2 contains SIYPSGGLTRYADSVKG (SEQ ID NO: 96) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxvi) HC CDR1 contains WYIMG (SEQ ID NO: 62), HC CDR2 contains YIYPSGGNTRYADSVKG (SEQ ID NO: 97) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxvii) HC CDR1 contains RYVMH (SEQ ID NO: 63), HC CDR2 contains SIWPSGGMTKYADSVKG (SEQ ID NO: 98) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxviii) HC CDR1 contains FYIMG (SEQ ID NO: 64), HC CDR2 contains RIYPSGGATQYADSVKG (SEQ ID NO: 99) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxix) HC CDR1 contains FYVMG (SEQ ID NO: 65), HC CDR2 contains RIYPSGGLTQYADSVKG (SEQ ID NO: 100) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxx) HC CDR1 contains FYSMH (SEQ ID NO: 66), HC CDR2 contains RIYPSGGITSYADSVKG (SEQ ID NO: 101) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxi) HC CDR1 contains MYIMH (SEQ ID NO: 67), HC CDR2 contains SIYPSGGMTKYADSVKG (SEQ ID NO: 102) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxii) HC CDR1 contains MYVMH (SEQ ID NO: 68), HC CDR2 contains SIYPSGGLTKYADSVKG (SEQ ID NO: 103) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxiii) HC CDR1 contains QYVMH (SEQ ID NO: 58), HC CDR2 contains RIYPSGGLTNYADSVKG (SEQ ID NO: 104) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxiv) HC CDR1 contains WYVMQ (SEQ ID NO: 69), HC CDR2 contains SIYPSGGMTKYADSVKG (SEQ ID NO: 102) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: - 37 045947 111); (xxxv) HC CDR1 contains WYIMG (SEQ ID NO: 62), HC CDR2 contains RIYPSGGSTHYADSVKG (SEQ ID NO: 105) and HC CDR3 contains QRYRGPRYYYYIDA (SEQ ID NO: 113); (xxxvi) HC CDR1 contains QYTMV (SEQ ID NO: 70), HC CDR2 contains RIYPSGGVTQYADSVKG (SEQ ID NO: 106) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxvii) HC CDR1 contains WYVMY (SEQ ID NO: 71), HC CDR2 contains RIYPSGGITHYADSVKG (SEQ ID NO: 107) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxviii) HC CDR1 contains FYVML (SEQ ID NO: 72), HC CDR2 contains SIWPSGGVTKYADSVKG (SEQ ID NO: 108) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxix) HC CDR1 contains WYVMQ (SEQ ID NO: 69), HC CDR2 contains YIYPSGGHTKYADSVKG (SEQ ID NO: 109) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); (xxxx) HC CDR1 contains RYSMN (SEQ ID NO: 73), HC CDR2 contains GIYPSGGKTKYADSVKG (SEQ ID NO: 110) and HC CDR3 contains QRYRGPKYYYYMDV (SEQ ID NO: 111); the light chain (LC) variable region contains LC CDR1 containing the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 114), LC CDR2 containing the amino acid sequence LGSNRAS (SEQ ID NO: 115), and LC CDR3 containing the amino acid sequence MQALQTPWT (SEQ ID NO : 116); and where the antibody is not a bispecific antibody. 2. The antibody according to claim 1, where the antibody is a full-length antibody or an antigen-binding fragment thereof. 3. The antibody of claim 2, wherein the antibody is a Fab. 4. An antibody according to any one of claims 1 to 3, wherein the antibody is a human antibody. 5. An antibody according to any one of claims 1 to 3, wherein the antibody is a humanized antibody. 6. An antibody according to any one of claims 1 to 5, wherein the heavy chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NO: 1-39 and SEQ ID NO: 125. 7. The antibody of claim 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 125. 8. An antibody according to any one of claims 1 to 7, wherein the light chain comprises a light chain variable region that contains the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 126. 9. An antibody according to any one of claims 1 to 8, wherein the heavy chain further comprises a heavy chain constant region. 10. The antibody of claim 9, wherein the heavy chain constant region contains at least one mutation that increases the half-life of the antibody compared to its wild-type counterpart, wherein the at least one mutation is at a position corresponding to positions 145, 147, or 149 of SEQ ID NO: 119. 11. The antibody of claim 10, wherein the at least one mutation is an amino acid substitution selected from the group consisting of M145Y, S147T and T149E. 12. The antibody of claim 10, wherein the heavy chain constant region comprises the amino acid sequence SEQ ID NO: 120. 13. An isolated nucleic acid that encodes an antibody according to any one of claims 1 to 12. 14. An expression vector containing the nucleic acid according to claim 13. 15. Host cell containing the vector of item 14. 16. The cell of claim 15, wherein the cell is a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. 17. A method for producing an antibody that binds to factor XIIa (FXIIa), comprising culturing cells according to claim 15 or 16 in a culture medium and collecting the cultured cells or culture medium to isolate the antibody. 18. Pharmaceutical composition containing: (a) an antibody according to any one of claims 1 to 12, a nucleic acid according to claim 13 or a vector according to claim 14; and (b) a pharmaceutically acceptable carrier. 19. A method of treating a disease associated with activation of the contact system, the method comprising administering to a subject in need thereof an antibody according to any one of claims 1 to 12, a nucleic acid according to claim 13, a vector according to claim 14, or a pharmaceutical composition according to claim 18. 20. The method according to claim 19, where the disease is hereditary angioedema (HAE). 21. The method according to claim 20, where the HAE is type 1, type 2 or type 3 HAE. 22. The method of claim 19, wherein the disease is an ocular disease. 23. The method of claim 22, wherein the subject has, is suspected of having, or is at risk of an eye disease -